bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the Asialoglycoprotein Receptor in Binding and Entry of Hepatitis C Virus Structural Proteins in Cultured Human Hepatocytes
Authors: Saunier Bertrand , Triyatni Miriam , Ulianich Luca , Maruvada Padma , Yen Paul , Kohn Leonard D.
Published: December 2002

We used a baculovirus-based system to prepare structural proteins of hepatitis C virus (HCV) genotype 1a. Binding of this preparation to cultured human hepatic cells was both dose dependent and saturable. This binding was decreased by calcium depletion and was partially prevented by ligands of the asialoglycoprotein receptor (ASGP-R), thyroglobulin, asialothyroglobulin, and antibody against a peptide in the carbohydrate recognition domain of ASGP-R but not preimmune antibody. Uptake by hepatocytes was observed with both radiolabeled and dye-labeled HCV structural proteins. With hepatocytes expressing the hH1 subunit of the ASGP-R fused to green fluorescent protein, we could show by confocal microscopy that dye stain cointernalized with the fusion protein in an area surrounding the nucleus. Internalization was more efficient with a preparation containing p7 than with one that did not. The two preparations bound to transfected 3T3-L1 cells expressing either both (hH1 and hH2) subunits of the ASGP-R (3T3-22Z cells) or both hH1 and a functionally defective variant of hH2 (3T3-24X cells) but not to parental cells. Additionally, uptake of dye-labeled preparation containing p7 was observed with 3T3-22Z cells but not with 3T3-L1 or 3T3-24X cells or with the preparation lacking p7, suggesting that p7 regulates the internalization properties of HCV structural proteins. Our observations suggest that HCV structural proteins bind to and cointernalize with the ASGP-R in cultured human hepatocytes.
Concordant Modulation of Neutralization Resistance and High Infectivity of the Primary Human Immunodeficiency Virus Type 1 MN Strain and Definition of a Potential gp41 Binding Site in gp120
Authors: Leavitt Maria , Park Eun Ju , Sidorov Igor A. , Dimitrov Dimiter S. , Quinnan, Gerald V.
Published: December 2002

Efforts to develop a vaccine against human immunodeficiency virus type 1 (HIV-1) are complicated by resistance of virus to neutralization. The neutralization resistance phenotype of HIV-1 has been linked to high infectivity. We studied the mechanisms determining this phenotype using clones of the T-cell-line-adapted (TCLA) MN strain (MN-TCLA) and the neutralization-resistant, primary MN strain (MN-P). Mutations in the amino- and carboxy-terminal halves of gp120 and the carboxy terminus of gp41 contributed to the neutralization resistance, high-infectivity phenotype but depended upon sequences in the leucine zipper (LZ) domain of gp41. Among 23 clones constructed to map the contributing mutations, there was a very strong correlation between infectivity and neutralization resistance (
R
2
= 0.81;
P
< 0.0001). Mutations that distinguished the gp120s of MN-P and MN-TCLA clones were clustered in or near the CD4 and coreceptor binding sites and in regions distant from those binding sites. To test the hypothesis that some of these distant mutations may interact with gp41, we determined which of them contributed to high infectivity and whether those mutations modulated gp120-gp41 association in the context of MN-P LZ sequences. In one clone, six mutations in the amino terminus of gp120, at least four of which clustered closely on the inner domain, modulated infectivity. This clone had a gp120-gp41 association phenotype like MN-P: in comparison to MN-TCLA, spontaneous dissociation was low, and dissociation induced by soluble CD4 binding was high. These results identify a region of the gp120 inner domain that may be a binding site for gp41. Our studies clarify mechanisms of primary virus neutralization resistance.
The Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Has Broad Signaling Effects in Primary Effusion Lymphoma Cells
Authors: Cannon Mark , Philpott Nicola J. , Cesarman Ethel
Published: December 2002

Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV-8]) is a gamma-2-herpesvirus responsible for Kaposi's sarcoma as well as primary effusion lymphoma (PEL). KSHV is a lymphotropic virus that has pirated many mammalian genes involved in inflammation, cell cycle control, and angiogenesis. Among these is the early lytic viral G protein-coupled receptor (vGPCR), a homologue of the human interleukin-8 (IL-8) receptor. When expressed, vGPCR is constitutively active and can signal via mitogen- and stress-activated kinases. In certain models it activates the transcriptional potential of NF-κB and activator protein 1 (AP-1) and induces vascular endothelial growth factor (VEGF) production. Despite its importance to the pathogenesis of all KSHV-mediated disease, little is known about vGPCR activity in hematopoietic cells. To study the signaling potential and downstream effects of vGPCR in such cells, we have developed PEL cell lines that express vGPCR under the control of an inducible promoter. The sequences required for tetracycline-mediated induction were cloned into a plasmid containing adeno-associated virus type 2 elements to enhance integration efficiency. This novel plasmid permitted studies of vGPCR activity in naturally infected KSHV-positive lymphocytes. We show that vGPCR activates ERK-2 and p38 in PEL cells. In addition, it increases the transcription of reporter genes under the control of AP-1, NF-κB, CREB, and NFAT, a Ca2+-dependent transcription factor important to KSHV lytic gene expression. vGPCR also increases the transcription of KSHV open reading frames 50 and 57, thereby displaying broad potential to affect viral transcription patterns. Finally, vGPCR signaling results in increased PEL cell elaboration of KSHV vIL-6 and VEGF, two growth factors involved in KSHV-mediated disease pathogenesis.
Structurally Conserved Amino Acid W501 Is Required for RNA Helicase Activity but Is Not Essential for DNA Helicase Activity of Hepatitis C Virus NS3 Protein
Authors: Kim Jong Won , Seo Mi Young , Shelat Anang , Kim Chon Saeng , Kwon Tae Woo , Lu Hui-hua , Moustakas Demetri Theodore , Sun Jeonghoon , Han Jang H.
Published: December 2002

Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU)
8
has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative π-facial hydrogen bond between the 2′-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the π-facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.
Rapid Prion Neuroinvasion following Tongue Infection
Authors: Bartz Jason C. , Kincaid Anthony E. , Bessen Richard A.
Published: December 2002

Food-borne transmission of prions can lead to infection of the gastrointestinal tract and neuroinvasion via the splanchnic and vagus nerves. Here we report that the transmission of transmissible mink encephalopathy (TME) is 100,000-fold more efficient by inoculation of prions into the tongues of hamsters than by oral ingestion. The incubation period following TME agent (hereinafter referred to as TME) inoculation into the lingual muscles was the shortest among the five nonneuronal routes of inoculation, including another intramuscular route. Deposition of the abnormal isoform of the prion protein, PrP
Sc
, was first detected in the tongue and submandibular lymph node at 1 to 2 weeks following inoculation of the tongue with TME. PrP
Sc
deposits in the tongue were associated with individual axons, and the initial appearance of TME in the brain stem was found in the hypoglossal nucleus at 2 weeks postinfection. At later time points, PrP
Sc
was localized to brain cell groups that directly project to the hypoglossal nucleus, indicating the transneuronal spread of TME. TME PrP
Sc
entry into the brain stem preceded PrP
Sc
detection in the rostral cervical spinal cord. These results demonstrate that TME can replicate in both the tongue and regional lymph nodes but indicate that the faster route of brain invasion is via retrograde axonal transport within the hypoglossal nerve to the hypoglossal nucleus. Topical application of TME to a superficial wound on the surface of the tongue resulted in a higher incidence of disease and a shorter incubation period than with oral TME ingestion. Therefore, abrasions of the tongue in livestock and humans may predispose a host to oral prion infection of the tongue-associated cranial nerves. In a related study, PrP
Sc
was detected in tongues following the intracerebral inoculation of six hamster-adapted prion strains, which demonstrates that prions can also travel from the brain to the tongue in the anterograde direction along the tongue-associated cranial nerves. These findings suggest that food products containing ruminant or cervid tongue may be a potential source of prion infection for humans.
Complement Regulation by Kaposi's Sarcoma-Associated Herpesvirus ORF4 Protein
Authors: Spiller O. Brad , Robinson Mairi , O'Donnell Elizabeth , Milligan Steven , Morgan B. Paul , Davison Andrew J. , Blackbourn David J.
Published: December 2002

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with three types of human tumor: Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. The virus encodes a number of proteins that participate in disrupting the immune response, one of which was predicted by sequence analysis to be encoded by open reading frame 4 (ORF4). The predicted ORF4 protein shares homology with cellular proteins referred to as regulators of complement activation. In the present study, the transcription profile of the ORF4 gene was characterized, revealing that it encodes at least three transcripts, by alternative splicing mechanisms, and three protein isoforms. Functional studies revealed that each ORF4 protein isoform inhibits complement and retains a C-terminal transmembrane domain. Consistent with the complement-regulating activity, we propose to name the proteins encoded by the ORF4 gene collectively as KSHV complement control protein (KCP). KSHV ORF4 is the most complex alternatively spliced gene encoding a viral complement regulator described to date. KCP inhibits the complement component of the innate immune response, thereby possibly contributing to the in vivo persistence and pathogenesis of this virus.
Disruption of CCL21-Induced Chemotaxis In Vitro and In Vivo by M3, a Chemokine-Binding Protein Encoded by Murine Gammaherpesvirus 68
Authors: Jensen Kristian K. , Chen Shu-Cheng , Hipkin R. William , Wiekowski Maria T. , Schwarz Martin A. , Chou Chuan-Chu , Simas J. Pedro , Alcami Antonio , Lira Sergio A.
Published: December 2002

Chemokine-binding proteins represent a novel class of antichemokine agents encoded by poxviruses and herpesviruses. One such protein is encoded by the
M3
gene present in the murine gammaherpesvirus 68 (MHV-68) genome. The
M3
gene encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and has been shown to block chemokine signaling in vitro. However, there has been no direct evidence that M3 blocks chemokine activity in vivo, nor has the nature of M3-chemokine interaction been defined. To better understand the ability of M3 to block chemokine activity in vivo, we examined its interaction with a specific subset of chemokines expressed in lymphoid tissues, areas where gammaherpesviruses characteristically establish latency. Here we show that M3 blocks in vitro chemotaxis induced by CCL19 and CCL21, chemokines expressed constitutively in secondary lymphoid tissues. Moreover, we provide evidence that chemokine M3 binding exhibits positive cooperativity. In vivo, the expression of M3 in the pancreas of transgenic mice inhibits recruitment of lymphocytes induced by transgenic expression of CCL21 in this organ. The ability of M3 to block the biological activity of chemokines may represent an important strategy used by MHV-68 to evade immune detection and favor viral replication in the infected host.
Disruption of Mitochondrial Networks by the Human Cytomegalovirus
UL37
Gene Product Viral Mitochondrion-Localized Inhibitor of Apoptosis
Authors: McCormick A. Louise , Smith Vanessa L. , Chow Dar , Mocarski Edward S.
Published: December 2002

