bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Soluble CD4 on Exposure of Epitopes on Primary, Intact, Native Human Immunodeficiency Virus Type 1 Virions of Different Genetic Clades
Authors: Mbah Henry A. , Burda Sherri , Gorny Miroslaw K. , Williams Constance , Revesz Kathy , Zolla-Pazner Susan , Nyambi Phillipe N.
Published: July 2002

We have used a virus-binding assay to examine conformational changes that occur when soluble CD4 (sCD4) binds to the surface of intact, native, primary human immunodeficiency virus type 1 virions. The isolates examined belong to seven genetic clades (A to H) and are representative of syncytium-inducing and non-syncytium-inducing phenotypes. Conformational changes in epitopes in the C2, V2, V3, C5, and CD4 binding domain (CD4bd) of gp120 and the cluster I and II regions of gp41 of these viruses were examined using human monoclonal antibodies that are directed at these regions. The studies revealed that sCD4 binding causes a marked increase in exposure of epitopes in the V3 loop, irrespective of the clade or the phenotype of the virus. Sporadic increases in exposure were observed in some epitopes in the V2 region, while no changes were observed in the C2, C5, or CD4bd of gp120 or the cluster I and II regions of gp41.
Differential Immunogenicity of Epstein-Barr Virus Latent-Cycle Proteins for Human CD4+T-Helper 1 Responses
Authors: Leen Ann , Meij Pauline , Redchenko Irina , Middeldorp Jaap , Bloemena Elisabeth , Rickinson Alan , Blake Neil
Published: July 2002

Human CD4+T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8+memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4+epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4+T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4+T-helper 1 cells (EBNA1, EBNA3C ≫ LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8+-T-cell responses (EBNA3C > EBNA1 > LMP2 ≫ LMP1). Furthermore, the range of CD4+memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8+-T-cell frequencies.
Inducible Expression of Inflammatory Chemokines in Respiratory Syncytial Virus-Infected Mice: Role of MIP-1α in Lung Pathology
Authors: Haeberle Helene A. , Kuziel William A. , Dieterich Hans-Juergen , Casola Antonella , Gatalica Zoran , Garofalo Roberto P.
Published: July 2002

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1β, MIP-1α, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (107PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1α, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1α gene (−/− mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of −/− mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.
Involvement of Toll-Like Receptor 4 in Innate Immunity to Respiratory Syncytial Virus
Authors: Haynes Lia M. , Moore Deborah D. , Kurt-Jones Evelyn A. , Finberg Robert W. , Anderson Larry J. , Tripp Ralph A.
Published: July 2002

The mammalian Toll-like receptor 4, TLR4, is an important component in the innate immune response to gram-negative bacterial infection. The role of TLR4 in antiviral immunity has been largely unexplored. In this study, the in vivo immune responses to respiratory syncytial virus (RSV) and influenza virus infection were examined in TLR4-deficient (C57BL/10ScNCr) and TLR4-expressing (C57BL/10Sn) mice. TLR4-deficient mice challenged with RSV, but not influenza virus, exhibited impaired natural killer (NK) cell and CD14+cell pulmonary trafficking, deficient NK cell function, impaired interleukin-12 expression, and impaired virus clearance compared to mice expressing TLR4. These findings suggest that Toll signaling pathways have an important role in innate immunity to RSV.
Multiple Effects of Codon Usage Optimization on Expression and Immunogenicity of DNA Candidate Vaccines Encoding the Human Immunodeficiency Virus Type 1 Gag Protein
Authors: Deml Ludwig , Bojak Alexandra , Steck Stephanie , Graf Marcus , Wild Jens , Schirmbeck Reinhold , Wolf Hans , Wagner Ralf
Published: July 2002

