bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue-Specific Transcriptional Targeting of a Replication-Competent Retroviral Vector
Authors: Logg Christopher R. , Logg Aki , Matusik Robert J. , Bochner Bernard H. , Kasahara Noriyuki
Published: November 2002

The inability of replication-defective viral vectors to efficiently transduce tumor cells in vivo has prevented the successful application of such vectors in gene therapy of cancer. To address the need for more efficient gene delivery systems, we have developed replication-competent retroviral (RCR) vectors based on murine leukemia virus (MLV). We have previously shown that such vectors are capable of transducing solid tumors in vivo with very high efficiency. While the natural requirement of MLV infection for cell division imparts a certain degree of specificity for tumor cells, additional means for confining RCR vector replication to tumor cells are desirable. Here, we investigated the parameters critical for successful tissue-specific transcriptional control of RCR vector replication by replacing various lengths of the MLV enhancer/promoter with sequences derived either from the highly prostate-specific probasin (PB) promoter or from a more potent synthetic variant of the PB promoter. We assessed the transcriptional specificity of the resulting hybrid long terminal repeats (LTRs) and the cell type specificity and efficiency of replication of vectors containing these LTRs. Incorporation of PB promoter sequences effectively restricted transcription from the LTR to prostate-derived cells and imparted prostate-specific RCR vector replication but required the stronger synthetic promoter and retention of native MLV sequences in the vicinity of the TATA box for optimal replicative efficiency and specificity. Our results have thus identified promoter strength and positioning within the LTR as important determinants for achieving both high transduction efficiency and strict cell type specificity in transcriptionally targeted RCR vectors.
A
cis
-Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging
Authors: Musatov Sergei , Roberts Jill , Pfaff Donald , Kaplitt Michael
Published: November 2002

A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB′), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential
cis
-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.
Upregulation of CD40 Expression on Endothelial Cells Infected with Human Cytomegalovirus
Authors: Maisch Tim , Kropff Barbara , Sinzger Christian , Mach Michael
Published: November 2002

CD40 has been identified as an important molecule for a number of processes, such as immune responses, inflammation, and the activation of endothelia. We investigated CD40 in endothelial cells (EC) following infection with an endotheliotropic strain of human cytomegalovirus (HCMV). Between 8 and 72 h postinfection, we observed a significant increase in CD40 levels on the surface of infected EC, as measured by fluorescence-activated cell sorting analysis. As a consequence of CD40 upregulation, increased levels of E-selectin were found on infected EC after stimulation with CD154-expressing T cells. Enhanced expression of CD40 was specific for EC, since infection of fibroblasts did not result in the upregulation of CD40. The addition of neutralizing antibodies as well as UV inactivation of virus completely prevented the upregulation of CD40 on EC. Also, laboratory-adapted HCMV strain AD169 was not able to induce CD40 on EC. De novo protein synthesis was necessary for the increased surface expression. At early times (4 to 24 h) postinfection, this change was not accompanied by increased levels of CD40 protein or mRNA. At late times (48 to 96 h) postinfection, increased amounts of CD40 protein and mRNA were detected. Immunohistochemical analysis of infected tissues demonstrated elevated levels of CD40 on HCMV-infected EC in vivo. Thus, infection of EC by HCMV may result in the activation of endothelia and in the augmentation of inflammatory processes.
The Complementary Strand of the Human T-Cell Leukemia Virus Type 1 RNA Genome Encodes a bZIP Transcription Factor That Down-Regulates Viral Transcription
Authors: Gaudray Gilles , Gachon Frederic , Basbous Jihane , Biard-Piechaczyk Martine , Devaux Christian , Mesnard Jean-Michel
Published: November 2002

