# Patent

## Title
Peptide aptamers that bind to the rep proteins of ssDNA viruses

## Abstract

Polypeptides and fusion proteins that bind to eukaryotic viruses, in particular, eukaryotic single-stranded DNA (ssDNA) viruses are provided. The polypeptides and fusion proteins bind to the replication initiation proteins (Rep) of ssDNA viruses and optionally inhibit viral replication and/or viral infection. The virus can be a plant pathogen or animal pathogen. Consensus sequences used to identify polypeptides that bind to eukaryotic viruses are also provided.

## Background
RELATED APPLICATION DATA
The present application is a divisional of U.S. patent application Ser. No. 11/995,973 filed Jun. 23, 2008, now U.S. Pat. No. 8,168,748 (now allowed), which is a 35 U.S.C. §371 national phase application of PCT Application No. PCT/US2006/030941, filed Aug. 4, 2006, which claims the benefit of U.S. provisional application Ser. No. 60/705,426, filed Aug. 4, 2005, the disclosures of which are incorporated herein by reference in their entireties.


STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5051-726DV_ST25.txt, 65,735 bytes in size, generated on Jan. 28, 2015 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
FIELD OF THE INVENTION
The present invention relates to products and methods for detecting viral infections and inhibiting viral replication and products resulting therefrom.
BACKGROUND OF THE INVENTION
Single-stranded DNA (ssDNA) viruses cause severe disease problems in plants and animals (Moffat (1999) Science 286:1835). Geminiviruses and nanoviruses infect many important crops worldwide, such as cassava, bean, pepper, tomato, sugar beet, cotton and maize (Brown and Bird (1992) Plant Disease 7:220-225; Czosnek and Laterrot (1997) Arch. Virol. 142:1391-1406; Lotrakul, et al. (1998) Plant Dis. 82:1253-1257; Zhou, et al. (1997) J. Gen. Virol. 78:2101-2111; Mansoor, et al. (1999) Virology 259:190-199; Polston, et al. (1999) Plant Dis. 83:984-988). Circoviruses cause significant disease losses among livestock and poultry (Allan, et al. (1998) J. Vet. Diagn. Invest. 10:3-10; Bassami, et al. (1998) Virology 249:453-9; Nayar, et al. (1999) Can. Vet. J. 40:277-8). A human circovirus in Hepatitis C patients has also been identified (Miyata, et al. (1999) J. Virol. 73:3582-3586; Mushahwar, et al. (1999) Proc. Natl. Acad. Sci. USA. 96:3177-3182). Even though these viruses have diverse host ranges and cause different diseases, they are highly related to each other.
Geminiviruses, nanoviruses, and circoviruses amplify their circular ssDNA genomes via a rolling circle mechanism through the combined action of a single viral protein, Rep, and the host DNA replication machinery (Laufs, et al. (1995) Biochimie 77:765-773; Mankertz, et al. (1997) J. Virol. 71:2562-2566; Katul, et al. (1998) J. Gen. Virol. 79:3101-3109; Mankertz, et al. (1998) J. Gen. Virol. 79:381-384; Hanley-Bowdoin, et al. (1999) Crit. Rev. Plant Sci. 18:71-106). Rep initiates plus-strand DNA synthesis by cleaving the viral origin within a hairpin structure at an invariant sequence, acts as a DNA ligase to terminate rolling circle replication, and hydrolyzes ATP. Because of the functional conservation, Rep proteins from all three ssDNA virus families are highly homologous.
The Geminiviridae family is classified into four genera based on genome structure, insect vector and type of host (Rybicki 1994; Briddon, Bedford et al. 1996). The four genera infect a broad range of plants and cause significant crop losses worldwide (Brown and Bird 1992; Brown 1994; Rybicki and Pietersen 1999; Morales and Anderson 2001; Mansoor, Briddon et al. 2003). All geminiviruses are characterized by twin icosahedral capsids (Zhang, Olson et al. 2001; Bottcher, Unseld et al. 2004) and single-stranded DNA (ssDNA) genomes that replicate through double-stranded DNA (dsDNA) intermediates (Hanley-Bowdoin, Settlage et al. 1999).
Geminiviruses replicate their small, circular DNA genomes using a combination of rolling circle and recombination-mediated replication (Gutierrez 1999; Jeske, Lutgemeier et al. 2001). They encode the proteins required for initiation of replication, Geminivirus Replication Initiation Protein (Rep), and depend on host polymerases for DNA synthesis (Gutierrez 2000; Hanley-Bowdoin, Settlage et al. 2004). Much of our knowledge of geminivirus replication comes from studies of TGMV, a typical begomovirus with a bipartite genome. Two of the seven proteins encoded by TGMV are involved in viral replication. AL1 is required for viral replication (Elmer, Brand et al. 1988; Hanley-Bowdoin, Elmer et al. 1990), whereas AL3 is an accessory factor that enhances viral DNA accumulation (Sunter, Hartitz et al. 1990). The AL1 protein shows conservation across all four genera. Different nomenclatures have been used to designate AL1, which is also known as Rep, AC1 or C1. As used herein, the Rep designation is employed because it is applicable to all geminiviruses.
Rep is a multifunctional protein that mediates both virus-specific recognition of its cognate origin (Fontes, Eagle et al. 1994) and transcriptional repression (Eagle, Orozco et al. 1994; Eagle and Hanley-Bowdoin 1997). Rep initiates and terminates (+) strand DNA synthesis within a conserved hairpin motif (Heyraud-Nitschke, Schumacher et al, 1995; Laufs, Traut et al. 1995; Orozco and Hanley-Bowdoin 1996). It also induces the accumulation of host replication factors in infected cells (Nagar, Pedersen et al. 1995). Rep binds to dsDNA at a repeated sequence in the origin (Fontes, Eagle et al. 1994; Fontes, Gladfelter et al. 1994), cleaves and ligates DNA within an invariant sequence of a hairpin loop (Laufs, Jupin et al. 1995; Orozco and Hanley-Bowdoin 1996), and is thought to unwind viral DNA in an ATP-dependent manner (Gorbalenya and Koonin 1993; Pant, Gupta et al. 2001). Rep interacts with itself and AL3 (Settlage, Miller et al. 1996). It binds to several host factors involved in DNA transactions, including the replicative clamp PCNA (Castillo, Collinet et al. 2003), the clamp loader RFC (Luque, Sanz-Burgos et al. 2002), histone H3 and a mitotic kinesin (Kong and Hanley-Bowdoin 2002). Rep also interacts with host regulatory factors, including the retinoblastoma protein (pRBR) which modulates the a cell cycle and differentiation (Xie, Suarezlopez et al. 1995; Grafi, Burnett et al. 1996; Ach, Durfee et al. 1997), a novel protein kinase (GRIK) associated with leaf development (Kong and Hanley-Bowdoin 2002), and Ubc9—a component of the sumoylation pathway (Castillo, Kong et al. 2004).
The functional domains of Rep have been mapped by deletion and mutational studies (FIG. 1). The N-terminal half of Rep contains overlapping domains for DNA cleavage/ligation, DNA binding, and protein interactions (Orozco, Miller et al. 1997; Orozco and Hanley-Bowdoin 1998). NMR spectroscopy revealed that the overlapping DNA binding/cleavage domains contain a β-sheet cluster that resemble other nucleic acid binding proteins (Campos-Olivas, Louis et al. 2002). The characterized Rep protein interactions fall into two classes—proteins that bind between amino acids 101-180 (Kong, Orozco et al. 2000; Settlage, Miller et al. 2001) and those that bind between amino acids 134-180. (Orozco, Kong et al. 2000; Kong and Hanley-Bowdoin 2002). The putative DNA helicase domain is in the C-terminus (Gorbalenya and Koonin 1993; Pant, Gupta et al. 2001).
Rep contains several conserved amino acid and structural motifs (FIG. 1). Motifs I, II and III are characteristic of rolling circle initiators (Ilyina and Koonin 1992; Koonin and Ilyina 1992). Motif I (FLTY) is a determinant of dsDNA binding specificity (Chatterji, Chatterji et al. 2000; Arguello-Astorga and Ruiz-Medrano 2001). Motif II (HLH) is a metal binding site that may impact protein conformation and/or catalysis. Motif III (YxxKD/E) is the catalytic site for DNA cleavage with the hydroxyl group of the Y residue forming a covalent bond with the 5′ end of the cleaved DNA strand (Laufs, Traut et al. 1995). The aromatic ring of the Y residue plays a role in dsDNA binding (Orozco and Hanley-Bowdoin 1998). The three motifs are exposed and in close proximity on the β-sheet surface in the Rep N-terminus (Campos-Olivas, Louis et al. 2002). Other conserved motifs include a sequence of near identity and unknown function immediately C-terminal of Motif III (Kong, Orozco et al. 2000), a helix-loop-helix motif that mediates pRBR binding (Arguello-Astorga, Lopez-Ochoa et al. 2004), and a NTP binding consensus (Walker, Saraste et al. 1982).
A variety of strategies have been applied to geminivirus resistance, including conventional breeding and transgenic approaches. Conventional breeding has been confounded by the limited sources of natural resistance, the multigenic nature of the resistance traits, and the time required for a breeding program (Miklas, Johnson et al. 1996; Pessoni, Zimmermann et al. 1997; Velez, Bassett et al. 1998; Welz, Schechert at al. 1998; Kyetere, Ming et al. 1999). TYLCV resistance genes have been introgressed from a wild Lycopersicon species (Pilowsky and Cohen 1990; Lapidot, Friedmann et al. 1997; Friedmann, Lapidot et al. 1998; Vidaysky and Czosnk 1998). This resistance is often unsatisfactory due to linkage with poor fruit quality, complex inheritance patterns, and the difficulty of transfer to commercial cultivars. Most conventional resistances collapse under early or severe infection pressure (Lapidot and Friedmann 2002). There is also evidence that host resistance genes are not equally effective against different geminiviruses (Pernet, Hoisington et al. 1999; Pernet, Hoisington et al. 1999), and many host genes only confer tolerance (Gilreath, Shuler at al. 2001; Lapidot, Friedmann et al. 2001; Gomez, Pinon et al. 2004). Tolerant plants, which support viral replication—albeit at lower levels, can serve as reservoirs for mutant and recombinant viruses that have the potential to overcome resistance.
Several transgenic strategies based on pathogen-derived resistance have also been tested. There is one report of transgenic tomatoes that contain a mutant begomovirus coat protein gene and display tolerance (Kunik, Salomon et al. 1994), but this result has not been reproduced by other researchers using wild type viral sequences (Azzam, Diaz et al. 1996; Sinisterra, Polston et al. 1999). Instead, expression of geminivirus sequences frequently results in the production of functional proteins that typically complement defective viruses or cause symptoms (Hanley-Bowdoin, Elmer et al. 1989; Hayes and Buck 1989; Hanley-Bowdoin, Elmer et al. 1990; Pascal, Goodlove et al. 1993; Latham, Saunders et al. 1997; Krake, Rezaian et al. 1998; Guevara-Gonzalez, Ramos at al. 1999; Hou, Sanders at al. 2000:Sunter, 2001 #7731). The reduced sensitivity to pathogen-derived resistance may reflect the lack of an RNA genomic form and the ability of geminiviruses to modulate host gene silencing (Ratcliff, Harrison et al. 1997; Voinnet, Pinto et al. 1999; Covey and AlKaff 2000; Noris, Lucioli et al. 2004; Vanitharani, Chellappan et al. 2004). Antisense RNA and defective-interfering replicon strategies have also been of limited success (Stanley, Fischmuth et al. 1990; Day, Bejarano et al. 1991; Frischmuth and Stanley 1994; Aragao, Ribeiro et al. 1998; Asad, Haris et al. 2003). Recent reports suggested that RNAi constructs can confer strong resistance, but this strategy is limited to homologous (or very closely related) geminiviruses (Pooggin, Shivaprasad et al. 2003; Pooggin and Hohn 2004). Transgenic plants that inducibly express dianthin upon geminivirus infection also display resistance (Hong, Saunders et al. 1996), but the safety of a toxic ribosome-inactivating protein has not been established. Expression of mutant begomovirus movement proteins in transgenic plants also resulted in resistance, but the phenotype is variable possibly because of the ability of the mutant proteins to confer symptoms in the absence of infection (Pascal, Goodbye et al. 1993; Duan, Powell et al. 1997; Duan, Powell et al. 1997; Hou, Sanders et al. 2000).
Unlike the strategies described above, Rep mutants have proven effective at interfering with geminivirus replication in cultured cells. Mutations in Motif III, the ATP binding site and the oligomerization domain (FIG. 1) interfere with virus replication in transient assays (Hanson and Maxwell 1999; Orozco, Kong et al. 2000; Chatterji, Beachy et al. 2001). However, plants that stably produce the Rep protein display de-velopmental defects (Brunetti, Tavazza et al. 1997; Brunetti, Tavazza et al. 2001), and expression is selected against during meiosis. The pRBR protein is required for gametogenesis (Ebel, Mariconti et al. 2004), suggesting that the Rep expression problem reflects its interaction with pRBR. Recent experiments showed that inclusion of a pRBR binding mutation in an interfering Rep transgene results in stable expression through at least 3 generations. Because Rep is highly specific for its cognate viral origin (Fontes, Gladfelter et al. 1994; Chatterji, Chatterji et al. 2000), the same plants were designed to also express a mutant AL3 in an effort to the broaden resistance. (AL3 functions in virus-nonspecific manner to enhance viral accumulation (Santer, Stenger et al. 1994; Sung and Coutts 1995)). The plants coexpressing the mutant Rep and AL3 proteins are immune to infection by the homologous virus through at least three generations. It is not yet known if they are resistant to unrelated begomoviruses. In contrast, other studies showed that infection with a homologous virus can lead to Rep transgene silencing. It is desirable to develop alternative resistance strategies.
Peptide aptamers resemble single chain antibodies, but because of their in vivo selection, are more likely to be stably expressed and correctly folded and to interact with their targets in an intracellular context (Crawford, Woodman et al. 2003). If an aptamer binds to residues critical for function, it can inactivate its target and interfere with cellular processes. For example, an aptamer that binds to the active site of the cell cycle regulator, cdk2, was isolated by screening a combinatorial peptide library in yeast dihybrid assays (Colas, Cohen et al. 1996). The aptamer blocks cdk2/cyclin E kinase activity in vitro and, when expressed in vivo, retards cell division (Cohen, Colas et al. 1998). An aptamer that interacts with the dimerization domain of cell cycle-associated transcription factor, E2F, also interferes with cell cycle progression in animal cells (Fabbrizio, LeCam et al. 1999). Aptamers have also been expressed in flies to study the specific roles of cdk1 and cdk2 during Drosophila organogenesis (Kolonin and Finley 1998). They have been used to distinguish between and selectively inactivate allelic variants of Ras and to inhibit Rho GTP exchange factors (Schmidt, Diriong et al. 2002; Xu and Luo 2002; Kurtz, Esposito et al. 2003) as well as interfere with the EGF signaling pathway, by binding to the downstream transcription factor Stat3 (Buerger, Nagel-Wolfrum et al. 2003; Nagel-Wolfrum, Buerger et al. 2004).
Peptide aptamers are especially well suited for targeting noncellular factors like viral proteins. An aptamer that binds to the hepatitis B virus core protein and inhibits viral capsid formation and replication has strong antiviral activity in liver cells (Butz, Denk et al. 2001). Aptamers that target the E6 or E7 proteins of human papillomavirus and block their anti-apoptotic activities result in specific elimination of HPV-positive cancer cells (Butz, Denk et al. 2000; Nauenburg, Zwerschke et al. 2001).
The present inventors have found that expression of aptamers that target essential, conserved Rep motifs can interfere with viral replication and confer broad resistance against geminivirus infection.
SUMMARY OF THE INVENTION
Single-stranded DNA (ssDNA) viruses cause severe disease problems in plants and animals. Geminiviruses and nanoviruses infect many important plant crops worldwide, whereas circoviruses cause significant disease losses among livestock and poultry. Even though these viruses have diverse host ranges and cause different diseases, their replication initiation proteins (Rep) are highly related to each other. The present invention can be used to develop a broad-based resistance strategy directed to eukaryotic viruses, in particular, eukaryotic ssDNA viruses.
A first aspect of the invention provides a polypeptide comprising, consisting essentially of or consisting of an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:169, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, or any combination thereof;
A further aspect of the invention provides a fusion protein comprising the foregoing polypeptides.
Additional aspects of the invention provide an isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide of the invention.
A further aspect of the present invention provides a vector or a cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding the polypeptide comprising an amino acid sequence as recited above.
Further aspects of the present invention provide a transgenic plant comprising transformed plant cells, the transformed plant cells comprising the isolated nucleic, acids of the invention. A plurality of transgenic plants, such as a crop of transgenic plants, comprising the transformed plant cells described herein is also provided.
A further aspect of the invention provides a transgenic plant having increased resistance to a geminivirus infection, a viral variant thereof and mixed infections. A plurality of transgenic plants, such as a crop of transgenic plants, having increased resistance to a geminivirus infection, a viral variant thereof and mixed infections is also provided.
Also provided are methods of making transgenic plants having increased resistance to a virus infection, said method comprising providing a plant cell capable of regeneration, transforming the plant cell with an isolated nucleic acid comprising an isolated nucleic acid as described herein, and regenerating a transgenic plant from said transformed plant cell, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus. Additional methods of making transgenic plants having increased resistance to a virus infection comprise introducing an isolated nucleic acid, as recited herein, into a cell to produce a transgenic plant, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus.
Another aspect of the present invention provides a method of inhibiting viral replication in a plant cell comprising introducing an isolated nucleic acid, as recited herein, into the plant cell in an amount effective to inhibit virus replication.
The present invention further provides methods of detecting a viral infection, comprising: (a) contacting a sample with a polypeptide as recited above or a fusion protein as recited herein; and (b) detecting the presence or absence of binding between the polypeptide or fusion protein and a target, wherein the binding of the polypeptide or fusion protein to the target in the sample indicates the presence of a virus.
A further aspect of the present invention provides polypeptides identified through a method comprising identifying polypeptides that correspond to consensus peptide sequences derived from statistical analysis of a library of peptide sequences.
The invention further provides polypeptides that target a ssDNA virus replication initiation protein and interfere with the function of the replication initiation protein in vivo.
Further aspects of the invention provide a method of treating a viral infection in a subject in need thereof comprising administering a polypeptide according to embodiments of the present invention to the subject. The polypeptide can be formulated in a suitable pharmaceutical or agricultural carrier.
These and other aspects of the invention are set forth in more detail in the description of the invention below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts functional domains and motifs of the Rep protein. Motifs I, II and III are associated with rolling circle replication initiator proteins. The solid box is the conserved element of unknown function. The conserved helix-loop-helix motif marked by the oval provides the primary pRBR contacts. The box marked “ATP” is the NTP binding site of the putative DNA helicase domain. The limits of the functional domains for DNA cleavage/ligation, DNA binding, and known protein interaction sites are indicated. The PCNA binding site has been localized to the Rep N-terminus but has not been finely mapped. The RFC and H3 binding sites have not been mapped. The numbers at the top indicate amino acid positions in TGMV Rep.
FIG. 2 presents baits for aptamer screens. FIG. 2A presents diagrams of the TGMV AL1 coding regions (TAL11-352 and TAL11-130) cloned downstream of the LexA DBD. Motifs I (SEQ ID NO:231), II and III (SEQ ID NO:232) associated with rolling circle replication initiator proteins are marked by the black boxes and their consensus are shown (Ilyina, T. V., and E. V. Koonin 1992. Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria. Nucleic Acids Res. 20:3279-3285; Koonin, E. V., and T. V. Ilyina. 1992. Geminivirus replication proteins are related to prokaryotic plasmid rolling circle DNA replication initiator proteins. J. Gen. Virol. 73:2763-2766). The oval corresponds to a conserved helix-loop-helix motif and the grey box is the ATP binding motif. In FIG. 2B, baits were tested for oligomerization activity using the positive (AD:TAL11-352) and the negative (AD:Jun) prey controls. The yeast transformants were [1] TAL11-352+AD:TAL11-352, [2] TAL11-352+AD:Jun, [3] TAL11-130 AD:TAL11-352, [4] TAL11-130+AD:Jun, [5] GUS+AD:TAL11-352, [6] GUS+AD:Jun, [7] CaAL11-349+AD:TAL11-352 and [8] CaAL11-349+AD:Jun. Interaction was monitored by growth on Gal-HWUL medium. Growth on Glu-HWU controlled for plasmid selection, whereas no growth on Glu-HWUL verified that interaction was dependent on induction of prey plasmid expression.
FIG. 3 shows that aptamers that bind to TAL11-130 also interact with TAL11-352. The 88 plasmids recovered from the screen of the JM-1 library using TAL11-130 as bait were retransformed into different bait strains to confirm specificity of interaction. FIG. 3A presents a key for N-TrxA-peptides on the plates shown in FIGS. 3B-3D. Controls in column 12 are numbered as in FIG. 2. The interaction assay was performed on Gal-HWUL (B), Glu-HWUL (C) and Glu-HWU (D) media with the TAL11-130, TAL11-352 and GUS baits as indicated at the top. Peptides that interfere with replication of TGMV are boxed in FIG. 3A.
FIG. 4 shows results of replication interference assays. FIG. 4A presents a diagram showing the input replicon cassette, the released TGMV A replicon, and the plant expression cassettes. The positions of primers (LLp1 and LLp2) used to distinguish input vector and replicated DNA are marked. In FIG. 4B, tobacco protoplasts were cotransfected with a TGMV A replicon (pMON1565; lanes 1-4) and a plant expression cassette. Total DNA was isolated 36 h post transfection, digested with DpnI and XhoI, and analyzed on DNA gel blots using a virus-specific probe for double-stranded DNA accumulation (dsDNA). The expression cassettes correspond to the trans-dominant TAL1 mutant FQ118 (pNSB866; lane 1), an empty cassette (pMON921; lane 2), aptamer FL-42 (pNSB1136; lane 3) and aptamer FL-60 (pNSB1144; lane 4). In FIG. 4C, released DNA was amplified from E. coli transfected with an AL1 mutant replicon cassette. Total DNA was isolated from E. coli transformed with either a wild type TGMV A replicon cassette (pMON1565; lanes 1-3) or a mutant replicon cassette carrying an AU frame-shift mutation (pMON1679; lanes 4-6) and amplified using primers LLp1 and LLp2 in (A). The methylation status of the template DNAs was assessed by digestion with DpnI (lanes 2 and 5) and MboI (lanes 3 and 6). PCR products corresponding to the replicon cassette and released TGMV A DNA are marked. Markers corresponding to 100-bp (lane 7) and 1-kb (lane 8) DNA ladders are shown. As shown in FIG. 4D, TGMV A replication required full length AL1 in plant cells. Tobacco protoplasts were transfected with a wild type TGMV A replicon (pMON1565; lanes 1-9) or the mutant AL1 replicon cassette (pMON1679; lanes 10-12). In lanes 1-9, plant expression cassettes corresponding to an empty cassette (pMON921; lanes 1-3), the trans-dominant AL1 mutant FQ118 (pNSB866; lane 4-6) and the TrxA-GST control (pNSB1166; lanes 7-9) were included in the transfections. Total DNA was isolated 36 h post transfection and analyzed directly by PCR or after digestion with DpnI or MboI.
FIG. 5 shows results of studies designed to study interference with TGMV replication for aptamers that bind to TAL11-130. The N-TrxA-peptides selected by screening with TAL11-130 and cloned into plant expression cassettes (Table 4) were tested in replication interference assays using the semi-quantitative PCR assay shown in FIG. 4D. Bands corresponding to the replicated TGMV A DNA (1.2 Kb) and the PCR internal control (700 bp) were quantified using imageJ software (Abramoff, M. D., P. J. Magelhaes, and S. J. Ram. 2004. Image processing with ImageJ. Biophotonics International 11:36-42; Rasband, W. S. 1997-2005. ImageJ. National Institutes of Health). Replication in the presence of the expression cassettes indicated on the left was normalized to amount of replicated DNA in the presence of the empty expression (set to 100). Cut off values of ≧25%, ≧50% and ≧65% indicate strong (black bars), moderate (gray bars), and weak interference (white bars), respectively. Some N-TrxA-peptides show no significant interference (also in white bars). Each assay was performed in triplicate with the error bars corresponding to 2 standard errors.
FIG. 6 shows results of studies designed to study interaction with replication proteins from a heterologous geminivirus. Selected N-TrxA-peptides were tested for interaction with CaLCuV AL1. FIG. 6A provides a key for the aptamers on the plates in FIG. 6 B. The negative prey control AD:Jun is marked by a “C”. FIG. 6B provides results of yeast cells containing the selected aptamers and the TAL11-130 (left), TAL11-352 (center) and CaAL11-349 (right) baits were analyzed for growth on Gal-HWUL medium.
FIG. 7 presents results showing the statistical significance of pairwise alignments. Pairwise alignments were performed for 100 sets of three random databases of computer-generated 20-mers containing 88, 31 or 57 members. The frequencies of hits were compared to equivalent alignments of the databases corresponding to All, Interfering or Non-interfering N-TrxA peptides, respectively. (A) The left panel shows the frequency distribution (expected mean=54) of a random 20-mer of having at least one hit against a database comprised of 88 random 20-mers. The right panel shows the frequency distribution (expected mean=101) of the total number of hits per peptide for all the 88 random 20-mers. The dashed lines represent the observed values for the All N-TrxA-peptides database for each analysis. Similar analyses were performed for the Interfering and Non-interfering TrxA peptide databases and their random 20-mer control databases (not shown). In FIG. 7B, the observed and expected means and standard errors of the pairwise alignments of the three TrxA peptide databases are given. The observed values for the three databases are significantly higher than the expected values derived from the random 20-mer databases (p values<0.0001).
FIG. 8 presents motifs involved in AL1 binding and replication interference. FIG. 8A presents consensus sequences corresponding to motifs (Motif 1, SEQ ID NO:9; Motif 4, SEQ ID NO:10; Motif 20, SEQ ID NO:11; Motif 24, SEQ ID NO:12; Motif 25, SEQ ID NO:13; Motif 27, SEQ ID NO:14; and Motif 28, SEQ ID NO:15) identified in pairwise alignments of the 88 TrxA-peptides described herein. Bold typeface indicates invariant residues, normal typeface marks amino acids conserved in a majority of group members, and X represents any amino acid. The number of members and interfering peptides in each group are listed on the right. (See FIGS. 9 and 10 for sequence alignments). FIG. 8B provides WebLogo representation of Motif 24 (SEQ ID NO:12). The amino acid type and position is shown in the X-axis. The overall height plotted on the Y-axis of the amino acid stacks indicates the sequence conservation at a given position, while the height of individual symbols within a stack indicates the relative frequency of an amino acid at that position (Crooks G E, G. Hon, J. M. Chandonia, S. E. Brenner 2004. WebLogo: A sequence logo generator. Genome Res. 14:1188-1190; Schneider T. D., and R. M. Stephens 1990. Sequence Logos: A new way to display consensus sequences. Nucleic Acids Res. 18:6097-6100). Amino acids are color coded according to their type as basic (blue), hydrophobic (black), polar/non polar (green) and acidic (red) (Bogan, A. A., and K. S. Thorn 1998. Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280:1-9; Glaser, F., D. Steinberg, I. Vakser, and N. Ben-Tal 2001. Residue frequencies and pairing preferences at protein-protein interfaces. Proteins 43:89-102).
FIG. 9 presents sequence alignment of Motif 24 peptides. Peptides containing Motif 24 were classified as interfering or non-interfering, and each class was aligned using Vector NTI-AlignX. Coding of amino acid similarities from Vector NTI-AlignX is: UPPERCASE normal—non-similar residues; UPPERCASE bold—a consensus residue derived from a block of similar residues; lowercase bold and shaded—a consensus residue derived from the occurrence of greater than 50% of a single residue; UPPERCASE bold and shaded—a consensus derived from a completely conserved residue; and lowercase bold—a residue weakly similar to a consensus residue at a given position.
FIG. 10 presents sequence alignments of selected motifs. Motifs 1, 4, 20, 25 and 27 include primarily interfering peptides (in bold). Motif 28 includes mostly noninterfering peptides (normal). Consensus color code presented above (FIG. 9).