We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt)gaggene flanked by essentialcis-acting sites such as the 5′-untranslated region and 3′-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with thesyngagplasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt)gagvector system. In contrast, intradermal immunization of both wtgagandsyngagvector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated withinsyngagby codon optimization do not contribute significantly to the high immunogenicity of thesyngagplasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of thesyngagDNA vaccine.
Common Origin of Four Diverse Families of Large Eukaryotic DNA Viruses
Authors: Iyer Lakshminarayan M. , Aravind L. , Koonin Eugene V.
Published: July 2002

Comparative analysis of the protein sequences encoded in the genomes of three families of large DNA viruses that replicate, completely or partly, in the cytoplasm of eukaryotic cells (poxviruses, asfarviruses, and iridoviruses) and phycodnaviruses that replicate in the nucleus reveals 9 genes that are shared by all of these viruses and 22 more genes that are present in at least three of the four compared viral families. Although orthologous proteins from different viral families typically show weak sequence similarity, because of which some of them have not been identified previously, at least five of the conserved genes appear to be synapomorphies (shared derived characters) that unite these four viral families, to the exclusion of all other known viruses and cellular life forms. Cladistic analysis with the genes shared by at least two viral families as evolutionary characters supports the monophyly of poxviruses, asfarviruses, iridoviruses, and phycodnaviruses. The results of genome comparison allow a tentative reconstruction of the ancestral viral genome and suggest that the common ancestor of all of these viral families was a nucleocytoplasmic virus with an icosahedral capsid, which encoded complex systems for DNA replication and transcription, a redox protein involved in disulfide bond formation in virion membrane proteins, and probably inhibitors of apoptosis. The conservation of the disulfide-oxidoreductase, a major capsid protein, and two virion membrane proteins indicates that the odd-shaped virions of poxviruses have evolved from the more common icosahedral virion seen in asfarviruses, iridoviruses, and phycodnaviruses.
Restoration of Wild-Type Infectivity to Human Immunodeficiency Virus Type 1 Strains Lacking
nef
by Intravirion Reverse Transcription
Authors: Khan Mahfuz , Garcia-Barrio Minerva , Powell Michael D.
Published: July 2002

Human immunodeficiency virus type 1 (HIV-1) Nef protein exerts several effects, both on infected cells and as a virion protein, which work together to enhance viral replication. One of these activities is the ability to enhance infectivity and the formation of proviral DNA. The mechanism of this enhancement remains incompletely understood. We show that virions with
nef
deleted can be restored to wild-type infectivity by stimulating intravirion reverse transcription. Particle composition and measures of reverse transcriptase activity remain the same for Nef
+
and Nef
−
virions both before and after natural endogenous reverse transcription (NERT) treatment. The effect of NERT treatment on virions pseudotyped with murine leukemia virus envelope protein was similar to that on particles pseudotyped with HIV-1 envelope protein. However, virions pseudotyped with vesicular stomatitis virus G envelope protein showed no influence of Nef on NERT enhancement of infectivity. These observations suggest that Nef may function at a level prior to reverse transcription. Since NERT treatment results in partial disassembly of the viral core, we speculate that Nef may function at the level of core particle disassembly.
Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus
Authors: Vieira Jeffrey , O'Hearn Patricia , Kimball Louise , Chandran Bala , Corey Lawrence
Published: July 2002