The RNA genome of the human T-cell leukemia virus type 1 (HTLV-1) codes for proteins involved in infectivity, replication, and transformation. We report in this study the characterization of a novel viral protein encoded by the complementary strand of the HTLV-1 RNA genome. This protein, designated HBZ (for HTLV-1 bZIP factor), contains a N-terminal transcriptional activation domain and a leucine zipper motif in its C terminus. We show here that HBZ is able to interact with the bZIP transcription factor CREB-2 (also called ATF-4), known to activate the HTLV-1 transcription by recruiting the viral
trans
-activator Tax on the Tax-responsive elements (TxREs). However, we demonstrate that the HBZ/CREB-2 heterodimers are no more able to bind to the TxRE and cyclic AMP response element sites. Taking these findings together, the functional inactivation of CREB-2 by HBZ is suggested to contribute to regulation of the HTLV-1 transcription. Moreover, the characterization of a minus-strand gene protein encoded by HTLV-1 has never been reported until now.
Theiler's Virus Infection of Primary Cultures of Bone Marrow-Derived Monocytes/Macrophages
Authors: Martinat Cécile , Mena Ignacio , Brahic Michel
Published: November 2002

Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR−/−mice were fully permissive.
Induction of Autoreactive CD8+Cytotoxic T Cells during Theiler's Murine Encephalomyelitis Virus Infection: Implications for Autoimmunity
Authors: Tsunoda Ikuo , Kuang Li-Qing , Fujinami Robert S.
Published: November 2002

Theiler's murine encephalomyelitis virus (TMEV) belongs to the familyPicornaviridaeand causes demyelinating disease in the spinal cords of infected mice. Although immune responses have been shown to play an important role in demyelination, the precise effector mechanism(s) is unknown. Potentially autoreactive cytotoxic cells could contribute to the destruction. We tested whether an autoreactive cell induced by TMEV infection mediated cytotoxicity by using a 5-h51Cr release assay in SJL/J mice. Spleen cells from TMEV-infected mice were stimulated with irradiated TMEV antigen-presenting cells and used as effector cells. The effector cells differed from conventional cytotoxic T cells since these cells could kill both TMEV-infected and uninfected syngeneic or semisyngenic cell lines (PSJLSV and BxSF11gSV) but could not kill an allogeneic cell line (C57SV). The TMEV-induced autoreactive cells were also different from conventional natural killer (NK) cells or lymphokine-activated killer (LAK) cells, because they could kill neither NK cell-sensitive YAC-1 nor NK cell-resistant P815 and EL4 cells. Induction of autoreactive cells was not detected in vaccinia virus infection. The autoreactive killing required direct cell-to-cell contact and was mediated by a Fas-FasL pathway but not by a perforin pathway. The phenotype of the killer cells was CD3+CD4−CD8+. Intracerebral inoculation of the effector cells into naive mice caused meningitis and perivascular cuffing not only in the brain parenchyma but also in the spinal cord, with no evidence of viral antigen-positive cells. This is the first report demonstrating that TMEV can induce autoreactive cytotoxic cells that induce central nervous system pathology.
Mamu-A*01
Allele-Mediated Attenuation of Disease Progression in Simian-Human Immunodeficiency Virus Infection
Authors: Zhang Zhi-Qiang , Fu Tong-Ming , Casimiro Danilo R. , Davies Mary-Ellen , Liang Xiaoping , Schleif William A. , Handt Larry , Tussey Lynda , Chen Minchun , Tang Aimin , Wilson Keith A. , Trigona Wendy L. , Freed Daniel C. , Tan Charles Y. , Horton Melanie , Emini Emilio A. , Shiver John W.
Published: November 2002

Expression of several major histocompatibility complex (MHC) class I alleles is associated with a protective effect against disease progression in both human immunodeficiency virus type 1 and simian immunodeficiency virus infection. To understand the mechanism underlying this effect, we investigated the expression of the MHC class I allele
Mamu-A*01
in simian-human immunodeficiency virus (SHIV) infection, one of the major models for evaluation of AIDS vaccine candidates. We found that disease progression was significantly delayed in
Mamu-A∗01
-positive rhesus monkeys infected with the highly pathogenic SHIV 89.6P. The delay corresponded not only to a noted
Mamu-A∗01
-restricted dominant cytotoxic T-lymphocyte (CTL) response but also to a lower viral load in lymph nodes (LN) and, importantly, to minimal destruction of LN structure during early infection. In contrast,
Mamu-A∗01
-negative monkeys exhibited massive destruction of LN structure with accompanying rapid disease progression. These data indicate that MHC class I allele-restricted CTL responses may play an important role in preservation of lymphoid tissue structure, thereby resulting in attenuation of disease progression in immunodeficiency virus infection.
Human Immunodeficiency Virus Envelope (gp120) Binding to DC-SIGN and Primary Dendritic Cells Is Carbohydrate Dependent but Does Not Involve 2G12 or Cyanovirin Binding Sites: Implications for Structural Analyses of gp120-DC-SIGN Binding
Authors: Hong Patrick W.-P. , Flummerfelt Karen B. , de Parseval Aymeric , Gurney Kevin , Elder John H. , Lee Benhur
Published: November 2002