DETAILED DESCRIPTION
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
Except as otherwise indicated, standard methods can be used for the production of viral and non-viral vectors, manipulation of nucleic acid sequences, production of transformed cells, and the like according to the present invention. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); F. M. AUSUBEL et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
As used herein, “aptamers” may be small peptide or nucleic acid molecules that specifically recognize and bind proteins, and in some instances, regulate a protein of interest, for example, decrease activity of the protein. In particular, peptide aptamers are recombinant proteins that have been selected for specific binding to a target protein (Hoppe-Seyler, Crnkovic-Mertens et al. 2004). They generally include a short peptide domain inserted into a supporting protein scaffold that enhances both specificity and affinity by conformationally constraining the peptide sequence (Colas, Cohen et al. 1996; Cohen, Colas et al. 1998; Buerger, Nagel-Wolfrum et al. 2003). Bacterial thioredoxin (Trx), which is rendered inactive by insertion of the peptide sequence into its active site, is the most commonly used scaffold because of its small size (12 kD), stability, solubility and known 3D structure. In some embodiments of the present invention, “aptamer” may be used to designate the peptide in the scaffold protein while “peptide” may refer to the inserted sequence.
“Amino acid sequence” as used herein, refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or partially or completely synthetic molecules. Where “amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence,” and like terms, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
A “functional fragment” of an amino acid sequence as used herein, refers to a portion of the amino acid sequence that retains at least one biological activity normally associated with that amino acid sequence.
In particular embodiments, a “functional variant” of an amino acid sequence as used herein, refers to no more than one, two, three, four, five, six, seven, eight, nine or ten amino acid substitutions in the sequence of interest. The functional variant retains at least one biological activity normally associated with that amino acid sequence. In particular embodiments, the “functional variant” retains at least about 40%, 50%, 60%, 75%, 85%, 90%, 95% or more biological activity normally associated with the full-length amino acid sequence. In other embodiments, a “functional variant” is an amino acid sequence that is at least about 60%, 70%, 80%, 90%, 95% 97% or 98% similar to the polypeptide sequence disclosed herein (or fragments thereof).
“Polypeptide” as used herein, is used interchangeably with “protein,” and refers to a polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the term “polypeptide” includes proteins, oligopeptides, protein fragments, protein analogs and the like. The term “polypeptide” contemplates polypeptides as defined above that are encoded by nucleic acids, are recombinantly produced, are isolated from an appropriate source, or are synthesized.
“Fusion protein” as used herein, refers to a protein produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides, or fragments thereof, are fused together in the correct translational reading frame. The two or more different polypeptides, or fragments thereof, include those not found fused together in nature and/or include naturally occurring mutants.
“Isolated” nucleic acid as used herein, refers to a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism or virus, such as for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid. Likewise, an “isolated” polypeptide means a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
“Vector” as used herein, refers to a viral or non-viral vector that is used to deliver a nucleic acid to a cell, protoplast, tissue or subject.
“Transgenic” as used herein, refers a plant that comprises a foreign nucleic acid incorporated into the genetic makeup of the plant, such as for example, by stable integration into the nuclear genome.
“Plant cell” as used herein, refers to plant cells, plant protoplasts and plant tissue cultures, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants, such as leaves, pollen, embryos, cotyledon, hypocotyl, roots, root tips, anthers, flowers and parts thereof, ovules, shoots, stems, stalks, pith, capsules, and the like.
“Resistance to a virus infection” as used herein, refers to the reduced susceptibility of a plant or animal subject to viral infection as compared with a control susceptible plant or animal subject under conditions of infestation. “Resistance” can refer to reduced onset, severity, duration and/or spread of viral infection.
In embodiments of the present invention, a polypeptide comprises, consists essentially of or consists of: (a) the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ. ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO: 137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, or any combination thereof; (b) a functional fragment of any of the amino acid sequences recited above that bind to a viral replication protein (Rep); and (c) a functional variant of any of the amino acid sequences of (a) or (b) that binds to a viral replication protein (Rep).
Moreover, polypeptides of the invention encompass those amino acids that have at least about 60%, 70%, 80%, 90%, 95%, 97%, 98% or higher amino acid sequence similarity with the polypeptide sequences specifically disclosed herein (or fragments thereof). As is known in the art, a number of different programs can be used to identify whether a nucleic acid or polypeptide has sequence identity or similarity to a known sequence. Sequence identity and/or similarity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85,2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux at al., Nucl. Acid Res. 12, 387-395 (1984), preferably using the default settings, or by inspection.
An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35, 351-360 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5, 151-153 (1989).
Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, which are preferably set to the default values. The parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values can be adjusted to increase sensitivity.
An additional useful algorithm is gapped BLAST as reported by Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402. A percentage amino acid sequence identity value can be determined by the number of matching identical residues divided by the total number of residues of the “longer” sequence in the aligned region. The “longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
The alignment can include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the polypeptides specifically disclosed herein, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than a sequence specifically disclosed herein, will be determined using the number of amino acids in the shorter sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
The present invention also encompasses functional fragments of the polypeptides disclosed herein. A functional fragment of an amino acid sequence recited above retains at least one of the biological activities of the unmodified sequence, for example, binding to the Rep protein and/or inhibiting viral replication. In some embodiments, the functional fragment of the amino acid sequence retains all of the activities possessed by the unmodified sequence. By “retains” biological activity, it is meant that the amino acid sequence retains at least about 10%, 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of one or more biological activities of the native amino acid sequence (and can even have a higher level of activity than the native amino acid sequence). A “non-functional” amino acid sequence is one that exhibits essentially no detectable biological activity normally associated with the amino acid sequence (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%).
The invention further provides functional variants of the polypeptides disclosed herein. In particular embodiments, a functional variant of an amino acid sequence recited above has no more than one, two, three, four, five or six amino acid substitutions in the amino acid sequence of interest. In some embodiments, one, more than one, or all of the amino acid substitutions are conservative substitutions. In other embodiments, amino acid substitutions facilitate binding affinity of the polypeptide to the Rep protein and/or improve inhibitory properties. In other embodiments, a functional variant has no more than 1, 2, 3, 4, 5 or 6 amino acid substitutions, insertions and/or deletions in the amino acid sequence of interest. In general, those skilled in the art will appreciate that minor deletions, insertions or substitutions may be made to the amino acid sequences of peptides of the present invention without unduly adversely affecting the activity thereof. Thus, polypeptides containing such deletions or substitutions are a further aspect of the present invention. In polypeptides containing substitutions or replacements of amino acids, one or more amino acids of a polypeptide sequence may be replaced by one or more other amino acids wherein such replacement does not affect the function of that sequence. Such changes can be guided by known similarities between amino acids in physical features such as charge density, hydrophobicity/hydrophilicity, size and configuration, so that amino acids are substituted with other amino acids having essentially the same functional properties. For example: Ala may be replaced with Val or Ser; Val may be replaced with Ala, Leu, Met, or Ile, preferably Ala or Leu; Leu may be replaced with Ala, Val or Ile, preferably Val or Ile; Gly may be replaced with Pro or Cys, preferably Pro; Pro may be replaced with Gly, Cys, Ser, or Met, preferably Gly, Cys, or Ser; Cys may be replaced with Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replaced with Pro or Cys, preferably Cys; His may be replaced with Phe or Gln, preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferably His or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe or Trp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may be replaced with Gln or Ser, preferably Gln; Gln may be replaced with His, Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced with Gln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser, preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replaced with Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced with Lys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced with Arg or Asp, preferably Asp. Once made, changes can be routinely screened to determine their effects on function.
In particular embodiments, the functional fragment or variant comprises one or more of the conserved structural motifs of the polypeptides specifically disclosed herein (See FIGS. 7-10).
In some embodiments, the polypeptides bind anywhere in the Rep protein. In some other embodiments, the polypeptide binds to the catalytic domain for DNA cleavage of the Rep protein. Binding of the polypeptide to the Rep protein can occur in one or more DNA cleavage motifs (Motif I, Motif II and Motif III) located in the Rep N-terminus (FIG. 1). In certain embodiments, the polypeptide binds to Motif III within the catalytic domain for DNA cleavage. In other representative embodiments, the polypeptide binds to the DNA binding domain of the Rep protein or any other conserved region of the Rep protein (e.g., the N-terminal portion). In still other embodiments, the polypeptide binds to the Rep protein and further inhibits viral replication.
The polypeptides and fusion proteins can bind to a viral Rep protein and optionally inhibit replication and/or infection. The viruses can include any single-stranded eukaryotic DNA virus employing a rolling circle replication mechanism. In representative embodiments, the virus is a plant pathogen or an animal pathogen. In certain embodiments, the virus is a geminivirus, a nanovirus, or a circovirus. In some embodiments, viral infection can be caused by a combination of viruses, i.e., is a mixed infection. In some embodiments, the virus is a tomato golden mosaic virus (TGMV), a cabbage leaf curl virus (CbLCV) or a combination thereof.
Infectious clones for a variety of geminiviruses, nanoviruses and circoviruses are available in the art. See, e.g., Table 1, which provides sequences for geminivirus, nanovirus and circovirus type members. The nucleic acid sequences of other infectious clones are available at http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/fr-fst-g.htm. See also Hill et al., Virology 250, 283-292 (1998); Kong and Hanley-Bowdoin, Plant Cell 14, 1817-1832 (2002) (CbLCV); Sangare et al., Mol Breeding 5, 95-102 (1999) (ACMV and EACMV); Petty et al., Virology 277, 429-438 (2000); Hanley-Bowdoin, Plant Cell 1, 1057-1067 (2002) (TGMV); and Kunik et al., BioTechnology 12, 500-504 (1994) (TYLCV).







