bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Monoclonal Antibodies against West Nile Virus Induced by Natural Infection Neutralize at a Postattachment Step
Authors: Vogt Matthew R. , Moesker Bastiaan , Goudsmit Jaap , Jongeneelen Mandy , Austin S. Kyle , Oliphant Theodore , Nelson Steevenson , Pierson Theodore C. , Wilschut Jan , Throsby Mark , Diamond Michael S.
Published: April 2009

West Nile virus (WNV) is a neurotropic flavivirus that is now a primary cause of epidemic encephalitis in North America. Studies of mice have demonstrated that the humoral immune response against WNV limits primary infection and protects against a secondary challenge. The most-potent neutralizing mouse monoclonal antibodies (MAbs) recognize an epitope on the lateral ridge of domain III (DIII-lr) of the envelope (E) protein. However, studies with serum from human patients show that antibodies against the DIII-lr epitope comprise, at best, a minor component of the human anti-WNV antibody response. Herein, we characterize in detail two WNV-specific human MAbs, CR4348 and CR4354, that were isolated from B-cell populations of convalescent patients. These MAbs strongly neutralize WNV infection of cultured cells, protect mice against lethal infection in vivo, and yet poorly recognize recombinant forms of the E protein. Instead, CR4348 and CR4354 bind determinants on intact WNV virions and subviral particles in a pH-sensitive manner, and neutralization is altered by mutations at the dimer interface in domain II and the hinge between domains I and II, respectively. CR4348 and CR4354 human MAbs neutralize infection at a postattachment step in the viral life cycle, likely by inhibiting acid-induced fusion within the endosome.
Structure and Expression Strategy of the Genome of
Culex pipiens
Densovirus, a Mosquito Densovirus with an Ambisense Organization
Authors: Baquerizo-Audiot Elizabeth , Abd-Alla Adly , Jousset Françoise-Xavière , Cousserans François , Tijssen Peter , Bergoin Max
Published: April 2009

The genome of all densoviruses (DNVs) so far isolated from mosquitoes or mosquito cell lines consists of a 4-kb single-stranded DNA molecule with a monosense organization (genus
Brevidensoviru
s, subfamily
Densovirinae
). We previously reported the isolation of a
Culex pipiens
DNV (CpDNV) that differs significantly from brevidensoviruses by (i) having a ∼6-kb genome, (ii) lacking sequence homology, and (iii) lacking antigenic cross-reactivity with
Brevidensovirus
capsid polypeptides. We report here the sequence organization and transcription map of this virus. The cloned genome of CpDNV is 5,759 nucleotides (nt) long, and it possesses an inverted terminal repeat (ITR) of 285 nt and an ambisense organization of its genes. The nonstructural (NS) proteins NS-1, NS-2, and NS-3 are located in the 5′ half of one strand and are organized into five open reading frames (ORFs) due to the split of both NS-1 and NS-2 into two ORFs. The ORF encoding capsid polypeptides is located in the 5′ half of the complementary strand. The expression of NS proteins is controlled by two promoters, P7 and P17, driving the transcription of a 2.4-kb mRNA encoding NS-3 and of a 1.8-kb mRNA encoding NS-1 and NS-2, respectively. The two NS mRNAs species are spliced off a 53-nt sequence. Capsid proteins are translated from an unspliced 2.3-kb mRNA driven by the P88 promoter. CpDNV thus appears as a new type of mosquito DNV, and based on the overall organization and expression modalities of its genome, it may represent the prototype of a new genus of DNV.
Single-Injection Vaccine Protects Nonhuman Primates against Infection with Marburg Virus and Three Species of Ebola Virus
Authors: Geisbert Thomas W. , Geisbert Joan B. , Leung Anders , Daddario-DiCaprio Kathleen M. , Hensley Lisa E. , Grolla Allen , Feldmann Heinz
Published: April 2009