The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-β-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding—and the full complement of complex glycans—significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.
Endocytosis Is a Critical Step in Entry of Subgroup B Avian Leukosis Viruses
Authors: Diaz-Griffero Felipe , Hoschander Steven Ari , Brojatsch Jürgen
Published: November 2002

The avian leukosis virus (ALV) entry mechanism is controversial, with evidence for and against a low-pH requirement for viral fusion. To further address this question, we tested the entry of human immunodeficiency virus type 1 (HIV-1) pseudotyped with the envelope protein of subgroup B ALV (ALV-B) in the presence of three different lysosomotropic agents. These lysosomotropic agents were able to block the entry of wild-type and pseudotyped ALV-B in two different cell lines, strongly suggesting that ALV-B requires a low-pH step for entry. ALV-B and pH-dependent Semliki Forest virus (SFV) entered cells with slower uptake kinetics than HIV-1, which is pH independent. These slow uptake rates support the theory that ALV-B utilizes endocytic pathways to enter cells. Using immunofluorescence and electron microscopy analysis, we visualized the colocalization of virus particles with the endosomal marker transferrin and demonstrated virus particles in clathrin-coated vesicles and endosome-like structures. Surprisingly, a low-pH treatment did not overcome the inhibition of ALV-B entry by lysosomotropic agents. This indicates that, in contrast to SFV, ALV-B is unable to fuse at the cellular surface, even at a low pH. Taken together, our findings suggest that endocytosis and a subsequent low-pH step are critical for successful ALV-B infection.
ICP27 Interacts with the RNA Export Factor Aly/REF To Direct Herpes Simplex Virus Type 1 Intronless mRNAs to the TAP Export Pathway
Authors: Chen I-Hsiung Brandon , Sciabica Kathryn S. , Sandri-Goldin Rozanne M.
Published: November 2002

Herpes simplex virus type 1 (HSV-1) protein ICP27 facilitates the export of viral intronless mRNAs. ICP27 shuttles between the nucleus and cytoplasm, which has been shown to require a leucine-rich nuclear export sequence (NES). ICP27 export was reported to be sensitive to the CRM1 inhibitor leptomycin B (LMB) in HSV-1-infected cells but not in
Xenopus
oocytes, where ICP27 interacts with the export factor Aly/REF to access the TAP export pathway. Here, we show that ICP27 interacts with Aly/REF in HSV-1-infected mammalian cells and that Aly/REF stimulates export of viral intronless RNAs but does not cross-link to these RNAs. During infection, Aly/REF was no longer associated with splicing factor SC35 but moved into structures that colocalized with ICP27, suggesting that ICP27 recruits Aly/REF from spliceosomes to viral intronless RNAs. Further, ICP27 was found to interact in vivo with TAP but not with CRM1. In vitro export assays showed that ICP27 export was not sensitive to LMB but was blocked by a dominant-negative TAP deletion mutant lacking the nucleoporin interaction domain. These data suggest that ICP27 uses the TAP pathway to export viral RNAs. Interestingly, the leucine-rich N-terminal sequence was required for efficient export, even though ICP27 export was LMB insensitive. Thus, this region is required for efficient ICP27 export but does not function as a CRM1-dependent NES.
Early Evolution of the Human Immunodeficiency Virus Type 1 Subtype C Epidemic in Rural Malawi
Authors: McCormack Grace P. , Glynn Judith R. , Crampin Amelia C. , Sibande Felix , Mulawa Dominic , Bliss Lyn , Broadbent Philip , Abarca Katia , Pönnighaus Jorg M. , Fine Paul E. M. , Clewley Jonathan P.
Published: November 2002