TABLE 1







Biological information for geminivirus,


nanovirus and circovirus type members



















NCBI Acc.


Family
Genus
Type Species
Abbrev
ICTV virus code
Motif 3
No.





Geminiviridae
Mastrevirus
Maize streak virus
(MSV)
00.029.0.01.001.
VRDYILKEPL
Y00514



Curtovirus
Beet curly top virus
(BCTV)
00.029.0.02.001
VKSYVDKDGD
X04144



Begomovirus
Bean golden mosaic virus
(BGMV-PR)
00.029.0.03.001
VKEYIDKDGV
M10070



Topocuvirus
Tomato pseudo-curly
(PCTV)
00.029.0.04.001
VNSYVDKDGD
X84735




top virus






 


Circoviridae
Circovirus
Porcine circovirus type 1
(PCV1)
00.016.0.01.001
NKEYCSKEGH
U49186



Gyrovirus
Chicken anaemia virus
(CAV)
00.016.0.02.001
NLTYVSKIGG
M55918


 


Nanoviridae
Nanovirus
Subterranean clover








stunt virus
(SCSV)
00.093.0.01.001.
AQLYAMKEDS
U16730



Babuvirus
Banana bunchy top virus
(BBTV)
00.093.0.02.001.
ARSYCMKEDT
S56276





Motif III sequences of virus type members Rep proteins. Motif III sequences of Rep proteins from geminiviruses, nanoviruses and circoviruses are shown. The invariant Y and K residues are in bold type. Geminiviruses are subgrouped as begomoviruses, curtoviruses, topocuviruses or mastreviruses. See Virus Taxonomy: The Seventh Report of the International Committee on Taxonomy of Viruses M.H. van Regenmortel, C.M. Fauquet, D.H.L. Bishop et al. (eds.)Academic Press,1024 pp. (2000) San Diego, Wien New York.