The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) and
neo
(conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.
Cell Cycle Regulation of Human Interleukin-8 Gene Expression by the Human Immunodeficiency Virus Type 1 Tat Protein
Authors: Mahieux R. , Lambert P. F. , Agbottah E. , Halanski M. A. , Deng L. , Kashanchi F. , Brady J. N.
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) Tat protein has been reported to transactivate several cellular genes, including the potent chemotactic factor interleukin-8 (IL-8). Consistent with these in vitro assays, elevated levels of IL-8 protein are found in the serum of HIV-infected individuals. We now extend these observations by demonstrating that Tat induction of IL-8 is linked to the cell cycle. Cells that constitutively express the Tat(1–86) protein (eTat) and control cells (pCEP) were reversibly blocked at the G1/S border with hydroxyurea or thymidine. The cells were subsequently released, and IL-8 expression was monitored by RNase protection assays and enzyme-linked immunosorbent assay (ELISA). RNase protection assays demonstrated that IL-8 mRNA expression is transiently induced, approximately fourfold, as the Tat-expressing cells enter S phase. Consistent with the RNase protection assay, an increase in IL-8 protein was observed in the cell supernatant using an IL-8 ELISA. Similar experiments were performed following a reversible block at the G2/M border with nocodazole and release into G1. Using the RNase protection assay and ELISA, little or no increase in IL-8 expression was observed during G1. Using gel shift as well as an immobilized DNA binding assay, we demonstrate that the increase in IL-8 gene expression correlates with a specific increase in p65 NF-κB binding activity only in the nucleus of the Tat-expressing cells. Moreover, the CREB-binding protein coactivator is present in the complex in the Tat cell line. Finally, we demonstrate that the presence of the proteasome inhibitor MG-132 inhibits the induction of NF-κB binding, as well as IL-8 expression, supporting the role of NF-κB.
RNA Triphosphatase Component of the mRNA Capping Apparatus of
Paramecium bursaria Chlorella
Virus 1
Authors: Ho C. Kiong , Gong Chunling , Shuman Stewart
Published: July 2002

Paramecium bursaria chlorella
virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5′ diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the γ-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that
Chlorella
virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid
Chlorella
virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses).
Chlorella
virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.
Interaction of Coxsackievirus A21 with Its Cellular Receptor, ICAM-1
Authors: Xiao Chuan , Bator Carol M. , Bowman Valorie D. , Rieder Elizabeth , He Yongning , Hébert Benoı̂t , Bella Jordi , Baker Timothy S. , Wimmer Eckard , Kuhn Richard J. , Rossmann Michael G.
Published: July 2002

Coxsackievirus A21 (CAV21), like human rhinoviruses (HRVs), is a causative agent of the common cold. It uses the same cellular receptor, intercellular adhesion molecule 1 (ICAM-1), as does the major group of HRVs; unlike HRVs, however, it is stable at acid pH. The cryoelectron microscopy (cryoEM) image reconstruction of CAV21 is consistent with the highly homologous crystal structure of poliovirus 1; like other enteroviruses and HRVs, CAV21 has a canyon-like depression around each of the 12 fivefold vertices. A cryoEM reconstruction of CAV21 complexed with ICAM-1 shows all five domains of the extracellular component of ICAM-1. The known atomic structure of the ICAM-1 amino-terminal domains D1 and D2 has been fitted into the cryoEM density of the complex. The site of ICAM-1 binding within the canyon of CAV21 overlaps the site of receptor recognition utilized by rhinoviruses and polioviruses. Interactions within this common region may be essential for triggering viral destabilization after attachment to susceptible cells.
Single Amino Acid Substitution in the V Protein of Simian Virus 5 Differentiates Its Ability To Block Interferon Signaling in Human and Murine Cells
Authors: Young D. F. , Chatziandreou N. , He B. , Goodbourn S. , Lamb R. A. , Randall R. E.
Published: July 2002

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N100D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N100D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N100D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N100D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N100D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.
Epstein-Barr Virus Nuclear Antigen 3C and Prothymosin Alpha Interact with the p300 Transcriptional Coactivator at the CH1 and CH3/HAT Domains and Cooperate in Regulation of Transcription and Histone Acetylation
Authors: Subramanian Chitra , Hasan Sameez , Rowe Martin , Hottiger Michael , Orre Rama , Robertson Erle S.
Published: July 2002