We have tracked the early years of the evolution of the human immunodeficiency virus type 1 (HIV-1) epidemic in a rural district of central east Africa from the first documented introductions of subtypes A, D, and C to the present predominance of subtype C. The earliest subtype C sequences ever reported are described. Blood samples were collected on filter papers from 1981 to 1984 and from 1987 to 1989 from more than 44,000 individuals living in two areas of Karonga District, Malawi. These samples included HIV-1-positive samples from 200 people. In 1982 to 1984, HIV-1 subtypes A, C, and D were all present, though in small numbers. By 1987 to 1989, 152 (90%) of a total of 168 sequences were subtype C and AC, AD, and DC recombinants had emerged. Four of the subtype C sequences from 1983 to 1984 were closely related and were found at the base of a large cluster of low diversity that by the late 1980s accounted for 40% of C sequences. The other two early C sequences fell into a separate and more diverse cluster. Three other clusters containing sequences from the late 1980s were identified. Each cluster contained at least one sample from a person who had recently arrived in the district. From 18 HIV-1-positive spouse pairs, 12 very closely related pairs of sequences were identified. We conclude that there were multiple introductions of HIV-1 with limited spread, followed by explosive growth of a subtype C cluster, probably arising from a single introduction in or before 1983.
Conjugate-Based Targeting of Recombinant Adeno-Associated Virus Type 2 Vectors by Using Avidin-Linked Ligands
Authors: Ponnazhagan Selvarangan , Mahendra Gandham , Kumar Sanjay , Thompson John A. , Castillas, Mark
Published: November 2002

The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1α receptor (FGFR1α), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1α-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.
Novel
N
Gene-Associated, Temperature-Independent Resistance to the Movement of
Tobacco Mosaic Virus
Vectors Neutralized by a
Cucumber Mosaic Virus
RNA1 Transgene
Authors: Canto Tomas , Palukaitis Peter
Published: November 2002

The
N
gene conditions for resistance to
Tobacco mosaic virus
(TMV) but only below 28°C. However, a TMV-based vector expressing green fluorescent protein (TMV-GFP) showed only limited movement at 33°C in tobacco plants harboring the
N
gene and other genes cointrogressed from
Nicotiana glutinosa
. TMV-GFP moved efficiently in tobacco plants that either lacked these genes or that contained the
N
gene but were transgenic for RNA1 of
Cucumber mosaic virus
. These findings identified novel temperature-independent resistance to the movement of TMV-GFP which could be neutralized by a different viral transgene. Using the
N
gene and
nahG
gene-transgenic tobacco, we show that this novel resistance is manifested specifically by the
N
gene itself and operates via a pathway independent of salicylic acid.
Chromosome Binding Site of Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Is Essential for Persistent Episome Maintenance and Is Functionally Replaced by Histone H1
Authors: Shinohara Hirohiko , Fukushi Masaya , Higuchi Masaya , Oie Masayasu , Hoshi Osamu , Ushiki Tatsuo , Hayashi Jun-Ichi , Fujii Masahiro
Published: November 2002

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (ΔN-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of ΔN-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.
RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted-Repeat RNA Derived from the Virus Genome
Authors: Adelman Zach N. , Sanchez-Vargas Irma , Travanty Emily A. , Carlson Jon O. , Beaty Barry J. , Blair Carol D. , Olson Ken E.
Published: November 2002