In still other embodiments, the invention provides a fusion protein comprising, consisting essentially of or consisting of the polypeptide recited above. In some embodiments, the polypeptide conformation is constrained. In certain embodiments, polypeptides wherein conformation is constrained can bind to the target with higher affinity as compared to polypeptides wherein conformation is random. In some embodiments, the fusion protein comprises thioredoxin (or a fragment thereof). In still other embodiments, the fusion proteins binds to Rep and/or inhibits viral replication. In certain embodiments, the fusion protein reduces geminivirus replication.
Embodiments of the present invention further provide an isolated nucleic acid comprising, consisting essentially of or consisting of a nucleotide sequence encoding the polypeptides and fusion proteins of the invention. In some embodiments, the isolated nucleic acid comprises a nucleotide sequence encoding the polypeptide recited above. The nucleic acid can be DNA, RNA or a chimera thereof, and can further include naturally occurring bases and/or analogs and derivatives of naturally occurring bases. Further, the isolated nucleic acid can be double-stranded, single-stranded or a combination thereof.
In other embodiments, the present invention further provides a vector comprising the isolated nucleic acid recited above. In particular embodiments, the vector is an expression vector. In other particular embodiments, the vector is compatible with bacterial, yeast, animal (e.g., mammalian, insect) or plant (e.g., monocot, dicot) cells. Exemplary vectors include but are not limited to plasmids (including the Ti plasmid from Agrobacteria), virus vectors, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophage and the like.
Also provided are cells comprising the isolated nucleic acids, vectors, polypeptides and fusion proteins of the invention. The cell can be any cell known in the art including plant cells and protoplasts, animal cells, bacterial cells, yeast cells, and the like. Further, in particular embodiments, the cell can be a cultured cell or a cell in an intact plant or subject in vivo.
Exemplary Viruses
1. Geminivirus
The geminiviruses are single-stranded plant DNA viruses. They possess a circular, single-stranded (ss) genomic DNA encapsidated in twinned “geminate” icosahedral particles. The encapsidated ssDNAs are replicated through circular double stranded DNA intermediates in the nucleus of the host cell, presumably by a rolling circle mechanism. Viral DNA replication, which results in the synthesis of both single and double stranded viral DNAs in large amounts, involves the expression of only a small number of viral proteins that are necessary either for the replication process itself or facilitates replication or viral transcription. The geminiviruses therefore appear to rely primarily on the machinery of the host for viral replication and gene expression.
Geminiviruses are subdivided on the basis of host range in either monocots or dicots and whether the insect vector is a leaf hopper, tree hopper or a whitefly species. Monocot-infecting geminiviruses, the mastreviruses, are transmitted by leaf hoppers and their genome comprises a single ss DNA component about 2.7 kb in size (monopartite geminivirus). This type of genome, the smallest known infectious DNA, is typified by wheat dwarf virus, which is one of a number from the subgroup that have been cloned and sequenced. A few mastreviruses infect dicot species as illustrated by bean yellow dwarf virus. Members of the geminivirus begomovirus genus infect dicot hosts and are transmitted by the whitefly. Many possess a bipartite genome comprising similarly sized DNAs (usually termed A and B) as illustrated by African cassava mosaic virus (ACMV), tomato golden mosaic virus (TGMV) and potato yellow mosaic virus. For successful infection of plants, both genomic components are required. Some begomoviruses possess single component genomes, as illustrated by tomato yellow leaf curl virus (TYLCV). Some single-component begomoviruses are associated with satellite DNAs, as illustrated by tomato leaf curl virus (TYLC). The curtoviruses, typified by beet curly top virus, occupy a unique intermediary position between the above two genera as they infect dicots but are transmitted by leaf hoppers. The fourth geminivirus genus, the topocuviruses, is comprised of a single virus, tomato pseudo-curly top virus, which has single component genome and is transmitted by tree hoppers.
The bipartite geminiviruses contain only the viruses that infect dicots. Exemplary is the African Cassava Mosaic Virus (ACMV) and the Tomato Golden Mosaic Virus (TGMV). TGMV, like ACMV, is composed of two circular DNA molecules of the same size, both of which are required for infectivity. Sequence analysis of the two genome components reveals six open reading frames (ORFs); four of the ORFs are encoded by DNA A and two by DNA B. On both components, the ORFs diverge from a conserved 230 nucleotide intergenic region (common region) and are transcribed bidirectionally from double stranded replicative form DNA. The ORFs are named according to genome component and orientation relative to the common region (i.e., left versus right). The AL2 gene product transactivates expression of the TGMV coat protein gene, which is also sometimes known as “AR1”. Functions have not yet been attributed to some of the ORFs in the geminivirus genomes. However, it is known that certain proteins are involved in the replication of viral DNA (REP genes). See, e.g., Elmer et al., Nucleic Acids Res. 16, 7043 (1988); Hatta and Francki, Virology 92, 428 (1979).
The A genome component contains all viral information necessary for the replication and encapsidation of viral DNA, while the B component encodes functions required for movement of the virus through the infected plant. The DNA A component of these viruses is capable of autonomous replication in plant cells in the absence of DNA B when inserted as a greater than full-length copy into the genome of plant cells, or when a copy is electroporated into plant cells. In monopartite geminivirus genomes, the single genomic component contains all viral information necessary for replication, encapsidation, and movement of the virus.
The geminivirus A component carries the Rep (also known as C1, AC1 or ALI), the AL2 (also known as C2 or TRAP), AL3 (also known as C3, AC3 or REN), and AR1 (also known as V1 or coat protein) sequences. The geminivirus B component carries the BR1 (also known as BV1) and BL1 (also known as BC1) sequences. Additionally, monopartite geminiviruses encode a protein that is homologous to the Rep protein of bipartite viruses.
As used herein, geminiviruses encompass viruses of the Genus Mastrevirus, Genus Curtovirus, Genus Topocuvirus and Genus Begomovirus. Exemplary geminiviruses include, but are not limited to, Abutilon Mosaic Virus, Ageratum Yellow Vein Virus, Bhendi Yellow Vein Mosaic virus, Cassava African Mosaic Virus, Chino del Tomato Virus, Cotton Leaf Crumple Virus, Croton Yellow Vein Mosaic Virus, Dolichos Yellow Mosaic Virus, Horsegram Yellow Mosaic Virus, Jatropha Mosaic virus, Lima Bean Golden Mosaic Virus, Melon Leaf Curl Virus, Mung Bean Yellow Mosaic Virus, Okra Leaf Curl Virus, Pepper Hausteco Virus, Potato Yellow Mosaic Virus, Rhynchosia Mosaic Virus, Squash Leaf Curl Virus, Tobacco Leaf Curl Virus, Tomato Australian Leaf Curl Virus, Tomato Indian Leaf Curl Virus, Tomato Leaf Crumple Virus, Tomato Pseudo-Curly Top Virus, Tomato Yellow Leaf Curl Virus, Tomato Yellow Mosaic Virus, Watermelon Chlorotic Stunt Virus, Watermelon Curly Mottle Virus, Bean Distortion Dwarf Virus, Cowpea Golden Mosaic Virus, Lupin Leaf Curl Virus, Solanum Apical Leaf Curling Virus, Soybean Crinkle Leaf Virus, Chloris Striate Mosaic Virus, Digitaria Striate Mosaic Virus, Digitaria Streak Virus, Miscanthus Streak Virus, Panicum Streak Virus, Pasalum Striate Mosaic Virus, Sugarcane Streak Virus, Tobacco Yellow Dwarf Virus, Cassava Indian Mosaic Virus, Serrano Golden Mosaic Virus, Tomato Golden Mosaic Virus, Cabbage Leaf Curl Virus, Bean Golden Mosaic Virus, Pepper Texas Virus, Tomato Mottle Virus, Euphorbia Mosaic Virus, African Cassava Mosaic Virus, Bean Calico Mosaic Virus, Wheat Dwarf Virus, Cotton Leaf Curl Virus, Maize Streak Virus, and any other virus designated as a Geminivirus by the International Committee on Taxonomy of Viruses (ICTV). In particular embodiments, the geminivirus is a Tomato Golden Mosaic Virus (TGMV), a Cabbage Leaf Curl Virus (CbLCV) or a combination thereof.
2. Nanovirus
Nanovirus Rep proteins differ from those of members of the Geminiviruses in being smaller (about 33 kDa), having a slightly distinct dNTP-binding motif and lacking the Rb-binding motif. Moreover, the Nanoviruses are distinct from Geminivirus particle morphology, genome size, number and size of DNA components, and mode of transcription. The Nanoviruses have a conserved nona-nucleotide motif at the apex of the stem-loop sequence, which is consistent with the operation of a rolling circle model for DNA replication.
As used herein, Nanoviruses include, but are not limited to, Banana Bunchy Top Virus (BBTV), Coconut Foliar Decay Virus, Faba Bean Necrotic Yellows Virus (FBNYN), Milk Vetch Dwarf Virus (MVDV), subterranean clover stunt virus (SCSV), and Ageratum yellow vein virus (AYVV) and any other virus designated as a Nanovirus by the International Committee on Taxonomy of Viruses (ICTV).
3. Circovirus
Circoviruses infect animal species and are characterized as round, non-enveloped virions with mean diameters from 17 to 23.5 nm containing circular ssDNA. The ssDNA genome of the circoviruses represent the smallest viral DNA replicons known. As disclosed in WO 99/45956, at least six viruses have been identified as members of the family according to The Sixth Report of the International Committee for the Taxonomy of Viruses (Lukert, et al. (1995) Arch. Virol. 10 Suppl.: 166-168).
As used herein, Circoviruses include, but are not limited to, members of the Circoviridae family including chicken anemia virus (CAV), beak and feather disease virus (BFDV), porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and pigeon circovirus and any other virus designated as a nanovirus by the ICTV. Embodiments of the present invention further provide a transgenic plant or plant cell comprising the isolated nucleic acid recited above. The plant or cell can be stably transformed with the isolated nucleic acid. Additionally, embodiments of the invention provide a plurality of plants or cells comprising the isolated nucleic acid recited above. In other representative embodiments, the isolated nucleic acid is flanked by a T-DNA border sequence, optionally by 5′ and 3′ T-DNA border sequences. In some embodiments, the invention provides a plant cell or plant comprising the polypeptides or fusion proteins of the present invention. In other embodiments, the invention provides a plurality of plant Cells or plants comprising the polypeptides or fusion proteins of the present invention.
Plants can be transformed according to the present invention using any suitable method known in the art. Intact plants, plant tissue, explants, meristematic tissue, protoplasts, callus tissue, cultured cells, and the like may be used for transformation depending on the plant species and the method employed. In a preferred embodiment, intact plants are inoculated using microprojectiles carrying a nucleic acid to be transferred into the plant. The site of inoculation will be apparent to one skilled in the art; leaf tissue is one example of a suitable site of inoculation. In some embodiments, intact plant tissues or plants are inoculated, without the need for regeneration of plants (e.g., from callus).
Exemplary transformation methods include biological methods using viruses and Agrobacterium, physicochemical methods such as electroporation, polyethylene glycol, ballistic bombardment, microinjection, floral dip method and the like.
In one form of direct transformation, the vector is microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA (Crossway, Mol. Gen. Genetics 202: 179 (1985)).
In another protocol, the genetic material is transferred into the plant cell using polyethylene glycol (Krens, et al. Nature 296, 72 (1982)).
In still another method, protoplasts are fused with minicells, cells, lysosomes, or other fusible lipid-surfaced bodies that contain the nucleotide sequence to be transferred to the plant (Fraley, et al., Proc. Natl. Acad. Sci. USA 79, 1859 (1982)).
DNA may also be introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide and regenerate. One advantage of electroporation is that large pieces of DNA, including artificial chromosomes, can be transformed by this method.
Viral vectors include RNA and DNA viral vectors (e.g., geminivirus, badnavirus, nanoviruses and caulimovirus vectors).
Ballistic transformation typically comprises the steps of: (a) providing a plant tissue as a target; (b) propelling a microprojectile carrying the heterologous nucleotide sequence at the plant tissue at a velocity sufficient to pierce the walls of the cells within the tissue and to deposit the nucleotide sequence within a cell of the tissue to thereby provide a transformed tissue. In some particular embodiments of the invention, the method further includes the step of culturing the transformed tissue with a selection agent. In particular embodiments, the selection step is followed by the step of regenerating transformed plants from the transformed tissue. As noted below, the technique may be carried out with the nucleotide sequence as a precipitate (wet or freeze-dried) alone, in place of the aqueous solution containing the nucleotide sequence.
Any ballistic cell transformation apparatus can be used in practicing the present invention. Exemplary apparatus are disclosed by Sandford et al. (Particulate Science and Technology 5, 27 (1988)), Klein et al. (Nature 327, 70 (1987)), and in EP 0 270 356. Such apparatus have been used to transform maize cells (Klein et al., Proc. Natl. Acad. Sci. USA 85, 4305 (1988)), soybean callus (Christou et al., Plant Physiol. 87, 671 (1988)), McCabe et al., BioTechnology 6, 923 (1988), yeast mitochondria (Johnston et al., Science 240, 1538 (1988)), and Chlamydomonas chloroplasts (Boynton et al., Science 240, 1534 (1988)).
Alternatively, an apparatus configured as described by Klein et al. (Nature 70, 327 (1987)) may be utilized. This apparatus comprises a bombardment chamber, which is divided into two separate compartments by an adjustable-height stopping plate. An acceleration tube is mounted on top of the bombardment chamber. A macroprojectile is propelled down the acceleration tube at the stopping plate by a gunpowder charge. The stopping plate has a borehole formed therein, which is smaller in diameter than the microprojectile. The macroprojectile carries the microprojectile(s), and the macroprojectile is aimed and fired at the borehole. When the macroprojectile is stopped by the stopping plate, the microprojectile(s) is propelled through the borehole. The target tissue is positioned in the bombardment chamber so that a microprojectile(s) propelled through the bore hole penetrates the cell walls of the cells in the target tissue and deposit the nucleotide sequence of interest carried thereon in the cells of the target tissue. The bombardment chamber is partially evacuated prior to use to prevent atmospheric drag from unduly slowing the microprojectiles. The chamber is only partially evacuated so that the target tissue is not desiccated during bombardment. A vacuum of typically between about 400 to about 800 millimeters of mercury is suitable.
In alternative embodiments, ballistic transformation is achieved without use of microprojectiles. For example, an aqueous solution containing the nucleotide sequence of interest as a precipitate may be carried by the macroprojectile (e.g., by placing the aqueous solution directly on the plate-contact end of the macroprojectile without a microprojectile, where it is held by surface tension), and the solution alone propelled at the plant tissue target (e.g., by propelling the macroprojectile down the acceleration tube in the same manner as described above). Other approaches include placing the nucleic acid precipitate itself (“wet” precipitate) or a freeze-dried nucleotide precipitate directly on the plate-contact end of the macroprojectile without a microprojectile. In the absence of a microprojectile, it is believed that the nucleotide sequence must either be propelled at the tissue target at a greater velocity than that needed if carried by a microprojectile, or the nucleotide sequenced caused to travel a shorter distance to the target tissue (or both).
The nucleotide sequence can be carried on a microprojectile. The microprojectile may be formed from any material having sufficient density and cohesiveness to be propelled through the cell wall, given the particle's velocity and the distance the particle must travel. Non-limiting examples of materials for making microprojectiles include metal, glass, silica, ice, polyethylene, polypropylene, polycarbonate, and carbon compounds (e.g., graphite, diamond). Metallic particles are currently preferred. Non-limiting examples of suitable metals include tungsten, gold, and iridium. The particles should be of a size sufficiently small to avoid excessive disruption of the cells they contact in the target tissue, and sufficiently large to provide the inertia required to penetrate to the cell of interest in the target tissue. Particles ranging in diameter from about one-half micrometer to about three micrometers are suitable. Particles need not be spherical, as surface irregularities on the particles may enhance their DNA carrying capacity.
The nucleotide sequence may be immobilized on the particle by precipitation. The precise precipitation parameters employed will vary depending upon factors such as the particle acceleration procedure employed, as is known in the art. The carrier particles may optionally be coated with an encapsulating agents such as polylysine to improve the stability of nucleotide sequences immobilized thereon, as discussed in EP 0 270 356 (column 8).
Alternatively, plants may be transformed using Agrobacterium tumefaciens or Agrobacterium rhizogenes, preferably Agrobacterium tumefaciens. Agrobacterium-mediated gene transfer exploits the natural ability of A. tumefaciens and A. rhizogenes to transfer DNA into plant chromosomes. Agrobacterium is a plant pathogen that transfers a set of genes encoded in a region called T-DNA of the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, into plant cells. The typical result of transfer of the Ti plasmid is a tumorous growth called a crown gall in which the T-DNA is stably integrated into a host chromosome. Integration of the Ri plasmid into the host chromosomal DNA results in a condition known as “hairy root disease”. The ability to cause disease in the host plant can be avoided by deletion of the genes in the T-DNA without loss of DNA transfer and integration. The DNA to be transferred is attached to border sequences that define the end points of an integrated T-DNA.
Gene transfer by means of engineered Agrobacterium strains has become routine for many dicotyledonous plants. Some difficulty has been experienced, however, in using Agrobacterium to transform monocotyledonous plants, in particular, cereal plants. However, Agrobacterium mediated transformation has been achieved in several monocot species, including cereal species such as rye (de la Pena et al., Nature 325, 274 (1987)), maize (Rhodes et al., Science 240, 204 (1988)), and rice (Shimamoto et al., Nature 338, 274 (1989)).
While the following discussion will focus on using A. tumefaciens to achieve gene transfer in plants, those skilled in the art will appreciate that this discussion also applies to A. rhizogenes. Transformation using A. rhizogenes has developed analogously to that of A. tumefaciens and has been successfully utilized to transform, for example, alfalfa, Solanum nigrum L., and poplar. U.S. Pat. No. 5,777,200 to Ryals et al. As described by U.S. Pat. No. 5,773,693 to Burgess et al., it is preferable to use a disarmed A. tumefaciens strain (as described below), however, the wild-type A. rhizogenes may be employed. An illustrative strain of A. rhizogenes is strain 15834.
The Agrobacterium strain is typically modified to contain the nucleotide sequences to be transferred to the plant. The nucleotide sequence to be transferred is incorporated into the T-region and is typically flanked by at least one T-DNA border sequence, preferably two T-DNA border sequences. A variety of Agrobacterium strains are known in the art, and can be used in the methods of the invention. See, e.g., Hooykaas, Plant Mol. Biol. 13, 327 (1989); Smith et al., Crop Science 35, 301 (1995); Chilton, Proc. Natl. Acad. Sci. USA 90, 3119 (1993); Mollony et al., Monograph Theor. Appl. Genet NY 19, 148 (1993); Ishida et al., Nature Biotechnol. 14, 745 (1996); and Komari et al., The Plant Journal 10, 165 (1996).
In addition to the T-region, the Ti (or Ri) plasmid contains a vir region. The vir region is important for efficient transformation, and appears to be species-specific.
Two exemplary classes of recombinant Ti and Ri plasmid vector systems are commonly used in the art. In one class, called “cointegrate,” the shuttle vector containing the gene of interest is inserted by genetic recombination into a non-oncogenic Ti plasmid that contains both the cis-acting and trans-acting elements required for plant transformation as, for example, in the PMLJ1 shuttle vector of DeBlock et al., EMBO J. 3, 1681 (1984), and the non-oncogenic Ti plasmid pGV2850 described by Zambryski et al., EMBO J. 2, 2143 (1983). In the second class or “binary” system, the gene of interest is inserted into a shuttle vector containing the cis-acting elements required for plant transformation. The other necessary functions are provided in trans by the non-oncogenic Ti plasmid as exemplified by the pBIN19 shuttle vector described by Bevan, Nucleic Acids Research 12, 8711 (1984), and the non-oncogenic Ti plasmid PAL4404 described by Hoekma, et al., Nature 303, 179 (1983).
Binary vector systems have been developed where the manipulated disarmed T-DNA carrying the heterologous nucleotide sequence of interest and the vir functions are present on separate plasmids. In this manner, a modified T-DNA region comprising foreign DNA (the nucleic acid to be transferred) is constructed in a small plasmid that replicates in E. coli. This plasmid is transferred conjugatively in a tri-parental mating or via electroporation into A. tumefaciens that contains a compatible plasmid with virulence gene sequences. The vir functions are supplied in trans to transfer the T-DNA into the plant genome. Such binary vectors are useful in the practice of the present invention.
Plant cells may be transformed with Agrobacteria by any means known in the art, e.g., by co-cultivation with cultured isolated protoplasts, or transformation of intact cells or tissues. The first generally utilizes an established culture system that allows for culturing protoplasts and subsequent plant regeneration from cultured protoplasts. Identification of transformed cells or plants is generally accomplished by including a selectable marker in the transforming vector, or by obtaining evidence of successful bacterial infection.
In plants stably transformed by Agrobacteria-mediated transformation, the nucleotide sequence of interest is incorporated into the plant genome, typically flanked by at least one T-DNA border sequence. In some embodiments, the nucleotide sequence of interest is flanked by two T-DNA border sequences.
Alternatively, transgenic plants may be produced using the well-established ‘floral dip’ method (See, e.g., Clough and Bent (1998) Plant Journal 16:735). In one representative protocol, plants are grown in soil until the primary inflorescence is about 10 cm tall. The primary inflorescence is cut to induce the emergence of multiple secondary inflorescences. The inflorescences of these plants are dipped in a suspension of Agrobacterium containing the vector of interest. After the dipping process, the plants are grown to maturity and the seeds are harvested. Transgenic seeds from these treated plants are selected by germination in soil under selective pressure (e.g., using the chemical bialaphos). Transgenic plants containing the selectable marker survive treatment and are transplanted to individual pots for subsequent analysis. See Bechtold, N. and Pelletier, G. Methods Mol Biol 82, 259-266 (1998); Chung, M. H. et al. Transgenic Res 9, 471-476 (2000); Clough, S. J. and Bent, A. F. Plant J 16, 735-743 (1998); Mysore, K. S. et al. Plant J 21, 9-16 (2000); Tague, B. W. Transgenic Res 10, 259-267 (2001); Wang, W. C. et al. Plant Cell Rep 22, 274-281 (2003); Ye, G. N. et al. Plant J., 19:249-257 (1999).
Plant cells, which have been transformed by any method known in the art, can also be regenerated to produce intact plants using known techniques.
Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMilan Publishing Co. New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II, 1986). It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugar-cane, sugar beet, cotton, fruit trees, and legumes.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently root. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
A large number of plants have been shown capable of regeneration from transformed individual cells to obtain transgenic whole plants.
The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner. The plants are grown and harvested using conventional procedures.
The particular conditions for transformation, selection and regeneration may be optimized by those of skill in the art. Factors that affect the efficiency of transformation include the species of plant, the tissue infected, composition of the media for tissue culture, selectable marker genes, the length of any of the above-described step, kinds of vectors, and light/dark conditions. Therefore, these and other factors may be varied to determine what is an optimal transformation protocol for any particular plant species. It is recognized that not every species will react in the same manner to the transformation conditions and may require a slightly different modification of the protocols disclosed herein. However, by altering each of the variables, an optimum protocol can be derived for any plant species.
The foregoing methods for transformation may be used for producing transgenic inbred or doubled-haploid lines. Transgenic inbred/doubled-haploid lines could then be crossed, with another (non-transformed or transformed) inbred or doubled-haploid line, in order to produce a transgenic hybrid plant. Alternatively, a genetic trait which has been engineered into a particular line using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts.
Plants that may be employed in practicing the present invention include any plant (angiosperm or gymnosperm; monocot or dicot).
Exemplary plants include, but are not limited to corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago saliva), rice (Oryza sativa), rape (Brassica napus), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), apple (Malus pumila), blackberry (Rubus), strawberry (Fragaria), walnut (Juglans regia), grape (Vitis vinifera), apricot (Prunus armeniaca), cherry (Prunus), peach (Prunus persica), plum (Prunus domestica), pear (Pyrus communis), watermelon (Citrullus vulgaris). duckweed (Lemna), oats, barley, vegetables, ornamentals, conifers, and turfgrasses (e.g., for ornamental, recreational or forage purposes).
Vegetables include Solanaceous species (e.g., tomatoes; Lycopersicon esculentum), lettuce (e.g., Lactuea sativa), carrots (Caucus carota), cauliflower (Brassica oleracea), celery (apium graveolens), eggplant (Solanum melongena), asparagus (Asparagus officinalis), ochra (Abelmoschus esculentus), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), members of the genus. Cucurbita such as Hubbard squash (C. Hubbard), Butternut squash (C. moschata), Zucchini (C. pepo), Crookneck squash (C. crookneck), C. argyrosperma, C. argyrosperma ssp sororia, C. digitata, C. ecuadorensis, C. foetidissima, C. lundelliana, and C. martinezii, and members of the genus Cucumis such as cucumber (Cucumis sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (dianthus caryophyllus), poinsettia (Euphorbia pulcherima), and chrysanthemum.
Conifers, which may be employed in practicing the present invention, include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
Turfgrass include, but are not limited to, zoysiagrasses, bentgrasses, fescue grasses, bluegrasses, St. Augustinegrasses, bermudagrasses, bufallograsses, ryegrasses, and orchardgrasses.
Also included are plants that serve primarily as laboratory models, e.g., Arabidopsis. 
In some embodiments, the transgenic plant can be a tobacco plant, a potato plant, a soybean plant, a peanut plant, a tomato plant, a melon plant, a cassava plant, a bean plant, a squash plant, a maize plant, a cotton plant or a vegetable plant. In certain embodiments, the plant is a cassava plant. In still other embodiments, the present invention provides methods of providing resistance against a plant virus infection, in an agricultural field, comprising planting the field with a crop of plants as recited above. Accordingly, the present invention provides a plurality of transgenic plants, such as a crop, having a resistance against a plant virus infection and fields of grasses having the same.
Embodiments of the present invention further provide transgenic plants having increased resistance to a virus infection from viruses such as a geminivirus, a nanovirus and combinations thereof as compared to a non-transgenic control. Resistance may be evaluated by any suitable method known in the art, e.g., measuring inhibition of viral replication, detecting specific mutations within the genome of the viral agent, detecting and quantifying viral load and measuring surrogate markers of viral replication. The term “resistant/resistance” is not intended to indicate that the subject is absolutely immune from viral infection. Those skilled in the art will appreciate that the degree of resistance may be assessed with respect to a population of subjects or an entire field of plants. A subject may be considered “resistant” to viral infection if the overall incidence of infection is reduced, even if particular subjects may be susceptible to disease.
In some embodiments of the present invention, methods of making transgenic plants having increased resistance to a virus comprise introducing an isolated nucleic acid recited above into a plant cell to produce a transgenic plant, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus. In particular embodiments, the present invention provides methods of making transgenic plants having increased resistance to a virus, wherein the method comprises providing a plant cell capable of regeneration; transforming the plant cell with an isolated nucleic acid comprising an isolated nucleic acid recited above; and regenerating a transgenic plant from that transformed plant cell, wherein expression of the isolated nucleic acid to produce the polypeptide increases resistance of the transgenic plant to infection by a virus. In some embodiments, the plant cell can be a tobacco plant cell, a potato plant cell, a soybean plant cell, a peanut plant cell, a tomato plant cell, a melon plant cell, a cassava plant cell, a bean plant cell, a squash plant cell, a maize plant cell, a cotton plant cell or a vegetable plant cell. In still other embodiments, the plant cell is stably transformed with the isolated nucleic acid. In certain embodiments, the plant cell is transformed by an Agrobacterium-mediated transformation method. In other embodiments, the plant cell is transformed by a biolistic transformation method.
In still other embodiments, the present invention provides methods of inhibiting viral replication in a plant cell (e.g., a cultured plant cell or protoplast or a plant cell in vivo) comprising introducing an isolated nucleic acid recited above into the plant cell in an amount effective to inhibit virus replication as compared to non-transgenic control. In some embodiments, virus replication is inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more. In certain embodiments, the virus can be a geminivirus, a nanovirus and combinations thereof.
In still other embodiments, the invention provides methods of inhibiting viral replication in a plant cell by introducing a polypeptide or fusion protein of the invention into a plant cell. In still other embodiments, the invention provides a method of providing increased resistance to a viral infection comprising introducing a polypeptide or fusion protein of the invention into a plant.
A further embodiment of the present invention is a method of detecting a viral infection. The method can involve contacting a sample with at least one polypeptide of the present invention or a fusion protein thereof and detecting the presence or absence of binding between the polypeptide and a target, wherein the binding of the polypeptide to the target in the sample indicates the presence of a virus.
A sample is intended to include biological or environmental material, which may be suspected of containing a virus of the family Geminiviridae, Nanoviridae or Circoviridae. A sample suspected of containing a virus is one that may have come in contact with a virus or may be at risk of having or acquiring a virus. When the virus being detected is of the family Circoviridae (or other animal virus as described above), a sample may be blood, plasma, serum, cell products, cell line cultures, cell extracts, cerebrospinal fluid (CSF), tissue homogenates, urine, organs for transplantation, or semen isolated preferably from a bird, such as chicken or pigeon, or a pig. When the virus being detected is of the family Geminiviridae or Nanoviridae (or other plant viruses as described above), the sample may be a plant tissue culture, fruit, leaf, root, stem, or seed. A sample of environmental origin may include, but not be limited to, soil, water, and food samples including canned goods, meats, and animal fodder. It is contemplated that the method of the invention may be useful in detecting viral contaminants in an environmental sample, viral presence in an organ being used in a transplantation, or viral infection of plant seeds or tissue cultures.
Upon, binding of a polypeptide with its viral target, excess polypeptide and/or targets that do not bind said polypeptide may be removed by washing the sample so that bound polypeptide-target complexes are isolated. Subsequently, the presence or absence of binding (i.e., the presence or absence of polypeptide-target complexes) is measured or detected. To facilitate the step of detecting the presence of absence of binding, the polypeptide of the present invention can be labeled, preferably with a fluorescent or bioluminescent tag. Fluorochromes such as Phycocyanine, Allophycocyanine, Tricolor, AMCA, Eosin, Erythrosin, Fluorescein, Fluorescein Isothiocyanate Hydroxycoumarin, Rhodamine, Texas Red, Lucifer Yellow, and the like may be attached directly to a polypeptide of the invention through standard groups such as sulfhydryl or primary amine groups. Methods of imaging and analyzing any of the above-mentioned labels are well-known in the art and the method employed will vary with the type of analysis being conducted, i.e. individual samples or multiple sample analyses in high-throughput screens. Measurement of the label can be accomplished using flow cytometry, laser confocal microscopy, spectrofluorometer, fluorescence microscopy, fluorescence scanners and the like.
Further, a polypeptide of the present invention may be biotinylated and detection of a biotinylated polypeptide may be performed using any of the well-known avidin or streptavidin reagents. Detection of biotin-avidin or biotin-streptavidin complexes typically involves conjugated forms of avidin or streptavidin including, but are not limited to, enzyme-conjugates (e.g., alkaline phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase) or fluorescent-conjugates (e.g., 7-amino-4-methylcoumarin-3-acetic (AMCA), fluorescein, phycoerythrin, rhodamine, TEXAS RED®, OREGON GREEN®) or antibodies which specifically bind to avidin or streptavidin.
Antibodies which specifically interact with a polypeptide of the present invention can also be used in the detection of binding between said polypeptide and its target. As will be understood by one of skill in the art, a bound polypeptide-target complex is contacted with an antibody specific for said polypeptide and standard methods for detecting antibodies are employed for detecting binding of the antibody to the polypeptide-target complex, e.g., spectrofluorometer, fluorescence microscopy, immunocytochemistry, western blotting, ELISA, fluorescence scanners, and the like. Other methods for detecting antibodies are well-known to those of skill in the art (see, e.g., “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., “Methods and Immunology”, W. A. Benjamin, Inc., 1964; and Oellerich, M. (1984) J. Clin. Chem. Clin. Biochem. 22:895-904).
Subsequently, the presence or absence of a bound polypeptide-target complex is then correlated with the presence or absence of a virus from which the target was derived.
As will be appreciated by one of skill in the art, the detection method of the invention may be used to detect one or more specific viruses, genera, or family of viruses depending on the specificity of the polypeptide being used.
In still other embodiments, the products of the present invention can be used for the preparation of a medicament, veterinary or agricultural product.
The present invention is applicable to animal, avian and plant subjects, where appropriate, for medicinal, diagnostic, drug screening, veterinary, or agricultural purposes. For example, geminiviruses and nanoviruses affect plants, circoviruses affect livestock and poultry, and a human circovirus has been identified in patients with Hepatitis C. Animal subjects include, but are not limited to, humans, primates, canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, and the like, and mammals in utero. Avian subjects include, but are not limited to, chickens, ducks, turkeys, geese, quail, pheasant, ratites (e.g., ostrich) and domesticated birds (e.g., parrots and canaries), and birds in ovo. Plant subjects are described above.
Methods of the present invention can be carried out in a manner suitable for administration or application to the suitable subject. Administration to a plant or a plant cell is described above. Additionally, an isolated nucleic acid vector, polypeptide or fusion protein of the present invention can be used to formulate pharmaceutical compositions comprising a vector of the invention in a pharmaceutically acceptable carrier and/or other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and will preferably be in solid or liquid particulate form. As an injection medium, it is preferred to use water that contains the additives usual for injection solutions, such as stabilizing agents, salts or saline, and/or buffers.
In general, a “physiologically acceptable carrier” is one that is not toxic or unduly detrimental to cells. Exemplary physiologically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. physiologically acceptable carriers include pharmaceutically acceptable carriers.
By “pharmaceutically acceptable” it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral particle or cell directly to a subject.
As used herein, the term “effective amount” refers to an amount of a compound or composition that is sufficient to produce the desired effect, which can be a therapeutic or agricultural effect, i.e., an effect on plant and plant matter as described herein. The effective amount will vary with the application for which the compound or composition is being employed, the subject, the age and physical condition of the subject, the severity of the condition, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically or agriculturally acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. An appropriate “effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example for pharmaceutical applications, Remington, The Science And Practice of Pharmacy (20th Ed. 2000).
Dosages will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, and can be determined in a routine manner. See e.g., Remington, The Science And Practice of Pharmacy (20th Ed. 2000).
In particular embodiments, more than one administration (e.g., two, three, four or more administrations) may be employed.
Exemplary modes of administration include oral, rectal, transmucosal, topical, transdermal, in utero (or in ovo), inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intraarticular) administration, and the like, as well as direct tissue or organ injection, alternatively, intrathecal, direct intramuscular, intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, administration may be by local rather than systemic manner, for example, in a depot or sustained-release formulation.
As used herein, the term “treat” refers to an action resulting in a reduction in the severity of the subject's condition or at least the condition is partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom (or agricultural index for plants) is achieved and/or there is a delay in the progression of the condition and/or prevention or delay of the onset of the condition. Thus, the term “treat” refers to both prophylactic and therapeutic treatment regimes.
As used herein, the term “agriculturally acceptable carrier” refers to adjuvants, e.g., inert components, dispersants, surfactants, tackifiers, binders, etc. that are ordinarily used in agricultural formulation technology.
Having now described the invention, the same will be illustrated with reference to certain examples, which are included herein for illustration purposes only, and which are not intended to be limiting of the invention.
Example 1
Viruses
Geminivirus Rep proteins have been studied extensively, and many of the assays for studying this protein are well-established in the art. Consequently, geminiviruses were used to test the capacity of aptamers to interfere with ssDNA virus replication and infection in eukaryotes. The tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CbLCV) were primarily studied. TGMV is a bipartite geminivirus that infects solanaceous species and encodes a typical Rep protein. CbLCV has a bipartite genome and is a severe pathogen in brassica. CbLCV is representative of a small group of dicot-infecting geminiviruses that encode an atypical Rep protein. Other viruses such as ACMV, BCTV, EACMV, MSV, ToMoV and TYLCV may also be useful in the analysis of selected aptamers. For example, TYLCV is a monopartite geminivirus that causes significant losses in tomato crops through out the world. Genomic clones as well as replication and infectivity assays are well established for these viruses. Together, these viruses can be used to establish the efficacy and breadth of the aptamer resistance strategy for ssDNA viruses.
Example 2
Materials and Methods
A. Yeast Plasmids.
The bait and prey plasmids used in this study are listed in Table 2. pNSB1118, the bait plasmid for full-length TGMV AL1 (TAL11-352), was generated by cloning a 1.2-Kb fragment with NdeI (trimmed) and BamHI ends from pNSB736 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122) into pEG202 (Golemis and Brent (1992) Mol. Cell. Biol. 12:3006-3014) with BamHI and EcoRI (repaired) ends. The same fragment was also ligated into pHybLex/Zeo (Invitrogen) digested with PvuII and BamHI to create pNSB1089. The TAL11-352 coding sequence from pNSB1089 was introduced into pYESTrp2 (Invitrogen) as a SacI/XhoI fragment to give the prey plasmid pNSB970. The bait plasmid for truncated TAL11-130 (pNSB1162) was generated in two steps. First, an 895-bp EcoRI/BamHI fragment from pNSB603 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122) was ligated into the same sites of pNSB1118 to create pNSB1153. Then, pNSB1153 was digested with NotI, repaired with E. coli DNA polymerase (Klenow fragment) and religated to delete an 861-bp sequence encoding the TAL1 C-terminus. The bait plasmid (pNSB1122) for full-length CaLCuV ALA (CaAL11-349) was built by cloning a 1.2-Kb BamHI/XhoI fragment from pNSB909 (Kong and Hanley-Bowdoin (2002) Plant Cell 14:1817-1832) into the same sites of pEG202.
The β-glucuronidase coding sequence (GUS) from pMON10018 (Monsanto) was cloned into pEG202 as a 2.2-Kb BglII-NotI fragment to give the control bait plasmid pNSB1120. An EcoRI/NotI fragment encoding TrxA-GST from pNSB1166 (described below) was cloned into the same sites of pYESTrp2 to generate pNSB1172, a negative control prey plasmid.