The filoviruses Marburg virus and Ebola virus cause severe hemorrhagic fever with high mortality in humans and nonhuman primates. Among the most promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (VSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Here, we performed a proof-of-concept study in order to determine the potential of having one single-injection vaccine capable of protecting nonhuman primates against
Sudan ebolavirus
(SEBOV),
Zaire ebolavirus
(ZEBOV),
Cote d'Ivoire ebolavirus
(CIEBOV), and
Marburgvirus
(MARV). In this study, 11 cynomolgus monkeys were vaccinated with a blended vaccine consisting of equal parts of the vaccine vectors VSVΔG/SEBOVGP, VSVΔG/ZEBOVGP, and VSVΔG/MARVGP. Four weeks later, three of these animals were challenged with MARV, three with CIEBOV, three with ZEBOV, and two with SEBOV. Three control animals were vaccinated with VSV vectors encoding a nonfilovirus GP and challenged with SEBOV, ZEBOV, and MARV, respectively, and five unvaccinated control animals were challenged with CIEBOV. Importantly, none of the macaques vaccinated with the blended vaccine succumbed to a filovirus challenge. As expected, an experimental control animal vaccinated with VSVΔG/ZEBOVGP and challenged with SEBOV succumbed, as did the positive controls challenged with SEBOV, ZEBOV, and MARV, respectively. All five control animals challenged with CIEBOV became severely ill, and three of the animals succumbed on days 12, 12, and 14, respectively. The two animals that survived CIEBOV infection were protected from subsequent challenge with either SEBOV or ZEBOV, suggesting that immunity to CIEBOV may be protective against other species of Ebola virus. In conclusion, we developed an immunization scheme based on a single-injection vaccine that protects nonhuman primates against lethal challenge with representative strains of all human pathogenic filovirus species.
Different Subcellular Localization of Theiler's Murine Encephalomyelitis Virus Leader Proteins of GDVII and DA Strains in BHK-21 Cells
Authors: Taniura Naoko , Saito Mineki , Okuwa Takako , Saito Kousuke , Ohara Yoshiro
Published: April 2009

The highly virulent GDVII strain of Theiler's murine encephalomyelitis virus causes acute and fatal encephalomyelitis, whereas the DA strain causes mild encephalomyelitis followed by a chronic inflammatory demyelinating disease with virus persistence. The differences in the amino acid sequences of the leader protein (L) of the DA and GDVII strains are greater than those for any other viral protein. We examined the subcellular distribution of DA L and GDVII L tagged with the FLAG epitope in BHK-21 cells. Wild-type GDVII L was localized predominantly in the cytoplasm, whereas wild-type DA L showed a nucleocytoplasmic distribution. A series of the L mutant experiments demonstrated that the zinc finger domain, acidic domain, and C-terminal region of L were necessary for the nuclear accumulation of DA L. A GDVII L mutant with a deletion of the serine/threonine (S/T)-rich domain showed a nucleocytoplasmic distribution, in contrast to the predominant cytoplasmic distribution of wild-type GDVII L. A chimeric DA/GDVII L, D/G, which encodes the N region of DA L including the zinc finger domain and acidic domain, followed by the GDVII L sequence including the S/T-rich domain, was distributed exclusively throughout the cytoplasm but not in the nucleus, as observed with wild-type GDVII L. Another chimeric L, G/D (which is the converse of the D/G construct), accumulated in the nucleus as well as the cytoplasm, as was observed for wild-type DA L. The findings suggest that the differential distribution of DA L and GDVII L is determined primarily by the S/T-rich domain. The S/T-rich domain may be important for the viral activity through the regulation of the subcellular distribution of L.
Critical Role of Cyclophilin A and Its Prolyl-Peptidyl Isomerase Activity in the Structure and Function of the Hepatitis C Virus Replication Complex
Authors: Liu Zhe , Yang Feng , Robotham Jason M. , Tang Hengli
Published: April 2009

Replication of hepatitis C virus (HCV) RNA occurs on intracellular membranes, and the replication complex (RC) contains viral RNA, nonstructural proteins, and cellular cofactors. We previously demonstrated that cyclophilin A (CyPA) is an essential cofactor for HCV infection and the intracellular target of cyclosporine's anti-HCV effect. Here we investigate the mechanism by which CyPA facilitates HCV replication. Cyclosporine treatment specifically blocked the incorporation of NS5B into the RC without affecting either the total protein level or the membrane association of the protein. Other nonstructural proteins or viral RNAs in the RC were not affected. NS5B from the cyclosporine-resistant replicon was resistant to this disruption of RC incorporation. We also isolated membrane fractions from both naïve and HCV-positive cells and found that CyPA is recruited into membrane fractions in HCV-replicating cells via an interaction with RC-associated NS5B, which is sensitive to cyclosporine treatment. Finally, we introduced point mutations in the prolyl-peptidyl isomerase (PPIase) motif of CyPA and demonstrated a critical role of this motif in HCV replication in cDNA rescue experiments. We propose a model in which the incorporation of the HCV polymerase into the RC depends on its interaction with a cellular chaperone protein and in which cyclosporine inhibits HCV replication by blocking this critical interaction and the PPIase activity of CyPA. Our results provide a mechanism of action for the cyclosporine-mediated inhibition of HCV and identify a critical role of CyPA's PPIase activity in the proper assembly and function of the HCV RC.
An 80-Kilodalton Protein That Binds to the Pre-S1 Domain of Hepatitis B Virus
Authors: Ryu Chun Jeih , Cho Dae-Yeon , Gripon Philippe , Kim Hee Sun , Guguen-Guillouzo Christiane , Hong Hyo Jeong
Published: April 2009