By 24 h after infection with human cytomegalovirus, the reticular mitochondrial network characteristic of uninfected fibroblasts was disrupted as mitochondria became punctate and dispersed. These alterations were associated with expression of the immediate-early (α) antiapoptotic
UL37x1
gene product viral mitochondrion-localized inhibitor of apoptosis (vMIA). Similar alterations in mitochondrial morphology were induced directly by vMIA in transfected cells. A 68-amino-acid antiapoptotic derivative of vMIA containing the mitochondrial localization and antiapoptotic domains also induced disruption, whereas a mutant lacking the antiapoptotic domain failed to cause disruption. These data suggest that the fission and/or fusion process that normally controls mitochondrial networks is altered by vMIA. Mitochondrial fission has been implicated in the induction of apoptosis and vMIA-mediated inhibition of apoptosis may occur subsequent to this event.
Fine Mapping of the Interaction of Neutralizing and Nonneutralizing Monoclonal Antibodies with the CD4 Binding Site of Human Immunodeficiency Virus Type 1 gp120
Authors: Pantophlet Ralph , Ollmann Saphire Erica , Poignard Pascal , Parren Paul W. H. I. , Wilson Ian A. , Burton Dennis R.
Published: December 2002

Alanine scanning mutagenesis was performed on monomeric gp120 of human immunodeficiency virus type 1 to systematically identify residues important for gp120 recognition by neutralizing and nonneutralizing monoclonal antibodies (MAbs) to the CD4 binding site (CD4bs). Substitutions that affected the binding of broadly neutralizing antibody b12 were compared to substitutions that affected the binding of CD4 and of two nonneutralizing anti-CD4bs antibodies (b3 and b6) with affinities for monomeric gp120 comparable to that of b12. Not surprisingly, the sensitivities to a number of amino acid changes were similar for the MAbs and for CD4. However, in contrast to what was seen for the MAbs, no enhancing mutations were observed for CD4, suggesting that the virus has evolved toward an optimal gp120-CD4 interaction. Although the epitope maps of the MAbs overlapped, a number of key differences between b12 and the other two antibodies were observed. These differences may explain why b12, in contrast to nonneutralizing antibodies, is able to interact not only with monomeric gp120 but also with functional oligomeric gp120 at the virion surface. Neutralization assays performed with pseudovirions bearing envelopes from a selection of alanine mutants mostly showed a reasonable correlation between the effects of the mutations on b12 binding to monomeric gp120 and neutralization efficacy. However, some mutations produced an effect on b12 neutralization counter to that predicted from gp120 binding data. It appears that these mutations have different effects on the b12 epitope on monomeric gp120 and functional oligomeric gp120. To determine whether monomeric gp120 can be engineered to preferentially bind MAb b12, recombinant gp120s were generated containing combinations of alanine substitutions shown to uniquely enhance b12 binding. Whereas b12 binding was maintained or increased, binding by five nonneutralizing anti-CD4bs MAbs (b3, b6, F105, 15e, and F91) was reduced or completely abolished. These reengineered gp120s are prospective immunogens that may prove capable of eliciting broadly neutralizing antibodies.
Structure of Isolated Nucleocapsids from Venezuelan Equine Encephalitis Virus and Implications for Assembly and Disassembly of Enveloped Virus
Authors: Paredes Angel , Alwell-Warda Kathy , Weaver Scott C. , Chiu Wah , Watowich Stanley J.
Published: December 2002

Venezuelan equine encephalitis virus (VEEV) is an important human and equine pathogen in the Americas, with widespread reoccurring epidemics extending from South America to the southern United States. Most troubling, VEEV has been made into a weapon by several countries and is currently restricted by the Centers for Disease Control and Prevention as a potential biological warfare and terrorism agent. To facilitate the development of antiviral compounds, the structure of the nucleocapsid isolated from VEEV has been determined by electron cryomicroscopy and image reconstruction and represents the first three-dimensional structure of a nucleocapsid isolated from a single-stranded enveloped RNA virus. The isolated VEEV nucleocapsid undergoes significant reorganization relative to its structure within VEEV. However, the isolated nucleocapsid clearly exhibits T=4 icosahedral symmetry, and its characteristic nucleocapsid hexons and pentons are preserved. The diameter of the isolated nucleocapsid is ∼11.5% larger than that of the nucleocapsid within VEEV, with radial expansion being greatest near the hexons. Significantly, this is the first direct structural evidence showing that a simple enveloped virus undergoes large conformational changes during maturation, suggesting that the lipid bilayer and the transmembrane proteins of simple enveloped viruses provide the energy necessary to reorganize the nucleocapsid during maturation.
Regulation of Human Polyomavirus JC Virus Gene Transcription by AP-1 in Glial Cells
Authors: Sadowska Beata , Barrucco Robert , Khalili Kamel , Safak Mahmut
Published: December 2002