TABLE 2







Yeast dihybrid plasmids










Insert
Cloning vector
Yeast selectiona
Plasmid





Bait (DBD)





TAL11-352
pEG202
(−) Histidine
pNSB1118


TAL11-130
pEG202
(−) Histidine
pNSB1162


CaAL11-349
pEG202
(−) Histidine
pNSB1122


GUS
pEG202
(−) Histidine
pNSB1120


Prey (AD)


TAL11-352
pYESTrp2
(−) Tryptophan
pNSB970


Jun
pYESTrp2
(−) Tryptophan
pYESTrp-Jun


TrxA-GST
pYESTrp2
(−) Tryptophan
pNSB1172





a(−) Histidine, medium lacking histidine; (−) Tryptophan, medium lacking tryptophan.






B. Plant Expression Plasmids

TrxA-peptide prey plasmids isolated in the TAL11-352 screen were digested with EcoRI-XbaI, and the resulting 412-bp fragments were gel purified and cloned into pMON921 (Fontes et al. (1994) J. Biol. Chem. 269:8459-8465). To eliminate the gel purification step during the cloning of aptamers derived from the AL11-130 screen, a polylinker was inserted into pMON921 and the β-lactamase gene was replaced by the aminoglycoside 3′-phosphotransferase (aphA) coding sequence, which confers kanamycin resistance. The polylinker was generated by ligating the annealed oligonucleotides LLp27 and LLp28 (Table 3) into pMON921 digested with BglII/BamH1 to create pNSB1208. A fragment carrying the aphA gene was amplified from pFGC5941 (Kerschen et al. (2004) FEBS Lett. 566:223-228) using the primers LLp29 and LLp30 (Table 3), digested with SmaI/AatII and cloned into pNSB1208 cut with DraI/AatII to generate pNSB1226. The N-TrxA peptides were cloned into pNSB1226 as EcoRI/BamHI fragments. N-TrxA aptamers with internal EcoRI or BamHI sites (N-3, N-71, N-99, N-123, N-149 and N-153) were cloned into pNSB1226 as PCR-generated EcoRI/SacI or Sad inserts using primers LLp41 and LLp42 (Table 3).
The TrxA-GST control was generated in two steps. First, the RsrII site in the active site of the thioredoxin coding sequence (TrxA) was reconstituted. Two fragments were generated using pJM-1 library DNA as template in PCR reactions with primer pairs LLp9/LLp16 and LLp15/LLp12 (Table 3.) The PCR products were digested with RsrII and ligated in vitro. The resulting 414-bp fragment was digested with EcoRI/BamHI and cloned into the same sites of pBSKSII(−) to give pNSB1151. Both constructs were sequenced to verify the integrity of the TrxA sequence. Oligonucleotides LLp55 and LLp56 (Table 3), carrying a 60-bp sequence of the glutathione S-transferase gene (GST), were annealed and ligated into RsrII site of pNSB1151, creating pNSB1166. An EcoRI/BamHI fragment from pNSB1166 was cloned into the same sites of pMON921 to generate pNSB1168. The expression cassette (pNSB866) corresponding to FQ118, a TAL1 transdominant negative mutant, has been described before (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122).







TABLE 3







Oligonucleotides










Oligo-





nucleotide
Target
Sequencea
Application





LLp1
5′-AL1 TGMV A
GATGTTTGGCAACCTCCTCTAG
Replication


 


LLp2
3′-CP TGMV A
GGTCGTTCTTTACCGTTGCAGTAC
Replication


 


LLp9
5′-pJM-1
TCAATGAGCTCGGTCCTACCCTTATGATGTG
Cloning


 


LLp10
5′-pJM-1
TTCACCTGACTGACGACAGT
Sequencing


 


LLp12
3′-pJM-1
ATGGATCCAGGCCTCTGGCGAAGAAGTCC
Cloning


 


LLp13
5′-pMON921
TCATTTCATTTGGAGAGGACACGC
Sequencing


 


LLp14
3′-pMON921
CCAATGCCATAATACTCGAACTCA
Sequencing


 


LLp15
TrxA-2
TACAGCGGTCCGTGCAAAATGATCGCC
Cloning


 


LLp16
TrxA-1
CGGACCGCACCACTCTGCCCAG
Cloning


 


LLp27
pMON921
GATCTGAATTCGCGATCTAGAGAGCTCG
Cloning


 


LLp28
pMON921
GATCCGAGCTCTCTAGATCGCGAATTCA
Cloning


 


LLp29
5′-aphA
AATTCGGACGTCGCTCCGTCGATACTATGTTATACGCC
Cloning


 


LLp30
3′-aphA
ATGACCCGGGGACGCTCAGTGGAACGAAAACTCACG
Cloning


 


LLp39
5′-Npt II
GGCGATAGAAGGCGATGCGCTGCG
Replication


 


LLp40
3′-Npt II
TGCACGCAGGTTCTCCGGCCGCT
Replication


 


LLp41
5′-pJM-1
AAGAGCTCAGTACTCCTACCCTTATGATGTGCCA
Cloning


 


LLp42
3′-pJM-1
TTGAGCTCCTCTGGCGAAGAAGTCCA
Cloning


 


LLp55
5′-GST 20mer
GTCCGGAGCTCCCTATACTAGGTTATTGGAAAATTAAGGGC
Cloning




CTTGTGCAACCCACTCGCG



 


LLp56
3T-GST 20mer
GACCGCGAGTGGGTTGCACAAGGCCCTTAATTTTCCAATAA
Cloning




CCTAGTATAGGGAGCTCCG





aNucleotides used to generate restriction sites for cloning are underlined.






C. Peptide Aptamer Screens

The pJM-1 library (Colas et al. (1996) Nature 380:548-550.) was amplified by transforming 5 μg plasmid DNA into 1×1010 electro-competent E. coli DH10B cells (Invitrogen) and stored at −80° C. in 40 mL aliquots containing 5×108 UFC/mL (Geyer and Brent (2000) Methods Enzymol. 328:171-208). Plasmid DNA was extracted using a QIAfilter plasmid maxi kit according to the manufacturer's protocols (QIAGEN), Saccharomyces cerevisiae strains EGY48 (MAT his3 trp1 ura3-52 leu2::LexA6op-) and EGY191 (MAT his3 trp1 ura3-52 leu2::LexA2op-LEU2) were used for the library screens (Estojak et al. (1995) Mol. Cell. Biol. 15:5820-5829). Plasmid DNA (50 μg) from the library was transformed into the bait strains containing the lacZ, reporter plasmid pSH18-34 (Invitrogen; Estojak et al. (1995) Mol. Cell. Biol. 15:5820-5829; Golemis and Brent (1992) Mol. Cell. Biol. 12:3006-3014) and the corresponding bait plasmids, Transformants were plated on synthetic dropout medium lacking histidine, tryptophan, uracil and leucine and supplemented with galactose/raffinose (Gal-HWUL) after heat shock and a 4 h incubation at 30° C. in liquid medium containing galactose/raffinose and lacking histidine and uracil (Gal-HU, Golemis and Brent (1992) Mol. Cell. Biol. 12:3006-3014). Recovered yeast colonies were also grown in medium lacking histidine, tryptophan and uracil and supplemented with glucose (Glu-HWU) to repress library expression. Activation of the leucine and β-galactosidase reporters was confirmed in growth assays (Gal-HWUL) and filter lift assays (Gal-HWU), respectively (Geyer and Brent (2000) Methods Enzymol. 328:171-208). pJM-1 plasmids containing the selected aptamers were recovered using the lyticase protocol and QIAGEN miniprep columns. The plasmids were transformed into E. coli KC8 strain (Clontech Yeast Protocols Manual PT3024-1) and selected on minimal M9 medium lacking tryptophan. Recovered plasmids were transferred into E. coli DH5α for isolation and retransformed into the yeast baits strains to confirm specific activation with the TAL11-352 and TAL11-130 baits and not with the GUS bait. For these assays, 4 μl droplets of 1×10−2 dilutions (OD600 adjusted to 0.08-0.12) of fresh yeast colonies were plated onto Gal-HWUL medium and incubated at 30° C. for 3-6 days. For sequencing, DNA minipreps were performed using the R.E.A.L. Prep 96 plasmid kit and a Biorobot 9600 (QIAGEN). Sequencing was performed according to the BigDye® Terminator v3.1 method (Applied Biosystems) using a Perkin Elmer Prism 3700 96-capillary automated DNA sequencer.
D. Replication Interference Assays.
Protoplasts were isolated from Nicotiana tabacum (BY-2) suspension cells, electroporated and cultured according to published methods (Fontes et al. (1994) J. Biol. Chem. 269:8459-8465). For the replication interference assays (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122), replicon DNA (2 μg) containing a partial tandem copy of TGMV A (pMON1565; Orozco and Hanley-Bowdoin. (1996) J. Virol. 270:148-158) was cotransfected with a plant expression cassette (40 μg). Viral DNA accumulation was monitored by either hybridization or semi-quantitative PCR. For the hybridization assays, total DNA was extracted 48 h post-transfection, digested with DpnI and XhoI, resolved on 1% agarose gels and probed with a 32P-labeled DNA corresponding to TGMV A. Double-stranded viral DNA accumulation was quantified by phosphorimager analysis in a minimum of three independent experiments.
For semi-quantitative PCR assays, BY-2 cells were harvested 36 h post-transfection and lysed by vortexing using 50 μL of glass beads in 400 μL lysis buffer (50 mM Tris-HCl pH 7.6, 100 mM NaCl, 50 mM EDTA, 0.5% SDS). The lysates were cleared by centrifugation at 14,000 g for 5 min and extracted using a QIAprep Spin Miniprep Kit according to the manufacturer protocols (QIAGEN). Total DNA was quantified by A260, and identical amounts were digested overnight with DpnI and subjected to PCR analysis using primers LLp1 and LLp2 (Table 3) for the TGMV A replicon. The amount (12.5-200 ng) of total DNA in the reactions was titrated for each experiment. pMON721 plasmid DNA (1 μg), which does not contain TGMV sequences (Lanahan et al. (1994) Plant Cell 6:521-530) was added to each PCR reaction as an internal control and amplified with primers LLp39 and LLp40 (Table 3). Bands were quantified using ImageJ software (Abramoff et al. (2004) Biophotonics International 11:36-42; Rasband, W. S. (2005) ImageJ. National Institutes of Health). PCR efficiency was standardized between reactions as a ratio of the band intensities corresponding to TGMV A DNA and the pMON721 control. Relative replication was determined as the ratio of the normalized intensity of each reaction versus the normalized intensity detected for protoplasts transfected with TGMV A replicon DNA and the empty expression cassette pMON921.
D. Sequence Alignments.
For each experimental database, the amino acid content of the peptide 20-mers was computed using a script aminocounter.pl that was coded using BioPerl Modules (Stajich et al. (2002) Genome Res. 12:1611-1618). Based on this information, 100 random databases of equivalent size and content were generated using the Perl script ranPEP.pl (Stajich et al. (2002) Genome Res. 12:1611-1618). The random and experimental peptide databases were formatted using NCBI formatdb.exe, and pairwise alignments were performed using the NCBI Basic Alignment Search Tool (BLASTP 2.2.10; Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) with a modified BLOSUM62 matrix (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The modified matrix removed the stringent gap restriction and included similarities based in amino acid hydrophobicity and charge. An E value of 20 (scores of 10 bits or more) was used as cutoff for the alignments, which were recorded as the number of peptides with hits and the sum of hits for all 20-mers in each database. These frequencies were used to calculate the expected or observed mean and the standard error of the mean for each database, which were compared in one-way T-tests in JMP 5.1 (SAS). The pairwise alignments of the experimental databases were analyzed further using the Vector NTI AlignX module (Invitrogen) to identify potential consensus motifs.
Example 3
Yeast Dihybrid Library Screen
Geminivirus replication proteins contain several conserved motifs that are essential for function. To develop new approaches to study and modulate these proteins, the pJM-1 library for interacting peptides using the well-characterized TGMV replication protein designated here as TAL1 was screened. The pJM-1 library encodes E. coli thioredoxin (TrxA) with 2.9×10a random 20-mer peptides in its active site (Colas et al. (1996) Nature 380:548-550). The TrxA-peptides are fused to the SV40 nuclear localization signal, the E. coli B42 activation domain (AD) and the hemagglutinin epitope tag and are expressed from the yeast Gall promoter (Colas et al. (1996) Nature 380:548-550). For the screens, two types of TAL1 bait plasmids (FIG. 2A) were constructed. TAL11-352 encodes the full-length TAL1 protein fused to the E. coli LexA DNA binding domain (DBD). TAL11-130 specifies a LexA DBD fusion with the first 130 amino acids of TAL1, which includes the conserved Motifs I, II and III (FIG. 1). A bait (CaAL11-349) containing the LexA DBD fused to the full-length coding sequence of the Cabbage leaf curl virus (CaLCuV) replication protein designated here as CaAL1 was also constructed. GUS encoding a DBD: β-glucuronidase fusion as a negative control was employed.
The advantages of the oligomerization properties of TAL1 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122) were used to test the functionality of our full-length AL1 baits (FIG. 2A). Yeast carrying the TAL11-352 bait and AD:TAL11-352 prey plasmids were able to activate the Leu reporter in the presence of galactose (Gal-HWUL plates, FIG. 2B-1), consistent with the ability of full-length TAL1 to form oligomers. Transformants carrying the CaAL11-349 bait and AD:TAL11-352 prey plasmids also grew (FIG. 2B-7), indicating that the heterologous CaAL1 protein is able to interact with TAU. In contrast, no growth was observed for the TAL11-130 bait (FIG. 2B-3), which lacks the oligomerization domain (Orozco et al. (1997) J. Biol. Chem. 272:9840-9846), or the GUS bait (FIG. 2B-5) in cotransfection assays with the AD:TAL11-352 prey plasmid. None of the baits interacted with the AD:Jun (FIGS. 2B-2, 4, 6 and 8) or the AD:TrxA-GST prey plasmids (data not shown). All of the bait/prey combinations also failed to grow on selective medium supplemented with glucose (Glu-HWUL plates, FIG. 2B). The presence of the bait and prey plasmids in the transformants was verified by growth on Glu-HWU plates. Similar results were obtained when activation of the LacZ reporter was used to detect interactions (data not shown). Together, these data established the specificity of the TAL11-352 and CaAL11-349 interactions. These results also verified that interaction is dependent on galactose induction of prey plasmid expression, and that none of the bait plasmids autoactivate the yeast reporters.
A two-step transformation protocol was used for the two-hybrid screens of the pJM-1 library (Geyer and Brent (2000) Methods Enzymol. 328:171-208). The yeast strain EGY48 was first co-transformed with the LacZ reporter pSH18-34 and the TAL11-352 bait plasmid. The recovered bait strains were transformed with 50 μg of the library DNA. A total of 5×106 colonies were plated on selective media (Gal-HWUL) in 2 transformation events, resulting in the recovery of 350 colonies. These colonies were transferred to Glu-HWU plates, grown for 2 days to repress the library expression and re-evaluated for induction of the Leu and LacZ reporters on HWUL and HWU media supplemented with either galactose or glucose. Prey plasmids were recovered from 350 colonies that grew only in the presence of galactose. Retransformation assays using bait strains carrying TAL11-352 or GUS verified the specificity of interaction for 170 of the recovered plasmids, 40 of which were sequenced. Eleven TrxA-peptides with unique sequences and no stop codons or frameshifts were selected for further analysis (Table 4).