Double-stranded RNA (dsRNA) initiates cellular posttranscriptional responses that are collectively called RNA silencing in a number of different organisms, including plants, nematodes, and fruit flies. In plants, RNA silencing has been associated with protection from virus infection. In this study, we demonstrate that dsRNA-mediated interference also can act as a viral defense mechanism in mosquito cells. C6/36 (
Aedes albopictus
) cells were stably transformed with a plasmid designed to transcribe an inverted-repeat RNA (irRNA) derived from the genome of dengue virus type 2 (DEN-2) capable of forming dsRNA. Clonal cell lines were selected with an antibiotic resistance marker and challenged with DEN-2. The cell lines were classified as either susceptible or resistant to virus replication, based on the percentage of cells expressing DEN-2 envelope (E) antigen 7 days after challenge. Eight out of 18 (44%) cell lines designed to express irRNA were resistant to DEN-2 challenge, with more than 95% of the cells showing no DEN-2 antigen accumulation. One of the DEN-2-resistant cell lines, FB 9.1, was further characterized. DEN-2 genome RNA failed to accumulate in FB 9.1 cells after challenge. Northern blot hybridization detected transcripts containing transgene sequences of both sense and antisense polarity, suggesting that DEN-2-specific dsRNA was present in the cells. In addition, a class of small RNAs 21 to 25 nucleotides in length was detected that specifically hybridized to labeled sense or antisense DEN-2 RNA derived from the target region of the genome. These observations were consistent with RNA silencing as the mechanism of resistance to DEN-2 in transformed mosquito cells.
A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells
Authors: Fujinaga Koh , Irwin Dan , Taube Ran , Zhang Fan , Geyer Matthias , Peterlin B. Matija
Published: November 2002

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn
2+
-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn
2+
-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex.
Mutations in the N-Terminal Domains of Nectin-1 and Nectin-2 Reveal Differences in Requirements for Entry of Various Alphaherpesviruses and for Nectin-Nectin Interactions
Authors: Struyf Frank , Martinez Wanda M. , Spear Patricia G.
Published: November 2002

Nectin-1 and nectin-2 are related molecules that can function with different specificities as entry receptors for mammalian alphaherpesviruses through interaction with viral glycoprotein D (gD). The normal function of members of the nectin family is to mediate cell-cell adhesion through homotypic and heterotypic nectin-nectin interactions in cadherin-based adherens junctions. We examined mutations in three equivalent regions of the N-terminal V-like domains of nectin-1 and nectin-2 to test the effects on entry of various alphaherpesviruses, nectin-nectin interactions, and interactions of the mutant nectins with gD. Mutations in region I previously shown to severely impair herpes simplex virus (HSV) entry activity, but not pseudorabies virus (PRV) or bovine herpesvirus 1 (BHV-1) entry, did not reduce homotypic
trans
interactions for either nectin-1 or nectin-2 or binding of nectin-3 to nectin-1. Mutations in region II, patterned after a reported single-nucleotide polymorphism in nectin-2, enhanced intracellular accumulation of both nectin-1 and nectin-2 and had a deleterious effect on all of the activities under study. Mutations in region III previously shown to reduce homotypic
trans
interactions of nectin-2 impaired the entry of PRV and BHV-1 when introduced into either nectin-1 or nectin-2, but only the nectin-2 mutation reduced HSV entry activity. Binding of nectin-1 to nectin-3 was not affected. Effects of the nectin-1 and nectin-2 mutations on interactions with gD did not necessarily correlate with entry activity of the mutant receptors. We can conclude that structural requirements for HSV entry, PRV and BHV-1 entry, and homotypic and heterotypic
trans
interactions are all different despite the previously reported ability of HSV and HSV gD to inhibit
trans
interactions.
Functional Replacement of the Carboxy-Terminal Two-Thirds of the Influenza A Virus NS1 Protein with Short Heterologous Dimerization Domains
Authors: Wang Xiuyan , Basler Christopher F. , Williams Bryan R. G. , Silverman Robert H. , Palese Peter , García-Sastre Adolfo
Published: November 2002

The NS1 protein of influenza A/WSN/33 virus is a 230-amino-acid-long protein which functions as an interferon alpha/beta (IFN-α/β) antagonist by preventing the synthesis of IFN during viral infection. In tissue culture, the IFN inhibitory function of the NS1 protein has been mapped to the RNA binding domain, the first 73 amino acids. Nevertheless, influenza viruses expressing carboxy-terminally truncated NS1 proteins are attenuated in mice. Dimerization of the NS1 protein has previously been shown to be essential for its RNA binding activity. We have explored the ability of heterologous dimerization domains to functionally substitute in vivo for the carboxy-terminal domains of the NS1 protein. Recombinant influenza viruses were generated that expressed truncated NS1 proteins of 126 amino acids, fused to 28 or 24 amino acids derived from the dimerization domains of either the
Saccharomyces cerevisiae
PUT3 or the
Drosophila melanogaster
Ncd (DmNcd) proteins. These viruses regained virulence and lethality in mice. Moreover, a recombinant influenza virus expressing only the first 73 amino acids of the NS1 protein was able to replicate in mice lacking three IFN-regulated antiviral enzymes, PKR, RNaseL, and Mx, but not in wild-type (Mx-deficient) mice, suggesting that the attenuation was mainly due to an inability to inhibit the IFN system. Remarkably, a virus with an NS1 truncated at amino acid 73 but fused to the dimerization domain of DmNcd replicated and was also highly pathogenic in wild-type mice. These results suggest that the main biological function of the carboxy-terminal region of the NS1 protein of influenza A virus is the enhancement of its IFN antagonist properties by stabilizing the NS1 dimeric structure.
RNA Interference Directed against Viral and Cellular Targets Inhibits Human Immunodeficiency Virus Type 1 Replication
Authors: Surabhi Rama M. , Gaynor Richard B.
Published: November 2002