It has been suggested that hepatitis B virus (HBV) binds to a receptor on the plasma membrane of human hepatocytes via the pre-S1 domain of the large envelope protein as an initial step in HBV infection. However, the nature of the receptor remains controversial. In an attempt to identify a cell surface receptor for HBV, purified recombinant fusion protein of the pre-S1 domain of HBV with glutathione
S
-transferase (GST), expressed in
Escherichia coli
, was used as a ligand. The surface of human hepatocytes or HepG2 cells was biotinylated, and the cell lysate (precleared lysate) which did not bind to GST and glutathione-Sepharose beads was used as a source of receptor molecules. The precleared lysate of the biotinylated cells was incubated with the GST–pre-S1 fusion protein, and the bound proteins were visualized by Western blotting and enhanced chemiluminescence. An approximately 80-kDa protein (p80) was shown to bind specifically to the pre-S1 domain of the fusion protein. The receptor binding assay using serially or internally deleted segments of pre-S1 showed that amino acid residues 12 to 20 and 82 to 90 are essential for the binding of pre-S1 to p80. p80 also bound specifically to the pre-S1 of native HBV particles. Analysis of the tissue and species specificity of p80 expression in several available human primary cultures and cell lines of different tissue origin showed that p80 expression is not restricted to human hepatocytes. Taken together the results suggest that p80 may be a component of the viral entry machinery.
Herpes Simplex Virus Type 1 U
L
34 Gene Product Is Required for Viral Envelopment
Authors: Roller Richard J. , Zhou Yuping , Schnetzer Renee , Ferguson John , DeSalvo Diana
Published: April 2009

The herpes simplex virus type 1 U
L
34 gene encodes a protein that is conserved in all human herpesviruses. The association of the U
L
34 protein with membranes in the infected cell and its expression as a gamma-1 gene suggest a role in maturation or egress of the virus particle from the cell. To determine the function of this gene product, we have constructed a recombinant virus that fails to express the U
L
34 protein. This recombinant virus, in which the U
L
34 protein coding sequence has been replaced by green fluorescent protein, forms minute plaques and replicates in single-step growth experiments to titers 3 to 5 log orders of magnitude lower than wild-type or repair viruses. On Vero cells, the deletion virus synthesizes proteins of all kinetic classes in normal amounts. Electron microscopic and biochemical analyses show that morphogenesis of the deletion virus proceeds normally to the point of formation of DNA-containing nuclear capsids, but electron micrographs show no enveloped virus particles in the cytoplasm or at the surface of infected cells, suggesting that the U
L
34 protein is essential for efficient envelopment of capsids.
Identification of the Minimal Essential RNA Sequences Responsible for Site-Specific Targeting of the
Leishmania
RNA Virus 1-4 Capsid Endoribonuclease
Authors: Ro Young-Tae , Patterson Jean L.
Published: April 2009

The
Leishmania
RNA virus 1-4 capsid protein possesses an endoribonuclease activity responsible for single-site-specific cleavage within the 450-nucleotide 5′ untranslated region of its own viral RNA transcript. To characterize the minimal essential RNA determinants required for site-specific cleavage, mutated RNA transcripts were examined for susceptibility to cleavage by the virus capsid protein in an in vitro assay. Deletion analyses revealed that all determinants necessary for accurate cleavage are encoded in viral nucleotides 249 to 342. Nuclease mapping and site-specific mutagenesis of the minimal RNA sequence defined a stem-loop structure that is located 40 nucleotides upstream from the cleavage site (nucleotide 320) and that is essential for accurate RNA cleavage. Abrogation of cleavage by disruption of base pairing within the stem-loop was reversed through the introduction of complementary nucleotide substitutions that reestablished the structure. We also provide evidence that divalent cations, essential components of the cleavage reaction, stabilized the stem-loop structure in solution. That capsid-specific antiserum eliminated specific RNA cleavage provides further evidence that the virus capsid gene encodes the essential endoribonuclease activity.
Mutations in the 5′ Trailer Region of a Respiratory Syncytial Virus Minigenome Which Limit RNA Replication to One Step
Authors: Peeples Mark E. , Collins Peter L.
Published: April 2009