TABLE 4







Aptamers isolated in screens with TAL11-352












Yeast
Expression


Aptamer
Peptide sequence
plasmida
cassetteb





FL-1
PLSGRQGVHLYFLLLMPA
B1118-001
pNSB1138


 


FL-7
FAVEYGSQGWGLWYCVWLDL
B1118-007
pNSB1141


 


FL-18
FQSRMGGGSGVVNAKLWAKE
B1118-018
pNSB1137


 


FL-19
VASRDSGAWRELHSFLNFAS
B1118-019
pNSB1135


 


FL-41
YYMALLYSQCPTVVLFRMTT
B1118-041
pNSB1139


 


FL-42
DFVCLCLFACTSDLSAFRVC
B1118-042
pNSB1136


 


FL-57
TAFRWDMFWMHTSGTWRKP
B1118-057
pNSB1143


 


FL-60
FASGSGEPVGLGLGSPLEKL
B1118-060
pNSB1144


 


FL-70
VYDSALCLVVGRCGLIRCR
B1118-070
pNSB1134


 


FL-90
LVWASM
B1118-090
pNSB1142


 


FL-99
LHESCWGWAGDSSPQGVLAG
B1118-099
pNSB1140





aThe yeast plasmids were cloned into pJM-1 with carbenicillin selection.


bThe plant expression cassettes were cloned into pMON921 with carbenicillin selection.






To facilitate the identification of TrxA-peptides that inhibit the essential DNA binding and cleavage activities mediated by the N-terminus of AL1 (Orozco et al. 1997), the pJM-1 library with the TAL11-130 bait (FIG. 2A) was also screened. The screens were performed as described above for the TAL11-352 bait except for the use of the stringent yeast strain EGY191 to ensure the identification of high affinity interactions (Golemis and Brent (1992) Mol. Cell. Biol. 12:3006-3014). In this experiment, 597 positive candidates were isolated from a screen of 2×107 yeast colonies. Interaction with TAL11-130 was confirmed for 287 yeast colonies displaying activation of both the Leu and LacZ reporters. Prey plasmids were recovered and sequenced for 130 colonies, out of which 88 unique TrxA-peptides were selected (Table 5).







TABLE 5







Aptamers isolated in screens with TAL11-130
















Yeast
Expression


Yeast
Expression


Aptamer
Peptide sequence
plasmida
cassetteb
Aptamer
Peptide sequence
plasmida
cassetteb





A-3
GFRAPGLSPTRPSCLICSTL
B1162-3
pNSB1228
A-114
VRSHRRYQRNWEPVVSWFSS
B1162-114
pNSB1279


 


A-5
NECLICHMLGIREFGLSA
B1162-5
pNSB1229
A-115
WCGPQVSARCK
B1162-115
pNSB1253


 


A-6
GTLWRRCASSWAFPPDCPSA
B1162-6
pNSB1230
A-116
SCDEAFDAASVASELFCQPY
B1162-116
pNSB1280


 


A-8
RRALRHCTGCMLSQRLGTAL
B1162-8
pNSB1258
A-117
ARMALSLREWEYLFFKDAPSGPGL
B1162-117
pNSB1228


 


A-9
HSMHSCSVGRCLVDVKVVVS
B1162-9
pNSB1231

QGLSLASRLNLVILRGYG




 


A-12
WMVCAGCGALRTRQVTLHPG
B1162-12
pNSB1232
A-119
RSYGGGEIPSVTMHCWIHCD
B1162-119
pNSB1281


 


A-15
GGFVPMRLCTCLLIVRLFI
B1162-15
pNSB1264
A-123
SSSRWVPFALQDPLFSSDDW
B1162-123
pNSB1282


 


A-16
VPQPLNCDLCVLMGGASSSR
B1162-16
pNSB1233
A-124
YLWSSKMDEWVAMDDVYAAC
B1162-124
pNSB1283


 


A-18
RRDYRKFFALNCQLCRLTVT
B1162-18
pNSB1234
A-127
TWGLVCTGTGWGLLDTVVRA
B1162-127
pNSB1284


 


A-22
CRTRGCGCHLCRMLSQFTGG
B1162-22
pNSB1235
A-129
VYEWGDVLCGGSMAIQWGL
B1162-129
pNSB1254


 


A-25
MRLGKGWNLMFLEEVSVLDA
B1162-25
pNSB1323
A-130
ASNGEIAYCVEQAMLLLCFH
B1162-130
pNSB1285


 


A-27
RDPQLGQVAQTWGCRLCLLE
B1162-27
pNSB1259
A-131
ELIVHEWPLILSRVGRIVL
B1162-131
pNSB1255


 


A-30
LVSESCGSWFCLCPWEVLNW
B1162-30
pNSB1263
A-132
GRVQLEILVSEAEEGVSPFL
B1162-132
pNSB1286


 


A-40
LQYSWNLYSVASFKTRRVSS
B1162-40
pNSB1236
A-135
RDAEWQDVLGRARAVHLRGR
B1162-135
pNSB1287


 


A-41
RLQESSIDLTPGIYLGMDFV
B1162-41
pNSB1265
A-136
GLKWKSDNGCVYVSFMRGGV
B1162-136
pNSB1288


 


A-46
CYMEVEGRPRRWADSFFVAW
B1162-46
pNSB1262
A-137
SSSPVPYSGGTCNLCSMRMW
B1162-137
pNSB1289


 


A-49
SESFVCKTCHMLRVSDAVGA
B1162-49
pNSB1260
A-140
EWEDPQYAGWELFSISDLVH
B1162-140
pNSB1256


 


A-50
MHVSLVFPWRLTGHIQQYKV
B1162-50
pNSB1261
A-141
PMVRTEWPLCAIIPLSMLYQ
B1162-141
pNSB1290


 


A-51
GRCNLQGMSFMGVGRSVWFE
B1162-51
pNSB1237
A-143
RAGWHERVRQWWAIECTLEV
B1162-143
pNSB1291


 


A-53
VVGGSLRDEWKWWREGRSLP
B1162-53
pNSB1267
A-145
SVRCWYVLRCSFLVGSGSSV
B1162-145
pNSB1292


 


A-59
AKDVERGAGGKIKACELCRL
B1162-59
pNSB1268
A-146
RSCVLCAYGSRTFNGSYLLF
B1162-146
pNSB1257


 


A-63
VETFKARARQTPSCDLCPKT
B1162-63
pNSB1238
A-147
GRGGCMLCDVDGSSAWLHTEGRLT
B1162-147
pNSB1266


 


A-64
TELWWADFAKMHMEGGKGMC
B1162-64
pNSB1239

GPITSQQCLSFQYLGNGEFIDG




 


A-67
RHRCTSRAPRQWFRPHRDSP
B1162-67
pNSB1269
A-149
TLETLDMGNPLYTCVLMDWM
B1162-149
pNSB1324


 


A-69
RYRVSAGPLCSLCSLWGSVG
B1162-69
pNSB1240
A-150
LVMGWRSEVSSLQGKTGTGGGPTL
B1162-150
pNSB1293


 


A-71
EEGLAAITHTWLTMCFAAGL
B1162-71
pNSB1241

RKCQLCRGSRYTLKYYPC




 


A-73
AAFLESVRSYWSRFVRHVQG
B1162-73
pNSB1242
A-153
RPGCPFCTSWRCG
B1162-153
pNSB1294


 


A-75
RAMCDKDKSVCSILALYVQV
B1162-75
pNSB1243
A-155
FCPECQMVAGAEDGDAIDLQ
B1162-155
pNSB1295


 


A-80
CWWLREIGTFRCVTLQHVAG
B1162-80
pNSB1244
A-158
RRCMLCTSDKPGGDQGALNM
B1162-158
pNSB1296


 


A-82
FESAWSTLMGAMTPMVLDET
B1162-82
pNSB1270
A-159
LWGGGTAWDFFVWGEDSAC
B1162-159
pNSB1297


 


A-83
QALVVSPETFLCLEALGVNS
B1162-83
pNSB1271
A-160
GMSGRIPEPDDWVVLFITGC
B1162-160
pNSB1298


 


A-84
GGRQTEPSLTLLADLTLLLS
B1162-84
pNSB1272
A-161
GGTNALLQKVFFGEVGVASM
B1162-161
pNSB1299


 


A-86
GSRAELSAPEVAWLLFCTPG
B1162-86
pNSB1245
A-164
ECCLFPIFAMADSFPCPSPV
B1162-164
pNSB1300


 


A-87
RYSAVCRDCYEGHGRGLWYM
B1162-87
pNSB1273
A-165
MLEGPLDQGLMMGTCCWECS
B1162-165
pNSB1301


 


A-89
GGWLVTIVEGPLAICCLRDD
B1162-89
pNSB1274
A-166
TPSVTWAEWCSCVFCRDAS
B1162-166
pNSB1302


 


A-90
PSIESGWVGDQAVAPCDLSV
B1162-90
pNSB1275
A-167
SWWWANNSLCREWEFAC
B1162-167
pNSB1303


 


A-91
TWGAWKRDIVLVSEIGFTWG
B1162-91
pNSB1246
A-168
WNMLAFGGALVASGLLRGWE
B1162-168
pNSB1304


 


A-94
RLGGGRPKLWHFSPNLMAGF
B1162-94
pNSB1247
A-169
DKCDDVEPFLWWGQQCFFDV
B1162-169
pNSB1305


 


A-97
ERVHVCFSRKCTALSVDSSV
B1162-97
pNSB1248
A-170
GSPSRISYTCLSPDVTLLFL
B1162-170
pNSB1306


 


A-99
RERGGDDYRRMMHPGAASGP
B1162-99
pNSB1249
A-172
MGIEACSITECTSQHCNEVA
B1162-172
pNSB1307


 


A-100
RLVVGCEWRIGCSTGSGPRG
B1162-100
pNSB1250
A-173
CLDNLCWELGGGFPVILIHC
B1162-173
pNSB1308


 


A-101
ASLIGVGIASMHGMQTDGIY
B1162-101
pNSB1251
A-174
HVHGSCPSMGWSSNSWCSVF
B1162-174
pNSB1309


 


A-108
VGLMEWAVWSLEVREKLYSC
B1162-108
pNSB1276
A-175
PLELEFAVCGCSWLVALDWS
B1162-175
pNSB1310


 


A-109
VLGRLGGAGGCSLCDQLEAL
B1162-109
pNSB1277
A-176
AWDSESLATVVASVMPWPYPT
B1162-176
pNSB1311


 


A-110
IWINPNGLWWTKVGLNPYAV
B1162-110
pNSB1252
A-177
TGCHYKGARCCRLTWDVLIL
B1162-177
pNSB1312


 


A-112
RHESALHKSCELCYCPWKVC
B1162-112
pNSB1278





aThe yeast plasmids were cloned into pJM-1 with carbenicillin selection.


bThe plant expression cassettes were cloned into pNSB1226 with kanamycin selection.