The Epstein-Barr virus nuclear antigen 3C (EBNA3C), encoded by Epstein-Barr virus (EBV), is essential for mediating transformation of human B lymphocytes. Previous studies demonstrated that EBNA3C interacts with a small, nonhistone, highly acidic, high-mobility group-like nuclear protein prothymosin alpha (ProTα) and the transcriptional coactivator p300 in complexes from EBV-infected cells. These complexes were shown to be associated with histone acetyltransferase (HAT) activity in that they were able to acetylate crude histones in vitro. In this report we show that ProTα interacts with p300 similarly to p53 and other known oncoproteins at the CH1 amino-terminal domain as well as at a second domain downstream of the bromodomain which includes the CH3 region and HAT domain. Similarly, EBNA3C also interacts with p300 at regions which include the CH1 and CH3/HAT domains, suggesting that ProTα and EBNAC3C may interact in a complex with p300. We also show that ProTα activates transcription when targeted to promoters by fusion to the GAL4 DNA binding domain and that this activation is enhanced by the addition of an exogenous source of p300 under the control of a heterologous promoter. This overall activity is down-modulated in the presence of EBNA3C. These results further establish the interaction of cellular coactivator p300 with ProTα and demonstrate that the associated activities resulting from this interaction, which plays a role in acetylation of histones and coactivation, can be regulated by EBNA3C. Furthermore, this study establishes for the first time a transcriptional role for ProTα in recruitment or stabilization of coactivator p300, as well as other basal transcription factors, at the nucleosomes for regulation of transcription.
cis
-
Acting Elements Important for Retroviral RNA Packaging Specificity
Authors: Beasley Benjamin E. , Hu Wei-Shau
Published: July 2002

Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Ψ and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Ψ. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5′-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.
Human Cytomegalovirus Gene Products US2 and US11 Differ in Their Ability To Attack Major Histocompatibility Class I Heavy Chains in Dendritic Cells
Authors: Rehm Armin , Engelsberg Arne , Tortorella Domenico , Körner Ida J. , Lehmann Insa , Ploegh Hidde L. , Höpken Uta E.
Published: July 2002

Human cytomegalovirus (HCMV) encodes several proteins that inhibit major histocompatibility complex (MHC) class I-dependent antigen presentation. The HCMV products US2 and US11 are each sufficient for causing the dislocation of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol, where the heavy chains are readily degraded. The apparent redundancy of US2 and US11 has been probed predominantly in cultured cell lines, where differences in their specificities were shown for murine and human MHC class I locus products. Here, we expressed US11 and US2 via adenovirus vectors and show that US11 exhibits a superior ability to degrade MHC class I molecules in primary human dendritic cells. MHC class II complexes are unaffected by US2- and US11-mediated attack. We suggest that multiple HCMV-encoded immunoevasions have evolved complementary functions in response to diverse host cell types and tissues.
Expression of Hepatitis C Virus Proteins Induces Distinct Membrane Alterations Including a Candidate Viral Replication Complex
Authors: Egger Denise , Wölk Benno , Gosert Rainer , Bianchi Leonardo , Blum Hubert E. , Moradpour Darius , Bienz Kurt
Published: July 2002

Plus-strand RNA viruses characteristically replicate their genome in association with altered cellular membranes. In the present study, the capacity of hepatitis C virus (HCV) proteins to elicit intracellular membrane alterations was investigated by expressing, in tetracycline-regulated cell lines, a comprehensive panel of HCV proteins individually as well as in the context of the entire HCV polyprotein. As visualized by electron microscopy (EM), expression of the combined structural proteins core-E1-E2-p7, the NS3-4A complex, and protein NS4B induced distinct membrane alterations. By immunogold EM (IEM), the membrane-altering proteins were always found to localize to the respective altered membranes. NS4B, a protein of hitherto unknown function, induced a tight structure, designated membranous web, consisting of vesicles in a membranous matrix. Expression of the entire HCV polyprotein gave rise to membrane budding into rough endoplasmic reticulum vacuoles, to the membranous web, and to tightly associated vesicles often surrounding the membranous web. By IEM, all HCV proteins were found to be associated with the NS4B-induced membranous web, forming a membrane-associated multiprotein complex. A similar web-like structure in livers of HCV-infected chimpanzees was previously described (Pfeifer et al., Virchows Arch. B.,
33:
233-243, 1980). In view of this finding and the observation that all HCV proteins accumulate on the membranous web, we propose that the membranous web forms the viral replication complex in HCV-infected cells.
Hepatitis B Virus of Genotype B with or without Recombination with Genotype C over the Precore Region plus the Core Gene
Authors: Sugauchi Fuminaka , Orito Etsuro , Ichida Takafumi , Kato Hideaki , Sakugawa Hiroshi , Kakumu Shinichi , Ishida Takafumi , Chutaputti Anuchit , Lai Ching-Ling , Ueda Ryuzo , Miyakawa Yuzo , Mizokami Masashi
Published: July 2002