The activating transcription factor 1 (AP-1) family of proteins consists of a large number of inducible factors that are implicated in many biological processes, including cellular and viral gene expression, cell proliferation, differentiation, and tumorigenesis. Here, we investigated the role of the AP-1 family members c-Jun and c-Fos in transcriptional regulation of the JC virus (JCV) promoter in glial cells. DNA binding studies demonstrated the specific association of c-Jun with its DNA sequences corresponding to the AP-1 site within the JCV promoter. Functional analysis of the promoter showed that ectopic expression of c-Jun and c-Fos results in an additive activation of the JCV early and late promoters. Further functional assays indicated that the JCV AP-1 binding site is sufficient to confer responsiveness to both c-Jun/c-Fos- and UV-induced activation when transposed to a heterologous promoter. Analysis of c-Jun expression during the viral infection cycle by Western blotting revealed that c-Jun is posttranslationally modified by phosphorylation and its protein level is substantially increased at the late phases of infection cycle. Altogether, our findings indicate that AP-1 family members may play a role in the pathogenesis of JCV-induced disease in the human brain by modulating JCV gene transcription.
The ND10 Component Promyelocytic Leukemia Protein Relocates to Human Papillomavirus Type 1 E4 Intranuclear Inclusion Bodies in Cultured Keratinocytes and in Warts
Authors: Roberts Sally , Hillman Michele L. , Knight Gillian L. , Gallimore Phillip H.
Published: December 2002

Human papillomavirus type 1 (HPV1) E4 protein is associated with cytoplasmic and nuclear inclusions in productively infected keratinocytes. Here we have used transient expression of HPV1 E4 (also known as E1^E4) protein in keratinocytes to reproduce formation of E4 inclusions. Immunofluorescence analysis showed that progressive formation of inclusions correlated with diminished colocalization between E4 and keratin intermediate filaments (IFs). Our results support a model in which the HPV1 E4-keratin IF association is transient, occurring only at an early stage of inclusion formation. We also demonstrate that E4 induces relocation of the promyelocytic leukemia protein (PML) from multiple intranuclear speckles (ND10 bodies) to the periphery of nuclear E4 inclusions and that this activity is specific to full-length E4 protein. Analysis of HPV1-induced warts demonstrated that nuclear PML-E4 inclusions were present in productively infected keratinocytes, indicating that reorganization of PML occurs during the virus's replication cycle. It has been suggested that ND10 bodies are the sites for papillomavirus genome replication and virion assembly. Our finding that E4 induces reorganization of ND10 bodies in vitro and in vivo is further strong evidence that these domains play an important role in the papillomavirus life cycle. This study indicates that HPV1 is analogous to other DNA viruses that disrupt or reorganize ND10 domains, possibly to increase efficiency of virus infection. We hypothesize that HPV1 E4-induced reorganization of PML is necessary for efficient replication of the virus during the virus-producing phase.
CD8+T Cells Mediate Viral Clearance and Disease Pathogenesis during Acute Hepatitis B Virus Infection
Authors: Thimme Robert , Wieland Stefan , Steiger Carola , Ghrayeb John , Reimann Keith A. , Purcell Robert H. , Chisari Francis V.
Published: December 2002

Although the CD4+- and CD8+-T-cell responses to the hepatitis B virus (HBV) are thought to be crucial for the control of HBV infection, the relative contribution of each T-cell subset as an effector of viral clearance is not known. To examine this question, we monitored the course of HBV infection in control, CD4-depleted, and CD8-depleted chimpanzees. Our results demonstrate that CD8+cells are the main effector cells responsible for viral clearance and disease pathogenesis during acute HBV infection, and they suggest that viral clearance is mediated by both noncytolytic and cytolytic effector functions of the CD8+-T-cell response.
Identification and Characterization of a New Class of Human Immunodeficiency Virus Type 1 Recombinants Comprised of Two Circulating Recombinant Forms, CRF07_BC and CRF08_BC, in China
Authors: Yang Rongge , Kusagawa Shigeru , Zhang Chiyu , Xia Xueshan , Ben Kunlong , Takebe Yutaka
Published: December 2002

We identified a new class of human immunodeficiency virus type 1 (HIV-1) recombinants (00CN-HH069 and 00CN-HH086) in which further recombination occurred between two established circulating recombinant forms (CRFs). These two isolates were found among 57 HIV-1 samples from a cohort of injecting drug users in eastern Yunnan Province of China. Informative-site analysis in conjunction with bootscanning plots and exploratory tree analysis revealed that these two strains were closely related mosaics comprised of CRF07_BC and CRF08_BC, which are found in China. The genotype screening based on
gag
-reverse transcriptase sequences of 57 samples from eastern Yunnan identified 47 CRF08_BC specimens (82.5%), 5 CRF07_BC specimens (8.8%), and 3 additional specimens with the novel recombinant structure. These new “second-generation” recombinants thus constitute a substantial proportion (5 of 57; 8.8%) of HIV-1 strains in this population and may belong to a new but yet-undefined class of CRF. This might be the first example of CRFs recombining with each other, leading to the evolution of second-generation inter-CRF recombinants.
Contribution of the Zinc Finger to Zinc and DNA Binding by a Suppressor of Posttranscriptional Gene Silencing
Authors: van Wezel Rene , Liu Huanting , Wu Zirong , Stanley John , Hong Yiguo
Published: December 2002