Because these TrxA-peptides were selected for binding to a truncated TAL1 protein that does not oligomerize, interaction with full-length TAL1 was investigated. FIG. 3B shows that yeast co-transformed with each of the 88 TrxA-peptides in the presence of either TAL11-130 or TAL11-352 baits grew on in Gal-HWUL medium. Cotransfection with the negative control bait GUS did not induce the Leu reporter and no growth occurred (FIG. 3B). No growth was seen on plates lacking leucine but supplemented with glucose (FIG. 3C). Growth on Glu-HWU medium confirmed the presence of the prey and bait plasmids in all of the transfections (FIG. 3D). Based on these results, it can be concluded that the 88 TrxA-peptides initially selected for interaction with TAL11-130 also bind to TAL11-352.
Example 4
Aptamer Interference with Viral DNA Replication
After the initial screening, a subsequent aptamer library screen was performed to identify aptamer sequences that specifically bind to the N-terminal domain of Rep and impact Rep function during viral replication.
A. Binding of TrxA-Peptides to TGMV AL1 and Interference with Viral DNA Replication.
The 11 FL-TrxA-peptides (Table 4) selected in the screen using the TAL11-352 bait were subcloned into a plant expression cassette containing the CaMV35S promoter with a duplicated enhancer and the rbcS E9 terminator. The constructs were co-transfected into tobacco protoplasts in the presence of a replicon plasmid containing a partial tandem copy of TGMV A that supports the release of unit length viral DNA (FIG. 4A). The TGMV A DNA, which encodes TAL1 and its replication accessory factor AL3 (FIG. 4A), replicates autonomously and accumulates to high copy number in plant cells (Orozco and Hanley-Bowdoin. (1996) J. Virol. 270:148-158). To determine whether expression of the FL-TrxA-peptides quantitatively impacts TGMV A DNA accumulation as an indicator of altered TAL1 activity, total DNA was isolated 48 h post transfection, and the levels of double-stranded TGMV A DNA were examined by DNA gel blot hybridization. Nine of the FL-TrxA-peptides had no detectable effect on viral DNA accumulation (data not shown). In contrast, cells containing the FL-42 (FIG. 4B, lane 3) and FL-60 (lane 4) cassettes only accumulated about 25% of the levels detected in a transfection containing an empty expression cassette (lane 2). The level of viral DNA detected in the presence of FL-42 and FL-60 was similar to that seen for FQ118 (FIG. 4B, lane 1), a strong trans-dominant negative mutant of TAL1 (Orozco et al. (2000) J. Biol. Chem. 275:6114-6122). These results indicated that two of the FL-TrxA-peptides interfere with the ability of TAL1 to support viral DNA replication.
The 88 N-TrxA-peptides were also tested in replication interference assays. For these experiments, a semi-quantitative PCR protocol was developed to facilitate the analysis of a large number of expression cassettes. The assay was based on primers that distinguish the input replicon cassette and nascent viral DNA by size and DpnI sensitivity. Primers LLp1 and LLp2 (Table 3 and FIG. 4A) amplify a 4.9 Kb DNA from the replicon cassette and a 1.2 Kb product from the released TGMV A component (FIG. 4C, lane 1) in DNA extracts from E. coli cells carrying the replicon cassette plasmid. Even though the replicon cassette is the prevalent form in E. coli (data not shown), it amplified less, efficiently than the released TGMV component because of its large size. The production of both products is sensitive to DpnI digestion (FIG. 4C, lane 2) and resistant to MboI digestion (lane 3), indicative of an E. coli Dam-methylated template. Interestingly, the same results (FIG. 4C, lanes 4-6) were obtained for the mutant TGMV A replicon cassette with a frame shift mutation at TAL1 amino acid position 120 (Elmer et al. (1988) Nucleic Acids Res. 16:7043-7060).
The amplification strategy was also tested with DNA extracted from tobacco cells co-transfected with various TGMV A replicon and expression cassettes. It was then determined whether the input and nascent viral DNA can be distinguished by comparing DNA samples isolated from cells transfected with the wild type TGMV A replicon cassette or the mutant AL1 cassette. The 1.2 Kb product (top band) was produced when uncut and MboI-digested DNA from cells transfected with both cassettes was amplified (FIG. 4D, lanes 1-3 and 10-12, top and bottom). In contrast, the 1.2 Kb product was only amplified from DpnI-treated DNA from cells with the wild type cassette (FIG. 4D, lanes 1-3, middle) but not the mutant cassette (lanes 10-12, middle). This result demonstrated that residual Dam-methylated E. coli DNA can be quantitatively removed by DpnI digestion, thereby allowing the detection of nascent DNA by PCR. Subsequent verification that replication interference can be monitored by PCR was obtained by showing that the level of the 1.2 Kb PCR product is reduced in cells carrying a TAL1 dominant negative mutant (FQ118) expression cassette (FIG. 4D, lanes 7-9) relative to cells with the empty (lanes 1-3) or TrxA-GST (lanes 7-9) cassettes. This difference was not apparent in uncut or MboI-treated DNA because of the presence of intact E. coli input DNA (FIG. 4D, lanes 1-9, upper and lower). Similar results were seen for the three biological replicas for each transfection condition. Together; these results establish that the PCR assay can be used to monitor viral DNA accumulation in a reproducible, semi-quantitative manner.
Expression cassettes corresponding to the 88 N-TrxA-peptides (Table 5) were transfected into tobacco protoplasts with the wild type TGMV A replicon cassette. Total DNA was isolated 36 h after transfection and analyzed in replication interference assays using the semi-quantitative PCR method. Because of the high number of samples, the N-TrxA-peptides were initially analyzed in triplicate in 3 separate experiments. 35 N-TrxA-peptide cassettes that reduced viral DNA accumulation relative to the empty expression cassette were selected. The selected cassettes were then assayed in a single transfection experiment (FIG. 5), with 31 of 35 showing statistically significant interference activity (p<0.05 in a one-tailed Students T-test). The experiment also included the FQ118 and TrxA-GST expression cassettes as positive and negative controls, respectively. The N-TrxA-peptides were classified as weak (50-65%), moderate (25-50%) and strong (<25%) interferers (FIG. 5, dotted lines) relative to the control transfection with an empty cassette (100%). Ten N-TrxA-peptides showed strong interference (black bars), fourteen exhibited moderate interference (grey bars), and seven were weak interferers (white bars). In total, fourteen aptamers displayed interference activity that was greater or equal to FQ118. TrxA-GST did not impact viral DNA accumulation, indicating the TrxA sequences per se do not contribute to interference.
B. Binding of N-TrxA-Peptides to CaAL1 and Interference with Viral DNA Replication.
Motifs I, II and III in TAL11-130 (FIG. 1) are conserved in all geminivirus replication proteins (Ilyina and Koonin (1992) Nucleic Acids Res. 20:3279-3285; Koonin and Ilyina (1992) J. Gen. Virol. 73:2763-2766). Hence, it was determined whether peptides that bind to TAL11-130 and inhibit TGMV replication are also able to interact with an AU protein from a heterologous geminivirus. Accordingly, experiments were conducted using CaLCuV AL1 protein because it only shares 42% identity and 58% similarity with TAL1 across the first 130 amino acids. Full-length CaAL1 fused to the LexA DBD (CaAL11-349) was used as bait in yeast two-hybrid assays with the 31 N-TrxA-peptides that displayed replication interference activity (FIG. 5). All of the peptides were positive for interaction with CaAL11-349 in growth assays (FIG. 6B, right). The prey control did not interact with any of the baits (FIG. 6). Comparison of the interactions with TAL11-130, TAL11-352 and CaAL11-349 baits on LacZ plates suggested that interaction is stronger with TAL11-130 than with the two full-length AU baits (data not shown).
Example 5
Pairwise Alignments of Peptide Aptamers
To determine whether the peptide aptamers of the present invention are random or related in view of their selection for binding to TAL1 the 20-mers were grouped into three database corresponding to the 88 selected for binding to TAL11-130 (All), the 31 positive for replication interference (Interfering), and the 57 negative for interference (Non-interfering), and their sequences were compared in pairwise alignments using BLASTP. The score values of the alignments were low (data not shown) because of the short length of the peptide sequences (20 amino acids). To address the possibility that the alignments were produced by chance, 100 sets of three random databases of the same size and amino acid content as the N-TrxA-peptide were compared using BLASTP. The distribution of the frequency of hits was analyzed for each database and used to determine the expected mean and standard error of the mean for random sequences.
The frequency distribution of the 100 databases comprising the 88 random 20-mers is shown in FIG. 7A. The left panel represents the expected distribution of peptides with at least one hit while the right panel shows the frequency distribution of the total number of hits for all the 20-mers in the database. The expected means of the two distributions (54 and 101) are lower than the observed means (67 and 221, respectively) for the All database (FIG. 7B). The observed means for the Interfering and Non-interfering databases are also higher than the expected means calculated using the corresponding random databases (FIG. 7B). The observed and expected means of all three databases differed by at least two standard deviations and gave p<0.0001 in one-way Students T-tests. These results established that even though N-TrxA-peptides were derived from a random library, their sequences are not random.
Inspection of the BLASTP alignments revealed that some pairs contained common sequences. Similar pairs were grouped and compared using the Vector NTI AlignX module. A total of 18 groups containing four or more sequences were identified among the 88 N-TrxA peptides. The putative motifs were filtered using four criteria—[1] include at least five members, [2] members interact with CaAL1, [3] contain amino acids typically involved in protein-protein interactions (Bogan and Thorn (1998) J. Mol. Biol. 280:1-9; Glaser et al. (2001) Proteins 43:89-102), and [4] related to a plant protein. Seven motifs that satisfied at least three criteria are shown in FIG. 8A. The sequence alignments are shown in FIGS. 9 and 10. Motifs 1, 4, 20, 25 and 27 consist primarily of interfering peptides, while Motif 28 is composed of mostly non-interfering peptides. Motif 24 includes 18 members that are distributed between interfering and non-interfering peptides, all of which contain a core CxLC sequence (FIG. 8). The interfering members also include conserved polar and nonpolar residues flanking the core sequence (FIG. 9). These residues occur individually in non-interfering peptides, but only the interfering peptides contain both sets of flanking residues.
Example 6
Characterization of Additional Peptide Aptamers
To expand the repertoire of aptamers, additional peptides selected in the dihybrid screen using full-length TGMV Rep were characterized. The locations of their binding sites were mapped using a series of baits corresponding to known Rep functional domains (FIG. 1). The baits contained Rep1-130 (DNA binding, cleavage and ligation), Rep101-180 (C3 and pRBR binding), Rep130-180 (GRIK, CRIMP, PCNA and Ubc9 binding and oligomerization), and Rep180-352 (helicase) and were designated as FL, NT, PI, OL and CT, respectively. Aptamers were further tested for binding to full-length CaLCuV Rep.
The results of the yeast two-hybrid experiments are summarized in Table 6. The peptide sequences and binding properties of 99 Trx-aptamers that bound to full-length TGMV Rep and do not contain a frameshift or stop condon in their coding sequence are shown in Table 7. A comprehensive listing of peptide sequences according to some embodiments of the present invention is provided in Table 8.







TABLE 6







Results summary











TGMV baits
TGMV Rep
CaLCuV Rep















FL
22
22



FL, NT
7
6



FL, PI
8
4



FL, OL
12
10



FL, CT
7
7



FL, NT, PI
7
7



FL, NT, OL
3
3



FL, NT, CT
5
4



FL, PI, OL
5
4



FL, OL, CT
11
10



FL, NT, PI, OL
1
1



FL, NT, PI, CT
2
1



FL, NT, OL, CT
5
4



FL, NT, PI, OL, CT
3
3

















TABLE 7







Binding properties and sequences of individual


peptide aptamers










Aptamer





name
TGMV Rep
CaLCuV



(FL#)
bait*
Rep bait*
peptide sequence













5
FL
YES
ELLVAHLITPWTSMGRTQAL


73
FL
YES
HKDRGYANLMLCSLLACFEP


76
FL
YES
GMLWTWLCDSPQSFVAPRGV


111
FL
YES
PVCRVGRGLLVQAKLVRAQS


119
FL
YES
DLEWDGNAYSGCCHCAFSIR


141
FL
YES
PNCEICYVARRLVLSMEACS


165
FL
YES
EGLPIDLLTNWHLTCWIALG


177
FL
YES
GVNLLQRCWGGPVHIFSYLM


184
FL
YES
CLMHMRFPLGGTWRMNLRAE


190
FL
YES
LVTALIMSESIVRMNPMYLT


193
FL
YES
EVERSRMLFNYGGMVASRVA


194
FL
YES
PWLSVDVTALIVDFLQDFSA


200
FL
YES
EGGEDSAWDWGSSGGIWCWF


206
FL
YES
CDVMEWRMMGLGLSKWLRGR


219
FL
YES
VYYHKECALDSYVRTCWVSG


225
FL
YES
PSAYEAVETLDMSEVKGLGQ


237
FL
YES
CEDMREAKVCRTLLAHSFLP


251
FL
YES
TSWRHRMPTGTDRCCFLVQL


275
FL
YES
TIDQRMVSLGAIWWSYPRCW


293
FL
YES
AQRSCWERLWTGQWRRSASD


322
FL
YES
WGYFGSFVGGVFDVWFSGVA


326
FL
YES
RNGRNICVLSVCSRFSHFNP


 


133
FL, NT
YES
FGVIVTNAASEFTTRVDDSC


199
FL, NT
YES
FNARALACKCDRGILILSQP


214
FL, NT
YES
IYDYTWAEEQGYVWRPAGGA


227
FL, NT
YES
LARCLCEIVGSCISYSNLPI


239
FL, NT
YES
RPWSSDTSVWWDGLFGMNYS


252
FL, NT
NO
RPWSSDTSVWWDGLFGMNYS


281
FL, NT
YES
TEACQVVLLGKRSLLPVVAG


 


52
FL, PI
YES
LARQPGKFIELPVLIRFATSGP





LLQSNSSSGHWLSDEHWTRR


180
FL, PI
YES
VDSIDKGEALVSLWGWHVQI


189
FL, PI
YES
LVRWSYTCSVLVGLRDSLDS


278
FL, PI
NO
PPWTKRPLTSGGVRGELWVW


280
FL, PI
NO
RVDSLGIKLDKSTLVTVHVV


294
FL, PI
NO
VSWSAAGRGYVFMYRWSPRC


325
FL, PI
NO
SRSDLWVSWCRNLLDGQSWS


381
FL, PI
YES
MLGIWRVLHEMVVPLKLGVD


 


58
FL, OL
NO
GKLTDTTRISVCCICVSVLD


72
FL, OL
YES
MDRREDLRSLLVLTLSDARG


100
FL, OL
YES
PLLLLAADSVELQRVLARR


107
FL, OL
YES
AMTYRAAWSPPPWGVLGIWH


154
FL, OL
YES
RLTVLEAAVVLWGWSLFQVP


197
FL, OL
YES
VIVDFLSTGVSTGEVRGGIV


221
FL, OL
YES
TLHTNRFCFRWVPALDSVTT


256
FL, OL
NO
LVTALIMSESIVRMNPMYLT


258
FL, OL
YES
GVNNGGTNDPEGVSEASWIP


300
FL, OL
YES
FGVIVTNAASEFTTRGYDSC


304
FL, OL
YES
SRSDLWVSWCRNLLDGQSWS


382
FL, OL
YES
CPDCPLSSVLRTATTAFFGG


 


121
FL, CT
YES
QSVRKMPMFWPLAGEVWCRPLGFR


181
FL, CT
YES
PPWTKRPLTSGGVRGELWVW


196
FL, CT
YES
ISSYLWWSEYCRPGSAMGDV


218
FL, CT
YES
RVWTFFVREAALELPSRDTL


222
FL, CT
YES
LNPWEGEWTRWDVFRVLGEF


265
FL, CT
YES
GIVSKQGADEGMLEIFASSW


292
FL, CT
YES
AQRSCWERLWTGQWRRLPLM


 


8
FL, NT, PI
YES
DEQESVCRSCKCRYVDNWLE


25
FL, NT, PI
YES
HKDRGYANLMLCSLLACFEP


117
FL, NT, PI
YES
SFVVQSFLGGKSIFNGPFAD


120
FL, NT, PI
YES
LGAPLLRCMVHHAMMVGEGY


135
FL, NT, PI
YES
RPWSSDTSVWWDGLFGMNYS


232
FL, NT, PI
YES
SFATANSEQVLRDMLLLASH


364
FL, NT, PI
YES
TGSGLTPCLHCRVQFQRSYL


 


216
FL, NT, OL
YES
QMNAAPPARSCADTWSLLLF


229
FL, NT, OL
YES
SGYPKMVWGEGPMLLDWKFV


246
FL, NT, OL
YES
WCSMCSVLRAFNCPYFCPWL


 


14
FL, NT, CT
YES
VGGMPPLPWYEPVGLVWSCM


21
FL, NT, CT
YES
GKLTDTRISVCCCICVSVLD


167
FL, NT, CT
YES
SFMMRLLRTGEMQFQADCVGGP





IPLKSPRALSLYNWGLLLWV


202
FL, NT, CT
YES
LLYPSLTLTLWRWLFADEGC


247
FL, NT, CT
NO
PWIFDRSVVCEEREAPRRHL


 


6
FL, PI, OL
YES
TNELPLTIVTDDVSQLVISRGP





ARHLYELMPEMLVLRSARLT


334
FL, PI, OL
YES
GVLFTFKKYPQGLSCTTSYG


341
FL, PI, OL
NO
LLACSVYWLWQRPCDGCLFM


365
FL, PI, OL
YES
TRMHSLCSGFCVICMGGPRV


380
FL, PI, OL
YES
RNGRNICVLSVCSRFSHFNP


 


109
FL, OL, CT
YES
GMLWTWLCDSPQSFVAPRGV


116
FL, OL, CT
YES
EVVETAEVYWSCGDWSCEGW


179
FL, OL, CT
YES
CAMCLDVFGWSASHWGGFTV


220
FL, OL, CT
YES
FNARALACKCDRRILILSQP


245
FL, OL, CT
YES
GRFGQGQCYQVADSTYWTFGPG





GPRKCEREPAGWSDTGWVC


254
FL, OL, CT
YES
RPWSSDTSVWWDGLFGMNYS


262
FL, OL, CT
YES
LLACSVYWLWQRPCDGCLFM


302
FL, OL, CT
NO
SFSSLFLAWLMQTGQEAGTV


312
FL, OL, CT
YES
HPYLITDIISMYRSPWSVPA


352
FL, OL, CT
YES
VVQNVRGWLVYCCADFHTYV


355
FL, OL, CT
YES.
LRGVSPWLQSFVSIAVQSCK


 


313
FL, NT, PI, OL
YES
RDALTNIGRSICALLLVLCK


 


153
FL, NT, PI, CT
YES
PNCEICYVARRLVLSMEACS


223
FL, NT, PI, CT
NO
QMFWFTDSEGKPGFCTFYGF


 


110
FL, NT, OL, CT
YES
ALKDEPFCDLPMVLVSWWRG


208
FL, NT, OL, CT
YES
DLEWDGNAYSGCCHCAGSIR


257
FL, NT, OL, CT
YES
LLACSVYWLWQRPCDGCLFM


328
FL, NT, OL, CT
NO
GGSDERYFWYQSFSSCAYEW


434
FL, NT, OL, CT
YES
RGRMEADKSFDSTCLRCGCS


 


236
FL, NT, PI, OL, CT
YES
VGPLIVGPPGMEMTANSWSC


242
FL, NT, PI, OL, CT
YES
DLRLPVYSEWVRVYSSDAWM


293
FL, NT, PI, OL, CT
YES
AQRSCWERLWTGQWRRLPLM





*TGMV baits


FL — full length (aa 1-352)


NT — N-terminus (aa 1-130)


PI — protein interaction (aa 101-180)


OL — oligomerization (aa 130-180)


CT — C-terminus (181-352)


CaLCuV bait


full length (aa 1-347)













TABLE 8







Listing of peptide sequences









SEQ ID NO.
Research Name
Peptide sequence












1
Motif 3
VRDYILKEPL


 


2
Motif 3
VKSYVDKDGD


 


3
Motif 3
VKEYIDKDGV


 


4
Motif 3
VNSYVDKDGD


 


5
Motif 3
NKEYCSKEGH


 


6
Motif 3
NLTYVSKIGG


 


7
Motif 3
AQLYAMKEDS


 


8
Motif 3
ARSYCMKEDT


 


9
Motif 1
WxDxxxAW


 


10
Motif 4
MHxxxxxG


 


11
Motif 20
(L/M)GGxxP


 


12
Motif 24
(G/S)CxLCxL


 


13
Motif 25
WxxxSLC


 


14
Motif 27
(S/A)FxxAxVAS


 


15
Motif 28
WfxVL


 


16
A-3
GFRAPGLSPTRPSCLICSTL


 


17
A-5
NECLICHMLGIREFGLSA


 


18
A-6
GTLWRRCASSWAFPPDCPSA


 


19
A-8
RRALRHCTGCMLSQRLGTAL


 


20
A-9
HSMHSCSVGRCLVDVKVVVS


 


21
A-12
WMVCAGCGALRTRQVTLHPG


 


22
A-15
GGFVPMRLCTCLLIVRLFI


 


23
A-16
VPQPLNCDLCVLMGGASSSR


 


24
A-18
RRDYRKFFALNCQLCRLTVT


 


25
A-22
CRTRGCGCHLCRMLSQFTGG


 


26
A-25
MRLGKGWNLMFLEEVSVLDA


 


27
A-27
RDPQLGQVAQTWGCRLCLLE


 


28
A-30
LVSESCGSWFCLCPWEVLNW


 


29
A-40
LQYSWNLYSVASFKTRRVSS


 


30
A-41
RLQESSIDLTPGIYLGMDFV


 


31
A-46
CYMEVEGRPRRWADSFFVAW


 


32
A-49
SESFVCKTCHMLRVSDAVGA


 


33
A-50
MHVSLVFPWRLTGHIQQYKV


 


34
A-51
GRCNLQGMSFMGVGRSVWFE


 


35
A-53
VVGGSLRDEWKWWREGRSLP


 


36
A-59
AKDVERGAGGKIKACELCRL


 


37
A-63
VETFKARARQTPSCDLCPKT


 


38
A-64
TELWWADFAKMHMEGGKGMC


 


39
A-67
RHRCTSRAPRQWFRPHRDSP


 


40
A-69
RYRVSAGPLCSLCSLWGSVG


 


41
A-71
EEGLAAITHTWLTMCFAAGL


 


42
A-73
AAFLESVRSYWSRFVRHVQG


 


43
A-75
RAMCDKDKSVCSILALYVQV


 


44
A-80
CWWLREIGTFRCVTLQHVAG


 


45
A-82
FESAWSTLMGAMTPMVLDET


 


46
A-83
QALVVSPETFLCLEALGVNS


 


47
A-84
GGRQTEPSLTLLADLTLLLS


 


48
A-86
GSRAELSAPEVAWLLFCTPG


 


49
A-87
RYSAVCRDCYEGHGRGLWYM


 