The entire nucleotide sequences of 70 hepatitis B virus (HBV) isolates of genotype B (HBV/B), including 38 newly determined and 32 retrieved from the international DNA database (DDBJ/EMBL/GenBank), were compared phylogenetically. Two subgroups of HBV/B were identified based on sequence divergence in the precore region plus the core gene, one with the recombination with genotype C and the other without it. The analysis over the entire genome of HBV/B by the SimPlot program located the recombination with genotype C in the precore region plus the core gene spanning nucleotide positions from 1740 to 1838 to 2443 to 2485. Within this genomic area, HBV/B strains with the recombination had higher nucleotide and amino acid homology to genotype C than those without the recombination (96.9 versus 91.1% in nucleotides and 97.0 versus 92.9% in amino acids). There were 29 HBV/B strains without the recombination, and they were all recovered from carriers in Japan. The remaining 41 HBV/B isolates having the recombination with genotype C were from carriers in China (12 strains), Hong Kong (3 strains), Indonesia (4 strains), Japan (3 strains), Taiwan (4 strains), Thailand (3 strains), and Vietnam (12 strains). Due to the frequency of the distribution of HBV/B without the recombination (29 of 32 isolates, or 91%) and the fact that it was exclusive to Japan, it was provisionally classified into the Bj (j standing for Japan) subgroup, and HBV/B with the recombination was classified into the Ba (a for Asia) subgroup. Virological differences between HBV/Bj and HBV/Ba may be reflected in the severity of clinical disease in the patients infected with HBV of genotype B, which seems to be under strong geographic influences in Asia.
Influence of a Putative Intermolecular Interaction between Core and the Pre-S1 Domain of the Large Envelope Protein on Hepatitis B Virus Secretion
Authors: Le Pogam Sophie , Shih Chiaho
Published: July 2002

Virion release of hepatitis B virus (HBV) from hepatocytes is a tightly regulated event. It is a dogma that only the mature HBV genome is preferentially allowed to export from the intracellular compartment (J. Summers and W. S. Mason, Cell 29:403-415, 1982). Recently, an “immature secretion” phenotype of a highly frequent naturally occurring HBV variant containing a leucine residue at amino acid 97 of the core protein was identified. Unlike wild-type HBV, this variant secretes almost equal amounts of mature and immature genomes. This phenomenon is not caused by any instability of core particles or by any deficiency in viral reverse transcription (T. T. Yuan, P. C. Tai, and C. Shih, J. Virol. 73:10122-10128, 1999). In this study, our kinetic analysis of virion secretion of the mutant F97L (phenylalanine to leucine) indicates that the secretion of its immature genome does not occur earlier than that of its mature genome. In addition, the secretion kinetics of the mature genomes are comparable between the wild-type HBV and the mutant F97L. Therefore, the immature secretion phenomenon of mutant F97L is not caused by premature secretion or more efficient secretion. Previously, we hypothesized that the immature secretion phenotype is probably caused by the aberrant interaction between its mutant core and wild-type envelope proteins. Here, we further demonstrated that a pre-S1 envelope mutation at position 119, changing an alanine (A) to a phenylalanine (F), can offset the immature secretion phenotype of the mutant I97L (isoleucine to leucine) and successfully restore the wild-type-like selective export of the mature genome of the double mutant pre-S1-A119F/core-I97L.
Localization of the Single-Stranded RNA-Binding Domains of Bluetongue Virus Nonstructural Protein NS2
Authors: Fillmore G. C. , Lin H. , Li J. K.-K.
Published: July 2002