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-κB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1
tat
or reverse transcriptase or the NF-κB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.
Hepatocyte Nuclear Factor 3β Inhibits Hepatitis B Virus Replication In Vivo
Authors: Banks Krista E. , Anderson Aimee L. , Tang Hong , Hughes Douglas E. , Costa Robert H. , McLachlan Alan
Published: November 2002

Hepatitis B virus (HBV) transgenic mice expressing rat hepatocyte nuclear factor 3β (HNF3β) were generated by breeding HBV transgenic mice with transgenic mice that constitutively overexpress the rat HNF3β polypeptide in the liver. HBV 3.5-, 2.4- and 2.1-kb transcripts were reduced 2- to 4-fold in these mice relative to the HBV transgenic mouse controls. In contrast, the abundance of viral replication intermediates was profoundly reduced in HBV transgenic mice by overexpression of HNF3β. This results, in part, from the preferential reduction in the level of the pregenomic 3.5-kb RNA relative to the precore 3.5-kb RNA. Therefore, it is apparent that increased expression of HNF3β modestly reduces viral transcription and dramatically inhibits replication in vivo in the HBV transgenic mouse. This suggests that altering the activity of this transcription factor in vivo in chronic HBV carriers might be therapeutically beneficial.
Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families
Authors: Yelina Natalia E. , Savenkov Eugene I. , Solovyev Andrey G. , Morozov Sergey Y. , Valkonen Jari P. T.
Published: November 2002

RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (
Barley stripe mosaic virus
[BSMV]) lacking the γ
b
gene was confined to inoculated leaves in
Nicotiana benthamiana
, but systemic infection was observed in transgenic
N. benthamiana
expressing the potyviral silencing suppressor protein HCpro, suggesting that the γb protein may be a long-distance movement factor and have antisilencing activity. This was shown for γb proteins of both BSMV and
Poa semilatent virus
(PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, γb and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the γb cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type
N. benthamiana
were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus γb proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.
Oligosaccharides as Receptors for JC Virus
Authors: Komagome Rika , Sawa Hirofumi , Suzuki Takashi , Suzuki Yasuo , Tanaka Shinya , Atwood Walter J. , Nagashima Kazuo
Published: November 2002

JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and in humans causes a demyelinating disease of the central nervous system, progressive multifocal leukoencephalopathy. Its hemagglutination activity and entry into host cells have been reported to depend on an N-linked glycoprotein containing sialic acid. In order to identify the receptors of JCV, we generated virus-like particles (VLP) consisting of major viral capsid protein VP1. We then developed an indirect VLP overlay assay to detect VLP binding to glycoproteins and a panel of glycolipids. We found that VLP bound to sialoglycoproteins, including α1-acid glycoprotein, fetuin, and transferrin receptor, and that this binding depended on α2-3-linked sialic acids and N-linked sugar chains. Neoglycoproteins were synthesized by using ovalbumin and conjugation with oligosaccharides containing the terminal α2-3- or α2-6-linked sialic acid or the branched α2-6-linked sialic acid. We show that the neoglycoprotein containing the terminal α2-6-linked sialic acid had the highest affinity for VLP, inhibited the hemagglutination activity of VLP and JCV, and inhibited the attachment of VLP to cells. We also demonstrate that VLP bound to specific glycolipids, such as lactosylceramide, and gangliosides, including GM3, GD2, GD3, GD1b, GT1b, and GQ1b, and that VLP bound weakly to GD1a but did not bind to GM1a, GM2, or galactocerebroside. Furthermore, the neoglycoprotein containing the terminal α2-6-linked sialic acid and the ganglioside GT1b inhibited JCV infection in the susceptible cell line IMR-32. These results suggest that the oligosaccharides of glycoproteins and glycolipids work as JCV receptors and may be feasible as anti-JCV agents.
Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication
Authors: Blight Keril J. , McKeating Jane A. , Rice Charles M.
Published: November 2002