The zinc finger C36-X1-C38-X7-C46-X6-H53of the nuclearly localized C2 protein ofTomato yellow leaf curl virusChina is involved in pathogenicity and suppression of posttranscriptional gene silencing (PTGS). Here, we demonstrate that the zinc finger is indispensable for the C2 protein to bind zinc and DNA. Mutation of cysteine residue C36, C38, or C46reduced the zinc and DNA binding capacity of C2 protein. When expressed from potato virus X, all three mutants, C2-C36R, C2-C38N, and C2-C46I, tagged with a green fluorescent protein (GFP) were still capable of transporting GFP into but aggregated abnormally in nuclei. Our data establish that zinc- and DNA-binding activity correlates with C2-mediated pathogenesis and PTGS suppression.
Responses of Herpes Simplex Virus Type 1-Infected Cells to the Presence of Extracellular Antibodies: gE-Dependent Glycoprotein Capping and Enhancement in Cell-to-Cell Spread
Authors: Rizvi Syed Monem , Raghavan Malini
Published: December 2002

Binding of anti-herpes simplex virus (HSV) immunoglobulin G (IgG) to HSV type 1 (HSV-1)-infected HEL and HEp-2 cells causes changes in surface viral glycoprotein distribution, resulting in a capping of all viral glycoproteins towards one pole of the cell. This occurs in a gE-dependent manner. In HEL cells, low concentrations of anti-HSV IgG also enhance cell-to-cell spread of wild-type HSV-1 but not of gE deletion mutant HSV-1. These observations raised the possibility that gE-dependent mechanisms exist that allow some HSV-1-infected cells to respond to the presence of extracellular antibodies by enhancing the antibody-resistant mode of virus transmission.
Expression of CXCR4 on Feline Peripheral Blood Mononuclear Cells: Effect of Feline Immunodeficiency Virus Infection
Authors: Willett Brian J. , Cannon Celia A. , Hosie Margaret J.
Published: December 2002

CXCR4 expression on feline peripheral blood mononuclear cells (PBMC) was analyzed. While monocytes and B lymphocytes expressed CXCR4, no CXCR4 was detected on T lymphocytes, in stark contrast to the expression pattern on T lymphocytes from humans. In spite of the important role that CXCR4 plays in infection with feline immunodeficiency virus, expression on PBMC in vivo was unaffected by infection with either a primary or a cell culture-adapted virus strain.
CD4 Binding Site Antibodies Inhibit Human Immunodeficiency Virus gp120 Envelope Glycoprotein Interaction with CCR5
Authors: Raja Aarti , Venturi Miro , Kwong Peter , Sodroski Joseph
Published: December 2002

The human immunodeficiency virus type 1 (HIV-1) gp120 exterior glycoprotein is conformationally flexible. Upon binding the host cell receptor, CD4, gp120 assumes a conformation that is able to bind the chemokine receptors CCR5 or CXCR4, which act as coreceptors for the virus. CD4-binding-site (CD4BS) antibodies are neutralizing antibodies elicited during natural infection that are directed against gp120 epitopes that overlap the binding site for CD4. Recent studies (S. H. Xiang et al., J. Virol. 76:9888-9899, 2002) suggest that CD4BS antibodies recognize conformations of gp120 distinct from the CD4-bound conformation. This predicts that the binding of CD4BS antibodies will inhibit chemokine receptor binding. Here, we show that Fab fragments and complete immunoglobulin molecules of CD4BS antibodies inhibit CD4-independent gp120 binding to CCR5 and cell-cell fusion mediated by CD4-independent HIV-1 envelope glycoproteins. These results are consistent with a model in which the binding of CD4BS antibodies limits the ability of gp120 to assume a conformation required for coreceptor binding.
Blotched Snakehead Virus Is a New Aquatic Birnavirus That Is Slightly More Related to Avibirnavirus Than to Aquabirnavirus
Authors: Da Costa Bruno , Soignier Stéphanie , Chevalier Christophe , Henry Celine , Thory Corinne , Huet Jean-Claude , Delmas Bernard
Published: December 2002

By different approaches, we characterized the birnavirus blotched snakehead virus (BSNV). The sequence of genomic segment A revealed the presence of two open reading frames (ORFs): a large ORF with a 3,207-bp-long nucleotide sequence and a 417-nucleotide-long small ORF located within the N-terminal half of the large ORF, but in a different reading frame. The large ORF was found to encode a polyprotein cotranslationally processed by the viral protease VP4 to generate pVP2 (the VP2 precursor), a 71-amino-acid-long peptide ([X]), VP4, and VP3. The two cleavage sites at the [X]-VP4 and VP4-VP3 junctions were identified by N-terminal sequencing. We showed that the processing of pVP2 generated VP2 and several small peptides (amino acids [aa] 418 to 460, 461 to 467, 468 to 474, and 475 to 486). Two of these peptides (aa 418 to 460 and 475 to 486) were positively identified in the viral particles with 10 additional peptides derived from further processing of the peptide aa 418 to 460. The results suggest that VP4 cleaves multiple Pro-X-Ala↓Ala motifs, with the notable exception of the VP4-VP3 junction. Replacement of the members of the predicted VP4 catalytic dyad (Ser-692 and Lys-729) confirmed their indispensability in the polyprotein processing. The genomic segment B sequence revealed a single large ORF encoding a putative polymerase, VP1. Our results demonstrate that BSNV should be considered a new aquatic birnavirus species, slightly more related to IBDV than to IPNV.
Human and Simian Immunodeficiency Virus Capsid Proteins Are Major Viral Determinants of Early, Postentry Replication Blocks in Simian Cells
Authors: Owens Christopher M. , Yang Peter C. , Göttlinger Heinrich , Sodroski Joseph
Published: December 2002