The 3′ termini of the genomic and antigenomic RNAs of human respiratory syncytial virus (RSV) are identical at 10 of the first 11 nucleotide positions and 21 of the first 26 positions. These conserved 3′-terminal sequences are thought to contain the genomic and antigenomic promoters. Furthermore, the complement of each conserved sequence (i.e., the 5′ end of the RNA it encodes) might contain an encapsidation signal. Using an RSV minigenome system, we individually mutated each of the last seven nucleotides in the 5′ trailer region of the genome. We analyzed effects of these mutations on encapsidation of the T7 polymerase-transcribed negative-sense genome, its ability to function as a template for RSV-driven synthesis of positive-sense antigenome and mRNA, and the ability of this antigenome to be encapsidated and to function as template for the synthesis of more genome. As a technical complication, mutations in the last five nucleotides of the trailer region were found to affect the efficiency of the adjoining T7 promoter over more than a 10-fold range, even though three nonviral G residues had been included between the core promoter and the trailer to maximize the efficiency of promoter activity. This was controlled in all experiments by monitoring the levels of total and encapsidated genome. The efficiency of encapsidation of the T7 polymerase-transcribed genome was not affected by any of the trailer mutations. Furthermore, neither the efficiency of positive-sense RNA synthesis from the genome nor the efficiency of encapsidation of the encoded antigenome was affected by the mutations. However, nucleotide substitution at positions 2, 3, 6, or 7 relative to the 5′ end of the trailer blocked the production of progeny genome, whereas substitution at positions 1 and 5 allowed a low level of genome production and substitutions at position 4 were tolerated. Position 4 is the only one of the seven positions examined that is not conserved between the 3′ ends of genomic and antigenomic RNA. The mutations that blocked the synthesis of progeny genome thus limited RNA replication to one step, namely, the synthesis and encapsidation of antigenome. Restoration of terminal complementarity for one of the trailer mutants by making a compensatory mutation in the leader region did not restore synthesis of genomic RNA, confirming that its loss was not due to reduced terminal complementarity. Interestingly, this leader mutation appeared to prevent antigenome synthesis with only a slight effect on mRNA synthesis, apparently providing a dissociation between these two synthetic activities. Genomes in which the terminal 24 or 325 nucleotides of the trailer have been deleted were competent for encapsidation and the synthesis of mRNA and antigenomic RNA, further confirming that terminal complementarity was not required for these functions.
Rainbow Trout Sleeping Disease Virus Is an Atypical Alphavirus
Authors: Villoing Stéphane , Béarzotti Monique , Chilmonczyk Stefan , Castric Jeannette , Brémont Michel
Published: April 2009

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10°C, while little virus was produced at 14°C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.
Mutagenesis of the Signal Sequence of Yellow Fever Virus prM Protein: Enhancement of Signalase Cleavage In Vitro Is Lethal for Virus Production
Authors: Lee Eva , Stocks Christine E. , Amberg Sean M. , Rice Charles M. , Lobigs Mario
Published: April 2009

Proteolytic processing at the C-prM junction in the flavivirus polyprotein involves coordinated cleavages at the cytoplasmic and luminal sides of an internal signal sequence. We have introduced at the COOH terminus of the yellow fever virus (YFV) prM signal sequence amino acid substitutions (VPQAQA mutation) which uncoupled efficient signal peptidase cleavage of the prM protein from its dependence on prior cleavage in the cytoplasm of the C protein mediated by the viral NS2B-3 protease. Infectivity assays with full-length YFV RNA transcripts showed that the VPQAQA mutation, which enhanced signal peptidase cleavage in vitro, was lethal for infectious virus production. Revertants or second-site mutants were recovered from cells transfected with VPQAQA RNA. Analysis of these viruses revealed that single amino acid substitutions in different domains of the prM signal sequence could restore viability. These variants had growth properties in vertebrate cells which differed only slightly from those of the parent virus, despite efficient signal peptidase cleavage of prM in cell-free expression assays. However, the neurovirulence in mice of the VPQAQA variants was significantly attenuated. This study demonstrates that substitutions in the prM signal sequence which disrupt coordinated cleavages at the C-prM junction can impinge on the biological properties of the mutant viruses. Factors other than the rate of production of prM are vitally controlled by regulated cleavages at this site.
Induction of Antibodies in Guinea Pigs and Rhesus Monkeys against the Human Immunodeficiency Virus Type 1 Envelope: Neutralization of Nonpathogenic and Pathogenic Primary Isolate Simian/Human Immunodeficiency Virus Strains
Authors: Liao Hua-Xin , Etemad-Moghadam Bijan , Montefiori David C. , Sun Ying , Sodroski Joseph , Scearce Richard M. , Doms Robert W. , Thomasch James R. , Robinson Suzanne , Letvin Norman L. , Haynes Barton F.
Published: April 2009