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-α) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.
A Novel Gene Expression Control System and Its Use in Stable, High-Titer 293 Cell-Based Adeno-Associated Virus Packaging Cell Lines
Authors: Qiao Chunping , Wang Bing , Zhu Xiaodong , Li Juan , Xiao Xiao
Published: November 2002

Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene expression since its promoter is located within the coding sequence of Rep78/68. To tightly regulate all four Rep proteins by using their own promoters, we have developed a novel gene control paradigm termed “dual splicing switch,” which disrupts all four Rep genes by inserting into their shared coding region an intron that harbors transcription termination sequences flanked the LoxP sites. As a result, the structure and activities of the Rep gene promoters, both p5 and p19, are not affected; however, all of the Rep transcripts are prematurely terminated and the genes were inactivated. Removal of the terminator by Cre protein reactivates the transcription of all four Rep proteins derived from their own promoters. This switch system was initially tested in the
lacZ
gene and a 600-fold induction of β-galactosidase activity was observed. Using the dual splicing switch strategy, we have subsequently established a number of AAV packaging cell lines from 293 cells, which showed a normal growth rate, high stability, and more importantly, high yields of AAV vectors. Such a gene control paradigm is also useful for other viruses, e.g., autonomous parvoviruses. Finally, the high-titer 293-based AAV packaging cell lines should greatly reduce the risk of wild-type adenovirus contamination and provide a scalable AAV vector production method for both preclinical and clinical studies.
Role of the Hemagglutinin-Neuraminidase Protein in the Mechanism of Paramyxovirus-Cell Membrane Fusion
Authors: Takimoto Toru , Taylor Garry L. , Connaris Helen C. , Crennell Susan J. , Portner Allen
Published: November 2002

Paramyxovirus infects cells by initially attaching to a sialic acid-containing cellular receptor and subsequently fusing with the plasma membrane of the cells. Hemagglutinin-neuraminidase (HN) protein, which is responsible for virus attachment, interacts with the fusion protein in a virus type-specific manner to induce efficient membrane fusion. To elucidate the mechanism of HN-promoted membrane fusion, we characterized a series of Newcastle disease virus HN proteins whose surface residues were mutated. Fusion promotion activity was substantially altered in only the HN proteins with a mutation in the first or sixth β sheet. These regions overlap the large hydrophobic surface of HN; thus, the hydrophobic surface may contain the fusion promotion domain. Furthermore, a comparison of the HN structure crystallized alone or in complex with 2-deoxy-2,3-dehydro-
N
-acetylneuraminic acid revealed substantial conformational changes in several loops within or near the hydrophobic surface. Our results suggest that the binding of HN protein to the receptor induces the conformational change of residues near the hydrophobic surface of HN protein and that this change triggers the activation of the F protein, which initiates membrane fusion.
Identification of a Second Major Site for CD46 Binding in the Hemagglutinin Protein from a Laboratory Strain of Measles Virus (MV): Potential Consequences for Wild-Type MV Infection
Authors: Massé Nicolas , Barrett Thomas , Muller Claude P. , Wild T. Fabian , Buckland Robin
Published: November 2002