The cells of most Old World monkey species exhibit early, postentry restrictions on infection by human immunodeficiency virus type 1 (HIV-1) but not by simian immunodeficiency virus of macaques (SIV
mac
). Conversely, SIV
mac
, but not HIV-1, infection is blocked in most New World monkey cells. By using chimeric HIV-1/SIV
mac
viruses capable of a single round of infection, we demonstrated that a major viral determinant of this restriction is the capsid (CA) protein. The efficiency of early events following HIV-1 and SIV
mac
entry is apparently determined by the interaction of the incoming viral CA and species-specific host factors.
Identification of a Novel Tripartite Complex Involved in Replication of Vesicular Stomatitis Virus Genome RNA
Authors: Gupta Ashim K. , Shaji Daniel , Banerjee Amiya K.
Published: December 2002

Our laboratory's recent observations that transcriptionally inactive phosphoprotein (P) mutants can efficiently function in replicating vesicular stomatitis virus (VSV) defective interfering particle in a three-plasmid-based (L, P, and N) reverse genetics system in vivo (A. K. Pattnaik, L. Hwang, T. Li, N. Englund, M. Mathur, T. Das, and A. K. Banerjee, J. Virol. 71:8167-8175, 1997) led us to propose that a tripartite complex consisting of L-(N-P) protein may represent the putative replicase for synthesis of the full-length genome RNA. In this communication we demonstrate that such a complex is indeed detectable in VSV-infected BHK cells. Furthermore, coexpression of L, N, and P proteins in Sf21 insect cells by recombinant baculovirus containing the respective genes also resulted in the formation of a tripartite complex, as shown by immunoprecipitation with specific antibodies. A basic amino acid mutant of P protein, P260A, previously shown to be inactive in transcription but active in replication (T. Das, A. K. Pattnaik, A. M. Takacs, T. Li, L. N. Hwang, and A. K. Banerjee, Virology 238:103-114, 1997) was also capable of forming the mutant [L-(N-Pmut)] complex in both insect cells and BHK cells. Sf21 extract containing either the wild-type P protein or the mutant P protein along with the L and N proteins was capable of synthesizing 42S genome-sense RNA in an in vitro replication reconstitution reaction. Addition of N-Pmut or wild-type N-P complex further stimulated the synthesis of the genome-length RNA. These results indicate that the transcriptase and replicase complexes of VSV are possibly two distinct entities involved in carrying out capped mRNAs and uncapped genome and antigenome RNAs, respectively.
G100R Mutation within 4070A Murine Leukemia Virus Env Increases Virus Receptor Binding, Kinetics of Entry, and Viral Transduction Efficiency
Authors: Lu Chi-Wei , O'Reilly Lucille , Roth Monica J.
Published: December 2002

Passage of 4070A murine leukemia virus (MuLV) in D17 cells resulted in a G-to-R change at position 100 within the VRA of the envelope protein (Env). Compared with 4070A MuLV, virus with the G100R Env displayed enhanced binding on target cells, internalized the virus more rapidly, and increased the overall viral titer in multiple cell types. This provides a direct correlation between binding strength and efficiency of viral entry. Deletion of a His residue at the SU N terminus eliminated the transduction efficiency by the G100R virus, suggesting that the G100R virus maintains the regulatory characteristics of 4070A viral entry. The improved transduction efficiency of G100R Env would be an asset for gene delivery systems.
Receptor Usage and Fetal Expression of Ovine Endogenous Betaretroviruses: Implications for Coevolution of Endogenous and Exogenous Retroviruses
Authors: Spencer Thomas E. , Mura Manuela , Gray C. Allison , Griebel Philip J. , Palmarini Massimo
Published: December 2002

Betaretroviruses of sheep include two exogenous viruses, Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), and a group of endogenous viruses known as enJSRVs. The exogenous JSRV and ENTV are the etiological agents of ovine pulmonary adenocarcinoma (OPA) and enzootic nasal tumor (ENT), respectively. Sheep affected by OPA or ENT do not show an appreciable antibody response to JSRV or ENTV. Consequently, it is conceivable that enJSRV expression in the fetal lamb tolerizes sheep to the related exogenous viruses. In this study, possible mechanisms of interference between the sheep exogenous and endogenous betaretroviruses were investigated. In situ hybridization detected enJSRV RNAs in lymphoid cells associated with the lamina propria of the small intestine and in the thymus of sheep fetuses. Low-level expression of enJSRVs was also detected in the lungs. In addition, expression of enJSRVs was found to block entry of the exogenous JSRV, presumably via mechanisms of receptor interference. Indeed, enJSRVs, like JSRV and ENTV, were found to utilize hyaluronidase-2 as a cellular receptor.
Functional Replacement of Nucleocapsid Flanking Regions by Heterologous Counterparts with Divergent Primary Sequences: Effects of Chimeric Nucleocapsid on the Retroviral Replication Cycle
Authors: Fu William , Hu Wei-Shau
Published: December 2002