We have compared the abilities of human immunodeficiency virus type 1 (HIV-1) envelope V3 peptides and recombinant gp120 to induce antibodies that neutralize simian/human immunodeficiency viruses (SHIVs). SHIV-89.6 is a nonpathogenic SHIV that expresses the envelope protein of primary HIV-1 isolate 89.6. SHIV-89.6P, clone KB9, is a pathogenic SHIV variant derived from SHIV-89.6. Infection of rhesus monkeys with these SHIVs rarely induces anti-V3 region antibodies. To determine the availability of the gp120 V3 loop for neutralizing antibody binding on SHIV-89.6 and KB9 virions, we have constructed immunogenic C4-V3 peptides from these SHIVs and induced anti-V3 antibodies in guinea pigs and rhesus monkeys. We found that both SHIV-89.6 and KB9 C4-V3 peptides induced antibodies that neutralized SHIV-89.6 but that only SHIV-KB9 C4-V3 peptide induced antibodies that neutralized SHIV-KB9. Immunoprecipitation assays demonstrated that SHIV-KB9 C4-V3 peptide-induced antibodies had a greater ability to bind SHIV-KB9 envelope proteins than did antibodies raised against SHIV-89.6 C4-V3 peptide. We have used a series of mutant HIV-1 envelope constructs to map the gp120 determinants that affect neutralization by anti-V3 antibodies. The residue change at position 305 of arginine (in SHIV-89.6) to glutamic acid (in SHIV-KB9) played a central role in determining the ability of peptide-induced anti-V3 antiserum to neutralize primary isolate SHIVs. Moreover, residue changes in the SHIV-89.6 V1/V2 loops also played roles in regulating the availability of the V3 neutralizing epitope on SHIV-89.6 and -KB9. Thus, SHIV-89.6 and -KB9 V3 region peptides are capable of inducing neutralizing antibodies against these primary isolate SHIVs, although the pathogenic SHIV-KB9 is less easily neutralized than its nonpathogenic variant SHIV-89.6. In contrast to natural infection with SHIV-89.6, in which few animals make anti-V3 antibodies, C4-V3 peptides frequently induced anti-V3 antibodies that neutralized primary isolate SHIV strains.
In Vitro Transcription by the Turnip Yellow Mosaic Virus RNA Polymerase: a Comparison with the Alfalfa Mosaic Virus and Brome Mosaic Virus Replicases
Authors: Deiman Birgit A. L. M. , Verlaan Paul W. G. , Pleij Cornelis W. A.
Published: April 2009

Recently, we showed that the main determinant in the tRNA-like structure of turnip yellow mosaic virus RNA to initiate minus-strand synthesis in vitro is the 3′ ACCA end. By mutational analysis of the 3′-terminal hairpin, we show here that only a non-base-paired ACCA end is functional and that the stability of the wild-type 3′-proximal hairpin is the most favorable, in that it has the lowest Δ
G
value and a high transcription efficiency. With a nested set of RNA fragments, we show that the minimum template length is 9 nucleotides and that transcription is improved with increasing the length of the template. The results also suggest that proper base stacking contributes to efficient transcription initiation. Internal initiation is shown to take place on every NPyCPu sequence of a nonstructured template. However, the position of the internal initiation site in the template is important, and competition between the different sites takes place. Internal initiation was also studied with the RNA-dependent RNA polymerase of brome mosaic virus (BMV) and alfalfa mosaic virus (AlMV). The BMV polymerase can start internally on ACCA sequences, though inefficiently. Unexpectedly, the polymerases of both AlMV and BMV can start efficiently on an internal AUGC sequence.
Foot-and-Mouth Disease Virus 3C Protease Induces Cleavage of Translation Initiation Factors eIF4A and eIF4G within Infected Cells
Authors: Belsham Graham J. , McInerney Gerald M. , Ross-Smith Natalie
Published: April 2009