Natural or wild-type (wt) measles virus (MV) infection in vivo which is restricted to humans and certain monkeys represents an enigma in terms of receptor usage. Although wt MV is known to use the protein SLAM (CD150) as a cell receptor, many human tissues, including respiratory epithelium in which the infection initiates, are SLAM negative. These tissues are CD46 positive, but wt MV strains, unlike vaccinal and laboratory MV strains, are not thought to use CD46 as a receptor. We have identified a novel CD46 binding site at residues S548 and F549, in the hemagglutinin (H) protein from a laboratory MV strain, which is also present in wt H proteins. Our results suggest that although wt MV interacts with SLAM with high affinity, it also possesses the capacity to interact with CD46 with low affinity.
A Mutant of Human Papillomavirus Type 16 E6 Deficient in Binding α-Helix Partners Displays Reduced Oncogenic Potential In Vivo
Authors: Nguyen Marie , Song Shiyu , Liem Amy , Androphy Elliot , Liu Yun , Lambert Paul F.
Published: November 2002

Human papillomaviruses (HPVs) are small DNA tumor viruses that are the causative agent of warts and are associated with many anogenital cancers. The viral gene encoding the E6 protein has been found to be involved in HPV oncogenesis. E6 is known to inactivate the cellular tumor suppressor, p53. In addition, E6 has been shown to bind to a variety of other cellular proteins. The focus of this study was to determine what role the interactions of E6 with a subset of cellular proteins which contain a common α-helical domain in their E6 binding region (α-helix partners) play in E6-mediated phenotypes. We generated transgenic mice expressing a mutant of E6, E6
I128T
, which is defective for binding at least a subset of the α-helix partners, including E6AP, the ubiquitin ligase that mediates E6-dependent degradation of the p53 protein, to determine whether binding of α-helix partners plays a role in E6-mediated activities in vivo. Unlike mice expressing the wild-type E6 (strain K14E6
WT
), the mice expressing E6
I128T
lacked the ability to alter the radiation-induced block to DNA synthesis and promote the formation of benign skin tumors in conjunction with chemical carcinogens. Additionally, they displayed reduced levels of skin hyperplasia, spontaneous skin tumors, and tumor progression activity compared to those of the K14E6
WT
mice. From these results, we conclude that a domain in E6 that mediates α-helix partner binding is critical for E6-induced phenotypes in transgenic mice.
Epitope-Tagged L* Protein of Theiler's Murine Encephalomyelitis Virus Is Expressed in the Central Nervous System in the Acute Phase of Infection
Authors: Asakura Kunihiko , Murayama Harunobu , Himeda Toshiki , Ohara Yoshiro
Published: November 2002

TO subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein from an alternative initiation codon. We first demonstrated L* expression in the central nervous system (CNS) of TMEV-infected mice during the acute phase of infection by immunoprecipitation and immunoblotting with anti-L* antibody. In addition, we generated mutant viruses which synthesize FLAG or 3xFLAG epitope-tagged L* protein. With a mutant virus expressing 3xFLAG epitope-tagged L*, designated DA/3xFLAGL*, we investigated L* in the CNS in the acute phase of infection. DA/3xFLAGL* did not change the virus tropism in comparison with wild-type virus, and L* was clearly identified in the CNS in both susceptible and resistant strains of mice. Double immunolabeling studies showed that L* is colocalized with TMEV polyprotein and exclusively expressed in neurons.
Restriction of Viral Replication by Mutation of the Influenza Virus Matrix Protein
Authors: Liu Teresa , Ye Zhiping
Published: November 2002

The matrix protein (M1) of influenza virus plays an essential role in viral assembly and has a variety of functions, including association with influenza virus ribonucleoprotein (RNP). Our previous studies show that the association of M1 with viral RNA and nucleoprotein not only promotes formation of helical RNP but also is required for export of RNP from the nucleus during viral replication. The RNA-binding domains of M1 have been mapped to two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105, which is also involved in RNP-binding activity. To further understand the role of the RNP-binding domain of M1 in viral assembly and replication, mutations in the coding sequences of RKLKR and the zinc finger motif of M1 were constructed using a PCR technique and introduced into wild-type influenza virus by reverse genetics. Altering the zinc finger motif of M1 only slightly affected viral growth. Substitution of Arg with Ser at position 101 or 105 of RKLKR did not have a major impact on nuclear export of RNP or viral replication. In contrast, deletion of RKLKR or substitution of Lys with Asn at position 102 or 104 of RKLKR resulted in a lethal mutation. These results indicate that the RKLKR domain of M1 protein plays an important role in viral replication.