Nucleocapsid (NC) proteins in most retroviruses have a well-conserved Cys-His box(es) as well as more divergent flanking regions that are rich in basic residues. Mutations in the flanking regions can affect RNA packaging, virus assembly, and reverse transcription of the viral RNA. To gain a further understanding of the roles of NC flanking regions in the retroviral replication cycle, we generated and characterized chimeric
gag-pol
expression constructs derived from murine leukemia virus and spleen necrosis virus by replacing an NC flanking region from one virus with the counterpart from the other virus. We found that all four chimeras were able to generate virions, package viral RNA, and complete the viral replication cycle. Two chimeras had mild defects in virus assembly that correlated with a decrease in the isoelectric points of NCs, suggesting that the basic nature of NC is important in virus assembly. This finding indicates that, although the primary sequences of these flanking regions have little homology, the heterologous sequences are functional both as part of the Gag polyprotein and as processed NC protein.
Rice Grassy Stunt Tenuivirus
Nonstructural Protein p5 Interacts with Itself To Form Oligomeric Complexes In Vitro and In Vivo
Authors: Chomchan Pritsana , Li Shi-Fang , Shirako Yukio
Published: December 2002

We investigated the interaction of
Rice grassy stunt tenuivirus
(RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).
Increased Virus Replication and Virulence after Serial Passage of Human Immunodeficiency Virus Type 2 in Baboons
Authors: Locher Christopher P. , Witt Stephanie A. , Herndier Brian G. , Abbey Nancy W. , Tenner-Racz Klara , Racz Paul , Kiviat Nancy B. , Murthy Krishna K. , Brasky Kathleen , Leland Michelle , Levy Jay A.
Published: December 2002

Similar to human immunodeficiency virus type 1 (HIV-1) infection of humans, the natural history of HIV-2 infection in baboons (Papio cynocephalus) is a slow and chronic disease that generally takes several years before an AIDS-like condition develops. To shorten the amount of time to the development of disease, we performed five serial passages of HIV-2UC2in baboons by using blood and bone marrow samples during the acute phase of infection when viral loads were at high levels. After these serial passages, virus levels in plasma, peripheral blood mononuclear cells (PBMC) and lymphatic tissues in the acutely infected baboons were increased. Within 1 year of the HIV-2 infection, all of the inoculated baboons showed specific signs of AIDS-related disease progression within the lymphatic tissues, such as vascular proliferation and lymphoid depletion. The HIV-2UC2recovered after four serial passages showed increased kinetics of viral replication in baboon PBMC and cytopathicity. This study suggests that the HIV-2 isolate recovered after several serial passages in baboons will be useful in future studies of AIDS pathogenesis and vaccine development by using this animal model.
Evidence for Packaging of
rep-cap
Sequences into Adeno-Associated Virus (AAV) Type 2 Capsids in the Absence of Inverted Terminal Repeats: a Model for Generation of
rep
-Positive AAV Particles
Authors: Nony Pascale , Chadeuf Gilliane , Tessier Jacques , Moullier Philippe , Salvetti Anna
Published: December 2002

We previously reported that a 350-bp region of the adeno-associated virus (AAV) type 2
rep
gene contains a
cis
-acting element responsible for the Rep-dependent replication of a transiently transfected
rep-cap
plasmid. In this study, we further report that replicated
rep-cap
sequences can be packaged into AAV capsids in the absence of the inverted terminal repeats.
Divergent Simian T-Cell Lymphotropic Virus Type 3 (STLV-3) in Wild-Caught
Papio hamadryas papio
from Senegal: Widespread Distribution of STLV-3 in Africa
Authors: Meertens Laurent , Gessain Antoine
Published: December 2002

Among eight samples obtained from a French primatology research center, six adult guinea baboons (
Papio hamadryas papio
), caught in the wild in Senegal, had a peculiar human T-cell leukemia virus type 2 (HTLV-2)-like Western blot seroreactivity (p24
+
, GD21
+
, K55
+/−
). Partial sequence analyses of the
tax
genes (433 bp) indicated that these baboons were infected by a novel divergent simian T-cell lymphotropic virus (STLV). Analyses of the complete proviral sequence (8,892 bp) for one of these strains (STLV-3/PPA-F3) indicate that this STLV was highly divergent from the HTLV-1 (61.6% of nucleotide similarity), HTLV-2 (61.2%), or STLV-2 (60.6%) prototype. It was, however, much more closely related to the few other known STLV-3 strains, exhibiting 87 and 89% of nucleotide similarity with STLV-3/PHA-PH969 (formerly PTLV-L/PH969) and STLV-3/CTO-604, respectively. The STLV-3/PPA-F3 sequence possesses the major HTLV or STLV open reading frames corresponding to the structural, enzymatic, and regulatory proteins. However, its long terminal repeat comprises only two 21-bp repeats. In all phylogenetic analyses, STLV-3/PPA-F3 clustered together in a highly supported single clade with the other known strains of STLV-3, indicating an independent evolution from primate T-cell lymphotropic virus type 1 (PTLV-1) and PTLV-2. The finding of a new strain of STLV-3 in a West African monkey (Guinea baboon) greatly enlarges the geographical distribution and the host range of species infected by this PTLV type in the African continent. The recent discovery of several different STLV-3 strains in many different African monkey species, often in contact with humans, strongly suggests potential interspecies transmission events, as it was described for STLV-1, between nonhuman primates but also to humans.
Poliovirus-Induced Apoptosis Is Reduced in Cells Expressing a Mutant CD155 Selected during Persistent Poliovirus Infection in Neuroblastoma Cells
Authors: Gosselin Anne-Sophie , Simonin Yannick , Guivel-Benhassine Florence , Rincheval Vincent , Vayssière Jean-Luc , Mignotte Bernard , Colbère-Garapin Florence , Couderc Thérèse , Blondel Bruno
Published: December 2002