Infection of cells by foot-and-mouth disease virus (FMDV) results in the rapid inhibition of host cell protein synthesis. This process is accompanied by the early cleavage of the translation initiation factor eIF4G, a component of the cap-binding complex eIF4F. This cleavage is mediated by the leader (L) protease. Subsequently, as the virus proteins accumulate, secondary cleavages of eIF4G occur. Furthermore, eIF4A (46 kDa), a second component of eIF4F, is also cleaved in these later stages of the infection cycle. The 33-kDa cleavage product of eIF4A has lost a fragment from its N terminus. Transient-expression assays demonstrated that eIF4A was not cleaved in the presence of FMDV L or with the poliovirus 2A protease (which also mediates eIF4G cleavage) but was cleaved when the FMDV 3C protease was expressed. The FMDV 3C protease was also shown in such assays to induce cleavage of eIF4G, resulting in the production of cleavage products different from those generated by the L protease. Consistent with these results, within cells infected with a mutant FMDV lacking the L protease or within cells containing an FMDV replicon lacking L-P1 coding sequences it was again shown that eIF4A and eIF4G were cleaved.
Development of an In Vivo Assay To Identify Structural Determinants in Murine Leukemia Virus Reverse Transcriptase Important for Fidelity
Authors: Halvas Elias K. , Svarovskaia Evguenia S. , Pathak Vinay K.
Published: April 2009

Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations. Structural features of retroviral RTs that are important for accuracy of DNA synthesis in vivo are not known. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P also contains an MLV-based retroviral vector (GA-1) which encodes a wild-type bacterial β-galactosidase gene (
lacZ
) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild-type or mutated MLV
gag-pol
expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with 5-bromo-4-chloro-3-indolyl-β-
d
-galactopyranoside (X-Gal), and the frequencies of inactivating mutations in
lacZ
were quantified. Three mutations in the YVDD motif (V223M, V223S, and V223A) and two mutations in the RNase H domain (S526A and R657S) exhibited frequencies of
lacZ
inactivation 1.2- to 2.3-fold higher than that for the wild-type MLV RT (
P
< 0.005). Two mutations (V223I and Y598V) did not affect the frequency of
lacZ
inactivation. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.
Successful Transmission of Three Mouse-Adapted Scrapie Strains to Murine Neuroblastoma Cell Lines Overexpressing Wild-Type Mouse Prion Protein
Authors: Nishida Noriyuki , Harris David A. , Vilette Didier , Laude Hubert , Frobert Yveline , Grassi Jacques , Casanova Danielle , Milhavet Ollivier , Lehmann Sylvain
Published: April 2009

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP
C
-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP
C
than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP
Sc
, in the agent-inoculated PrP-overexpressing cells, while no PrP
Sc
was detectable in the untransfected cells after 3 passages. Production of PrP
Sc
in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP
Sc
-A strains Chandler, 139A, and 22L but not to PrP
Sc
-B strains 87V and 22A. We further demonstrate the successful transmission of PrP
Sc
from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP
C
and the sequence of PrP
Sc
. This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.
Sequential CD4-Coreceptor Interactions in Human Immunodeficiency Virus Type 1 Env Function: Soluble CD4 Activates Env for Coreceptor-Dependent Fusion and Reveals Blocking Activities of Antibodies against Cryptic Conserved Epitopes on gp120
Authors: Salzwedel Karl , Smith Erica D. , Dey Barna , Berger Edward A.
Published: April 2009

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems.
A Cysteine-Rich Motif in Poliovirus Protein 2C
ATPase
Is Involved in RNA Replication and Binds Zinc In Vitro
Authors: Pfister Thomas , Jones Keith W. , Wimmer Eckard
Published: April 2009

Protein 2C
ATPase
of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C
ATPase
engages in these processes are not known. To characterize functional domains of 2C
ATPase
, we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C
ATPase
. This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C
ATPase
as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.
Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells
Authors: Von Seggern Dan J. , Huang Shuang , Fleck Shonna Kaye , Stevenson Susan C. , Nemerow Glen R.
Published: April 2009

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.βgal.ΔF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.βgal.ΔF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types.
Vaccination of Macaques against Pathogenic Simian Immunodeficiency Virus with Venezuelan Equine Encephalitis Virus Replicon Particles
Authors: Davis Nancy L. , Caley Ian J. , Brown Kevin W. , Betts Michael R. , Irlbeck David M. , McGrath Kathryn M. , Connell Mary J. , Montefiori David C. , Frelinger Jeffrey A. , Swanstrom Ronald , Johnson Philip R. , Johnston Robert E.
Published: April 2009

Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.
The Differentiation-Specific Factor CDP/Cut Represses Transcription and Replication of Human Papillomaviruses through a Conserved Silencing Element
Authors: O'Connor Mark J. , Stünkel Walter , Koh Choon-Heng , Zimmermann Holger , Bernard Hans-Ulrich
Published: April 2009

The life cycles of human papillomaviruses (HPVs) are intimately linked to the differentiation program of infected stratified epithelia, with both viral gene expression and replication being maintained at low levels in undifferentiated basal cells and increased upon host cell differentiation. We recently identified, in HPV-16, a negative regulatory element between the epithelial-cell-specific enhancer and the E6 promoter that is capable of silencing E6 promoter activity, and we termed this element a papillomavirus silencing motif (PSM) and the unknown cellular factor that bound to it PSM binding protein (PSM-BP). Here we show that the homologous genomic segments of six other distantly related genital HPV types contain a PSM that binds PSM-BP and is capable of repressing transcription. Conservation of the PSM suggests that it is indispensable for the HPV life cycle. Purification, electrophoretic mobility shift assay experiments, and the use of specific antibodies proved that the cellular factor PSM-BP is identical to a previously described transcriptional repressor, the CCAAT displacement protein (CDP), also referred to as the human Cut protein (Cut). CDP/Cut repression of HPV-16 may stem from the modification of specifically positioned nucleosomes, as suggested by transcriptional stimulation under the influence of the histone deacetylase inhibitor trichostatin A. CDP/Cut is an important developmental regulator in several different tissues. It was recently shown that CDP/Cut is expressed in basal epithelial cells but not in differentiated primary keratinocytes. This suggests the possibility that repression by PSM couples HPV transcription to the stratification of epithelia. In each of the studied HPV types, the two CDP/Cut binding sites of PSM overlap with the known or presumed binding sites of the replication initiator protein E1. Transfection of CDP/Cut expression vectors into cells that support HPV-16 or HPV-31 replication leads to the elimination of viral episomes. Similarly, two PSM-like motifs overlapping the E1 binding site of bovine papillomavirus type 1 bind CDP/Cut, and CDP/Cut overexpression reduces the copy number of episomally replicating BPV-1 genomes in mouse fibroblasts. CDP/Cut appears to be a master regulator of HPV transcription and replication during epithelial differentiation, and PSMs are important
cis
-responsive targets of this repressor.
Polyuridylated mRNA Synthesized by a Recombinant Influenza Virus Is Defective in Nuclear Export
Authors: Poon Leo L. M. , Fodor Ervin , Brownlee George G.
Published: April 2009

The poly(A) tail of influenza virus mRNA is synthesized by reiterative copying of a U track near the 5′ end of the virion RNA (vRNA) template by the viral RNA polymerase. We have engineered a novel influenza A/WSN/33 virus which contains a neuraminidase (NA) vRNA with its U track mutated into an A track. Instead of synthesizing poly(A)-tailed NA mRNA, this novel virus synthesizes poly(U)-tailed NA mRNA. In infected cells, most poly(U)-tailed NA mRNA was retained in the nucleus, while most control polyadenylated NA mRNA was transported to the cytoplasm. These results suggest that the poly(A) tail is important for efficient nuclear export of NA mRNA. The mutant virus produced a reduced amount of NA and showed an attenuated phenotype, suggesting that poly(A) signal mutants of this type might be useful as potential live attenuated virus vaccines. In addition, this virus mutant might provide a useful model to further elucidate the basic mechanisms of mRNA nuclear export.
Identification of the Novel K15 Gene at the Rightmost End of the Kaposi's Sarcoma-Associated Herpesvirus Genome
Authors: Choi Joong-Kook , Lee Bok-Soo , Shim Sung N. , Li Mengtao , Jung Jae U.
Published: April 2009