50
A-89
GGWLVTIVEGPLAICCLRDD


 


51
A-90
PSIESGWVGDQAVAPCDLSV


 


52
A-91
TWGAWKRDIVLVSEIGFTWG


 


53
A-94
RLGGGRPKLWHFSPNLMAGF


 


54
A-97
ERVHVCFSRKCTALSVDSSV


 


55
A-99
RERGGDDYRRMMHPGAASGP


 


56
A-100
RLVVGCEWRIGCSTGSGPRG


 


57
A-101
ASLIGVGIASMHGMQTDGIY


 


58
A-108
VGLMEWAVWSLEVREKLYSC


 


59
A-109
VLGRLGGAGGCSLCDQLEAL


 


60
A-110
IWINPNGLWWTKVGLNPYAV


 


61
A-112
RHESALHKSCELCYCPWKVC


 


62
A-114
VRSHRRYQRNWEPVVSWFSS


 


63
A-115
WCGPQVSARCK


 


64
A-116
SCDEAFDAASVASELFCQPY


 


65
A-117
ARMALSLREWEYLFFKDAPSGPGLQGLSLASRLNLVILRGYG


 


66
A-119
RSYGGGEIPSVTMHCWIHCD


 


67
A-123
SSSRWVPFALQDPLFSSDDW


 


68
A-124
YLWSSKMDEWVAMDDVYAAC


 


69
A-127
TWGLVCTGTGWGLLDTVVRA


 


70
A-129
VYEWGDVLCGGSMATQWGL


 


71
A-130
ASNGEIAYCVEQAMLLLCFH


 


72
A-131
ELIVHEWPLILSRVGRIVL


 


73
A-132
GRVQLEILVSEAEEGVSFL


 


74
A-135
RDAEWQDVLGRARAVHLRGR


 


75
A-136
GLKWKSDNGCVYVSFMRGGV


 


76
A-137
SSSPVPYSGGTCNLCSMRMW


 


77
A-140
EWEDPQYAGWELFSISDLVH


 


78
A-141
PMVRTEWPLCATIPLSMLYQ


 


79
A-143
RAGWHE RVRQWWAIECTLEV


 


80
A-145
SVRCWYVLRCSFLVGSGSSV


 


81
A-146
RSCVLCAYGSRTFNGSYLLF


 


82
A-147
GRGGCMLCDVDGSSAWLHTEGRLTGPITSQQCLSFQYLGNGEFIDG


 


83
A-149
TLETLDMGNPLYTCVLMDWM


 


84
A-150
LVMGWRSEVSSLQGKTGTGGGPTLRKCQLCRGSRYTLKYYPC


 


85
A-153
RPGCPFCTSWRCG


 


86
A-155
FCPECQMVAGAEDGDAIDLQ


 


87
A-158
RRCMLCTSDKPGGDQGALNM


 


88
A-159
LWGGGTAWDFFVWGEDSAC


 


89
A-160
GMSGRIPEPDDWVVLFITGC


 


90
A-161
GGTNALLQKVFFGEVGVASM


 


91
A-164
ECCLFPIFAMADSFPCPSPV


 


92
A-165
MLEGPLDQGLMMGTCCWECS


 


93
A-166
TPSVTWLAEWCSCVFCRDAS


 


94
A-167
SWWWANNSLCREWEFAC


 


95
A-168
WNMLAFGGALVASGLLRGWE


 


96
A-169
DKCDDVEPFLWWGQQCFFDV


 


97
A-170
GSPSRISYTCLSPDVTLLFL


 


98
A-172
MGIEACSITECTSQHCNEVA


 


99
A-173
CLDNLCWELGGGFPVILIHC


 


100
A-174
HVHGSCPSMGWSSNSWCSVF


 


101
A-175
PLELEFAVCGCSWLVALDWS


 


102
A-176
AWDSESLATWASVMPWPYPT


 


103
A-177
TGCHYKGARCCRLTWDVLIL


 


104
FL-1
PLSGRQGVHLYFLLLMPA


 


105
FL-7
FAVEYGSQGWGLWYCVWLDL


 


106
FL-18
FQSRMGGGSGVVNAKLWAKE


 


107
FL-19
VASRDSGAWRELHSFLNFAS


 


108
FL-41
YYMALLYSQCPTVVLFRMTT


 


109
FL-42
DFVCLCLFACTSDLSAFRVC


 


110
FL-57
TAFRWDMFWMHTSGTWRKP


 


111
FL-60
FASGSGEPVGLGLGSPLEKL


 


112
FL-70
VYDSALCLVVGRCGLIRCR


 


113
FL-90
LVWASM


 


114
FL-99
LHESCWGWAGDSSPQGVLAG


 


115
FL-5
ELLVAHLITPWTSMGRTQAL


 


116
FL-6
TNELPLTIVTDDVSQLVISRGPARHLYELMPEMLVLRSARLT


 


117
FL-8
DEQESVCRSCKCRYVDNWLE


 


118
FL-14
VGGMPPLPWYEPVGLVWSCM


 


119
FL-21
GKLTDTRISVCCCICVSVLD


 


120
FL-25
HKDRGYANLMLCSLLACFEP


 


121
FL-52
LARQPGKFIELPVLIRFATSGPLLQSNSSSGHWLSDEHWTRR


 


122
FL-58
GKLTDTTRISVCCICVSVLD


 


123
FL-72
MDRREDLRSLLVLTLSDARG


 


124
FL-73
HKDRGYANLMLCSLLACFEP


 


125
FL-76
GMLWTWLCDSPQSFVAPRGV


 


126
FL-100
PLLLLAADSVELQRVLARR


 


127
FL-107
AMTYRAAWSPPPWGVLGIWH


 


128
FL-109
GMLWTWLCDSPQSFVAPRGV


 


129
FL-110
ALKDEPFCDLPMVLVSWWRG


 


130
FL-111
PVCRVGRGLLVQAKLVRAQS


 


131
FL-116
EVVETAEVYWSCGDWSCEGW


 


132
FL-117
SFVVQSFLGGKSIFNGPFAD


 


133
FL-119
DLEWDGNAYSGCCHCAFSIR


 


134
FL-120
LGAPLLRCMVHHAMMVGEGY


 


135
FL-121
QSVRKMPMFWPLAGEVWCRPLGFR


 


136
FL-133
FGVIVTNAASEFTTRVDDSC


 


137
FL-135
RPWSSDTSVWWDGLFGMNYS


 


138
FL-141
PNCEICYVARRLVLSMEACS


 


139
FL-153
PNCEICYVARRLVLSMEACS


 


140
FL-154
RLTVLEAAVVLWGWSLFQVP


 


141
FL-165
EGLPIDLLTNWHLTCWIALG


 


142
FL-167
SFMMRLLRTGEMQFQADCVGGPIPLKSPRALSLYNWGLLLWV


 


143
FL-177
GVNLLQRCWGGPVHIFSYLM


 


144
FL-179
CAMCLDVFGWSASHWGGFTV


 


145
FL-180
VDSIDKGEALVSLWGWHVQI


 


146
FL-181
PPWTKRPLTSGGVRGELWVW


 


147
FL-184
CLMHMRFPLGGTWRMNLRAE


 


148
FL-189
LVRWSYTCSVLVGLRDSLDS


 


149
FL-190
LVTALIMSESIVRMNPMYLT


 


150
FL-193
EVERSRMLFNYGGMVASRVA


 


151
FL-194
PWLSVDVTALIVDFLQDFSA


 


152
FL-196
ISSYLWWSEYCRPGSAMGDV


 


153
FL-197
VIVDFLSTGVSTGEVRGGIV


 


154
FL-199
FNARALACKCDRGILILSQP


 


155
FL-200
EGGEDSAWDWGSSGGIWCWF


 


156
FL-202
LLYPSLTLTLWRWLFADEGC


 


157
FL-206
CDVMEWRMMGLGLSKWLRGR


 


158
FL-208
DLEWDGNAYSGCCHCAGSIR


 


159
FL-214
IYDYTWAEEQGYVWRPAGGA


 


160
FL-216
QMNAAPPARSCADTWSLLLF


 


161
FL-218
RVWTFFVREAALELPSRDTL


 


162
FL-219
VYYHKECALDSYVRTCWVSG


 


163
FL-220
FNARALACKCDRRILILSQP


 


164
FL-221
TLHTNRFCFRWVPALDSVTT


 


165
FL-222
LNPWEGEWTRWDVFRVLGEF


 


166
FL-223
QMFWFTDSEGKPGFCTFYGF


 


167
FL-225
PSAYEAVETLDMSEVKGLGQ


 


168
FL-227
LARCLCEIVGSCISYSNLPI


 


169
FL-229
SGYPKMVWGEGPMLLDWKFV


 


170
FL-232
SFATANSEQVLRDMLLLASH


 


171
FL-236
VGPLIVGPPGMEMTANSWSC


 


172
FL-237
CEDMREAKVCRTLLAHSFLP


 


173
FL-239
RPWSSDTSVWWDGLFGMNYS


 


174
FL-242
DLRLPVYSEWVRVYSSDAWM


 


175
FL-245
GRFGQGQCYQVADSTYWTFGPGGPRKCEREPAGWSDTGWVC


 


176
FL-246
WCSMCSVLRAFNCPYFCPWL


 


177
FL-247
PWIFDRSVVCEEREAPRRHL


 


178
FL-251
TSWRHRMPTGTDRCCFLVQL


 


179
FL-252
RPWSSDTSVWWDGLFGMNYS


 


180
FL-254
RPWSSDTSVWWDGLFGMNYS


 


181
FL-256
LVTALIMSESIVRMNPMYLT


 


182
FL-257
LLACSVYWLWQRPCDGCLFM


 


183
FL-258
GVNNGGTNDPEGVSEASWIP


 


184
FL-262
LLACSVYWLWQRPCDGCLFM


 


185
FL-265
GIVSKQGADEGMLEIFASSW


 


186
FL-275
TIDQRMVSLGAIWWSYPRCW


 


187
FL-278
PPWTKRPLTSGGVRGELWVW


 


188
FL-280
RVDSLGIKLDKSTLVTVHVV


 


189
FL-281
TEACQVVLLGKRSLLPVVAG


 


190
FL-292
AQRSCWERLWTGQWRRLPLM


 


191
FL-293
AQRSCWERLWTGQWRRSASD


 


192
FL-294
VSWSAAGRGYVFMYRWSPRC


 


193
FL-300
FGVIVTNAASEFTTRGYDSC


 


194
FL-302
SFSSLFLAWLMQTGQEAGTV


 


195
FL-304
SRSDLWVSWCRNLLDGQSWS


 


196
FL-312
HPYLITDIISMYRSPWSVPA


 


197
FL-313
RDALTNIGRSICALLLVLCK


 


198
FL-322
WGYFGSFVGGVFDVWFSGVA


 


199
FL-325
SRS DLWVSWCRNLLDGQSWS


 


200
FL-326
RNGRNICVLSVCSRFSHFNP


 


201
FL-328
GGSDERYFWYQSFSSCAYEW


 


202
FL-334
GVLFTFKKYPQGLSCTTSYG


 


203
FL-341
LLACSVYWLWQRPCDGCLFM


 


204
FL-352
VVQNVRGWLVYCCADFHTYV


 


205
FL-355
LRGVSPWLQSFVSIAVQSCK


 


206
FL-364
TGSGLTPCLHCRVQFQRSYL


 


207
FL-365
TRMHSLCSGFCVICMGGPRV


 


208
FL-380
RNGRNICVLSVCSRFSHFNP


 


209
FL-381
MLGIWRVLHEMVVPLKLGVD


 


210
FL-382
CPDCPLSSVLRTATTAFFGG


 


211
FL-434
RGRMEADKSFDSTCLRCGCS









The peptide aptamers of the present invention were isolated in a stringent in vivo screen and, as such, are likely to bind to viral replication initiation proteins with high affinity, fold correctly and be stably expressed in a cellular environment. Some of the peptide aptamers were selected for binding to the N-terminus of AL1, which does not resemble plant proteins. Consequently, the peptide aptamers are unlikely to interact with host proteins, minimizing the risk that their expression will be toxic to plants. Further, the peptide aptamers are ca. 12 kD—a size typical of a small, stable protein that can move passively into the nucleus where replication proteins are localized. Additionally, several of the aptamer peptides reduced TGMV DNA accumulation more strongly than a trans-dominant negative TAL1 mutant that can confer immunity to TGMV infection when expressed in transgenic plants. Finally, the ability of the aptamer peptides to bind to the divergent TAL1 and CaAL1 proteins suggests that these peptides recognize conserved features in the N-termini of geminivirus replication proteins. This observation represents a key difference from interference strategies based on viral sequences like trans-dominant negative mutants, antisense RNAs and RNAi constructs, all of which are only effective against the cognate geminivirus or closely related viruses. In contrast, a resistance strategy based on the interfering aptamer peptides could be broadly applicable to all geminivirus genera and other eukaryotic single-stranded DNA viruses with related replication proteins and could confer resistance to mixed infections and viral variants.
The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is described by the following claims, with equivalents of the claims to be included therein.

## Claims

1. A polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:38;
(b) a fragment of the amino acid sequence of SEQ ID NO:38, wherein the fragment comprises MHXXXXXG; and wherein the fragment binds to a viral replication (Rep) protein; and
(c) an amino acid sequence having at least 95% sequence identity to the amino acid sequence of (a) or (b) and binds to a viral Rep protein selected from the group consisting of a geminivirus Rep protein, a nanovirus Rep protein, a circovirus Rep protein and combinations thereof;
wherein X represents any amino acid.

2. The polypeptide according to claim 1, wherein the polypeptide binds to a Rep protein selected from the group consisting of a nanovirus Rep protein, a circovirus Rep protein and combinations thereof.

3. The polypeptide according to claim 1, wherein the polypeptide binds to a geminivirus Rep protein.

4. The polypeptide according to claim 1, wherein the polypeptide binds to a Rep protein selected from the group consisting of a tomato golden mosaic virus (TGMV) Rep protein, a cabbage leaf curl virus (CbLCV) Rep protein, a tomato yellow leaf curl virus (TYLCV) Rep protein, a tomato mottle virus (ToMoV) Rep protein, an African cassava mosaic virus (ACMV) Rep protein, a maize streak virus (MSV) Rep protein, and a cotton leaf curl virus (CLCuV) Rep protein, and combinations thereof.

5. The polypeptide according to claim 1, wherein the amino acid sequence comprises MHXXXXXG.

6. The polypeptide according to claim 1 consisting of the amino acid sequence of SEQ ID NO:38.

7. A fusion protein comprising the polypeptide according to claim 1.

8. The fusion protein according to claim 7, wherein the fusion protein comprises thioredoxin.

9. The polypeptide according to claim 1, wherein the polypeptide comprises a fragment of the amino acid sequence of SEQ ID NO:38, wherein the fragment comprises MHXXXXXG, and wherein the fragment binds to a viral Rep protein.

10. The polypeptide according to claim 1, wherein the amino acid sequence comprises no more than three amino acid substitutions, insertions and/or deletions as compared with the amino acid sequence of (a).

11. The polypeptide according to claim 1, wherein the amino acid sequence comprises no more than two amino acid substitutions as compared with the amino acid sequence of (a), and wherein all of the amino acid substitutions are conservative substitutions.

12. The polypeptide according to claim 1, wherein the polypeptide inhibits viral replication.

13. The polypeptide according to claim 12, wherein the polypeptide inhibits geminivirus, nanovirus and/or circovirus replication.

14. The polypeptide according to claim 12, wherein the polypeptide inhibits geminivirus replication.

15. The polypeptide according to claim 14, wherein the polypeptide inhibits tomato golden mosaic virus (TGMV), tomato yellow leaf curl virus (TYLCV) replication, cabbage leaf curl virus (CbLCV) replication, tomato mottle virus (ToMoV) replication, African cassava mosaic virus (ACMV) replication, maize streak virus (MSV) replication and/or cotton leaf curl virus (CLCuV) replication.

16. The polypeptide according to claim 1, wherein the polypeptide inhibits viral infection.

17. The polypeptide according to claim 16, wherein the polypeptide inhibits geminivirus, nanovirus and/or circovirus infection.

18. The polypeptide according to claim 16, wherein the polypeptide inhibits geminivirus replication.

19. The polypeptide according to claim 18, wherein the polypeptide inhibits tomato golden mosaic virus (TGMV), tomato yellow leaf curl virus (TYLCV) infection, cabbage leaf curl virus (CbLCV) infection, tomato mottle virus (ToMoV) infection, African cassava mosaic virus (ACMV) infection, maize streak virus (MSV) infection and/or cotton leaf curl virus (CLCuV) infection.

20. A method of detecting a viral infection, the method comprising:
(a) contacting a sample with a polypeptide according to claim 1; and
(b) detecting the presence or absence of binding between the polypeptide and a viral Rep protein, wherein the binding of the polypeptide to the viral Rep protein in the sample indicates the presence of a geminivirus, nanovirus and/or circovirus.

21. A method of detecting a viral infection, the method comprising:
(a) contacting a sample with a fusion protein according to claim 7; and
(b) detecting the presence or absence of binding between the polypeptide and a viral Rep protein, wherein the binding of the polypeptide to the viral Rep protein in the sample indicates the presence of a geminivirus, nanovirus and/or circovirus.

22. A method of inhibiting viral replication in a plant cell, the method comprising introducing the polypeptide of claim 1 into the plant cell in an amount effective to inhibit geminivirus, nanovirus and/or circovirus replication.

23. A method of inhibiting viral replication in a plant cell, the method comprising introducing the fusion protein of claim 7 into the plant cell in an amount effective to inhibit geminivirus, nanovirus and/or circovirus replication.

24. A method of providing increased resistance to viral infection in a plant, the method comprising introducing the polypeptide of claim 1 into the plant in an amount effective to increase resistance to geminivirus, nanovirus and/or circovirus infection.

25. A method of providing increased resistance to viral infection in a plant, the method comprising introducing the fusion protein of claim 7 into the plant in an amount effective to increase resistance to geminivirus, nanovirus and/or circovirus infection.

26. The polypeptide of claim 1, wherein the fragment of the amino acid sequence of SEQ ID NO:38 binds to a single-stranded DNA (ssDNA) viral replication (Rep) protein.