Poliovirus (PV) can establish persistent infections in human neuroblastoma IMR-32 cells. We previously showed that during persistent infection, specific mutations were selected in the first extracellular domain of the PV receptor (CD155) of these cells (N. Pavio, T. Couderc, S. Girard, J. Y. Sgro, B. Blondel, and F. Colbère-Garapin, Virology
274:
331-342, 2000). These mutations included the Ala 67 → Thr substitution, corresponding to a previously described allelic form of the PV receptor. The mutated CD155
Thr67
and the nonmutated IMR-32 CD155 (CD155
IMR
) were expressed independently in murine LM cells lacking the CD155 gene. Following infection of the cells with PV, we analyzed the death of cells expressing these two forms of CD155. Levels of DNA fragmentation, caspase activity, and cytochrome
c
release were lower in LM-CD155
Thr67
cells than in LM-CD155
IMR
cells. Thus, the level of apoptosis was lower in cells expressing mutated CD155 selected during persistent PV infection in IMR-32 than in cells expressing the wild-type receptor.
Overcoming Immunity to a Viral Vaccine by DNA Priming before Vector Boosting
Authors: Yang Zhi-yong , Wyatt Linda S. , Kong Wing-pui , Moodie Zoe , Moss Bernard , Nabel Gary J.
Published: December 2002

Replication-defective adenovirus (ADV) and poxvirus vectors have shown potential as vaccines for pathogens such as Ebola or human immunodeficiency virus in nonhuman primates, but prior immunity to the viral vector in humans may limit their clinical efficacy. To overcome this limitation, the effect of prior viral exposure on immune responses to Ebola virus glycoprotein (GP), shown previously to protect against lethal hemorrhagic fever in animals, was studied. Prior exposure to ADV substantially reduced the cellular and humoral immune responses to GP expressed by ADV, while exposure to vaccinia inhibited vaccine-induced cellular but not humoral responses to GP expressed by vaccinia. This inhibition was largely overcome by priming with a DNA expression vector before boosting with the viral vector. Though heterologous viral vectors for priming and boosting can also overcome this effect, the paucity of such clinical viral vectors may limit their use. In summary, it is possible to counteract prior viral immunity by priming with a nonviral, DNA vaccine.
Genetic Interaction between Distinct Dobrava Hantavirus Subtypes in
Apodemus agrarius
and
A. flavicollis
in Nature
Authors: Klempa Boris , Schmidt Heiko A. , Ulrich Rainer , Kaluz Stefan , Labuda Milan , Meisel Helga , Hjelle Brian , Krüger Detlev H.
Published: December 2002

Dobrava virus (DOBV) occurs in two different rodent species,
Apodemus flavicollis
(DOBV-Af) and
A. agrarius
(DOBV-Aa). We sequenced the S and M genomic segments from sympatric DOBV-Af and DOBV-Aa strains which fell into two distinct genetic lineages. Molecular phylogenetic analyses gave evidence for genetic reassortment between S and M segments of DOBV-Af and DOBV-Aa and indicated homologous recombination events in DOBV evolution. DOBV-Af and DOBV-Aa are distinct but also subject to genetic exchanges that affect their evolutionary trajectories.
Sequences Downstream of the 5′ Splice Donor Site Are Required for both Packaging and Dimerization of Human Immunodeficiency Virus Type 1 RNA
Authors: Russell Rodney S. , Hu Jing , Bériault Véronique , Mouland Andrew J. , Kleiman Lawrence , Wainberg Mark A. , Liang Chen
Published: December 2002

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5′ splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a “kissing-loop” mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5′ splice donor site, are required for efficient RNA packaging and dimerization.
Heterodimerization of the Two Major Envelope Proteins Is Essential for Arterivirus Infectivity
Authors: Snijder Eric J. , Dobbe Jessika C. , Spaan Willy J. M.
Published: December 2002

The two major envelope proteins of arteriviruses, the membrane protein (M) and the major glycoprotein (GP
5
), associate into a disulfide-linked heterodimer that is incorporated into the virion and has been assumed to be a prerequisite for virus assembly. Using an equine arteritis virus (EAV) infectious cDNA clone, we have analyzed the requirement for GP
5
-M heterodimerization and have identified the Cys residues involved in the formation of the GP
5
-M disulfide bond. The single Cys residue (Cys-8) in the M ectodomain was crucial for heterodimerization and virus infectivity. Mutagenesis of any of the five Cys residues in the GP
5
ectodomain or removal of the single GP
5
N-glycosylation site also rendered the full-length clone noninfectious. However, an analysis of revertants yielded an exceptional pseudorevertant in which residues 52 to 79 of the GP
5
ectodomain had been deleted and the original Cys-80→Ser mutation had been maintained. Consequently, this revertant lacked the GP
5
N-glycosyation site (Asn-56) and retained only a single cysteine residue (Cys-34). By using this GP
5
deletion, we confirmed that Cys-34 of GP
5
and Cys-8 of M are essential for GP
5
-M heterodimerization, a key event in the assembly of the EAV envelope.