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a distinct open reading frame called K15 at a position equivalent to the gene encoding LMP2A of Epstein-Barr virus (EBV). K15 isolates from body cavity-based lymphoma (BCBL) cells exhibited a dramatic sequence variation and a complex splicing pattern. However, all K15 alleles are organized similarly with the potential SH2 and SH3 binding motifs in their cytoplasmic regions. Northern blot analysis showed that K15 was weakly expressed in latently infected BCBL-1 cells, and the level of its expression was significantly induced by tetradecanoyl phorbol acetate stimulation. K15 encoded 40- to 55-kDa proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was localized at the cytoplasm and plasma membrane. To demonstrate the signal-transducing activity of the K15 protein, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8α polypeptide was replaced with that of KSHV K15. While the CD8-K15 chimera was not capable of eliciting cellular signal transduction upon stimulation with an anti-CD8 antibody, it significantly inhibited B-cell receptor signaling, as evidenced by a suppression of tyrosine phosphorylation and intracellular calcium mobilization. This inhibition required the putative SH2 or SH3 binding motif in the cytoplasmic region of K15. Biochemical study of CD8-K15 chimeras showed that the cytoplasmic region of K15 was constitutively tyrosine phosphorylated and that the tyrosine residue within the putative SH2 binding motif of K15 was a primary site of phosphorylation. These results demonstrate that KSHV K15 resembles LMP2A in genomic location, splicing pattern, and protein structure and by the presence of functional signal-transducing motifs in the cytoplasmic region. Thus, KSHV K15 is likely a distant evolutionary relative of EBV LMP2A.
Role of the Cytoplasmic Tail of Ecotropic Moloney Murine Leukemia Virus Env Protein in Fusion Pore Formation
Authors: Melikyan Grigory B. , Markosyan Ruben M. , Brener Sofya A. , Rozenberg Yanina , Cohen Fredric S.
Published: April 2009

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*—a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding—resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4°C and the time until fusion measured after the temperature was raised to 37°C or whether cells were first brought into contact at 37°C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.
Small Dense Nuclear Bodies Are the Site of Localization of Herpes Simplex Virus 1 U
L
3 and U
L
4 Proteins and of ICP22 Only When the Latter Protein Is Present
Authors: Markovitz Nancy S. , Roizman Bernard
Published: April 2009

The herpes simplex virus 1 U
L
3 and U
L
4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U
L
4 protein colocalizes with the products of the α22/U
S
1.5 genes in small nuclear dense bodies. Here we report that the U
L
3 protein also colocalized in these small nuclear dense bodies and the localization of U
L
3 and U
L
4 proteins in these bodies required the presence of α22/U
S
1.5 genes. In cells infected with a mutant lacking intact α22/U
S
1.5 genes, U
L
3 was diffused throughout the nucleus even though the overall accumulation of the γ2 U
L
3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U
L
3 accumulation and as the structural component and anchor of these small dense nuclear bodies.
env
Sequences of Simian Immunodeficiency Viruses from Chimpanzees in Cameroon Are Strongly Related to Those of Human Immunodeficiency Virus Group N from the Same Geographic Area
Authors: Corbet Sylvie , Müller-Trutwin Michaela C. , Versmisse Pierre , Delarue Severine , Ayouba Ahidjo , Lewis John , Brunak Soren , Martin Paul , Brun-Vezinet Françoise , Simon François , Barre-Sinoussi Françoise , Mauclere Philippe
Published: April 2009

Human immunodeficiency virus type 1 (HIV-1) group N from Cameroon is phylogenetically close, in
env
, to the simian immunodeficiency virus (SIV) cpz-gab from Gabon and SIVcpz-US of unknown geographic origin. We screened 29 wild-born Cameroonian chimpanzees and found that three (Cam3, Cam4, and Cam5) were positive for HIV-1 by Western blotting. Mitochondrial DNA sequence analysis demonstrated that Cam3 and Cam5 belonged to
Pan troglodytes troglodytes
and that Cam4 belonged to
P. t. vellerosus
. Genetic analyses of the viruses together with serological data demonstrated that at least one of the two
P. t. troglodytes
chimpanzees (Cam5) was infected in the wild, and revealed a horizontal transmission between Cam3 and Cam4. These data confirm that
P. t. troglodytes
is a natural host for HIV-1-related viruses. Furthermore, they show that SIVcpz can be transmitted in captivity, from one chimpanzee subspecies to another. All three SIVcpz-cam viruses clustered with HIV-1 N in
env
. The full Cam3 SIVcpz genome sequence showed a very close phylogenetic relationship with SIVcpz-US, a virus identified in a
P. t. troglodytes
chimpanzee captured nearly 40 years earlier. Like SIVcpz-US, SIVcpz-cam3 was closely related to HIV-1 N in
env
, but not in
pol
, supporting the hypothesis that HIV-1 N results from a recombination event. SIVcpz from chimpanzees born in the wild in Cameroon are thus strongly related in
env
to HIV-1 N from Cameroon, demonstrating the geographic coincidence of these human and simian viruses and providing a further strong argument in favor of the origin of HIV-1 being in chimpanzees.