The S2 gene of bluetongue virus, serotype 17, has been cloned, and the nonstructural protein NS2 has been expressed. Synthetic peptides matching regions within the amino acid sequence of NS2 were used to map three single-stranded RNA (ssRNA)-binding regions within the protein. A prokaryotic expression system was then used to generate a series of deletion mutants with the ssRNA-binding domains of NS2 removed, singly and in different combinations. These truncated proteins were expressed on a large scale and purified to near homogeneity. The affinity of each truncated protein towards ssRNA was then assayed by electrophoretic mobility shift assays. As a result, the three ssRNA-binding domains of BTV nonstructural protein NS2 have been conclusively localized, and removal of these three domains completely abrogates the ability of NS2 to bind to ssRNA.
Cross-Packaging of a Single Adeno-Associated Virus (AAV) Type 2 Vector Genome into Multiple AAV Serotypes Enables Transduction with Broad Specificity
Authors: Rabinowitz Joseph E. , Rolling Fabienne , Li Chengwen , Conrath Hervè , Xiao Weidong , Xiao Xiao , Samulski R. Jude
Published: July 2002

The serotypes of adeno-associated virus (AAV) have the potential to become important resources for clinical gene therapy. In an effort to compare the role of serotype-specific virion shells on vector transduction, we cloned each of the serotype capsid coding domains into a common vector backbone containing AAV type 2 replication genes. This strategy allowed the packaging of AAV2 inverted terminal repeat vectors into each serotype-specific virions. Each of these helper plasmids (pXR1 through pXR5) efficiently replicated the transgene DNA and expressed helper proteins at nearly equivalent levels. In this study, we observed a correlation between the amount of transgene replication and packaging efficiency. The physical titer of these hybrid vectors ranged between 1.3 × 10
11
and 9.8 × 10
12
/ml (types 1 and 2, respectively). Of the five serotype vectors, only types 2 and 3 were efficiently purified by heparin-Sepharose column chromatography, illustrating the high degree of similarity between these virions. We analyzed vector transduction in reference and mutant Chinese hamster ovary cells deficient in heparan sulfate proteoglycan and saw a correlation between transduction and heparan sulfate binding data. In this analysis, types 1 and 5 were most consistent in transduction efficiency across all cell lines tested. In vivo each serotype was ranked after comparison of transgene levels by using different routes of injection and strains of rodents. Overall, in this analysis, type 1 was superior for efficient transduction of liver and muscle, followed in order by types 5, 3, 2, and 4. Surprisingly, this order changed when vector was introduced into rat retina. Types 5 and 4 were most efficient, followed by type 1. These data established a hierarchy for efficient serotype-specific vector transduction depending on the target tissue. These data also strongly support the need for extending these analyses to additional animal models and human tissue. The development of these helper plasmids should facilitate direct comparisons of serotypes, as well as begin the standardization of production for further clinical development.
NS1 Protein of Influenza A Virus Down-Regulates Apoptosis
Authors: Zhirnov O. P. , Konakova T. E. , Wolff T. , Klenk H.-D.
Published: July 2002

Wild-type (WT) influenza A/PR/8/34 virus and its variant lacking the NS1 gene (delNS1) have been compared for their ability to mediate apoptosis in cultured cells and chicken embryos. Cell morphology, fragmentation of chromatin DNA, and caspase-dependent cleavage of the viral NP protein have been used as markers for apoptosis. Another marker was caspase cleavage of the viral M2 protein, which was also found to occur in an apoptosis-specific manner. In interferon (IFN)-competent host systems, such as MDCK cells, chicken fibroblasts, and 7-day-old chicken embryos, delNS1 virus induced apoptosis more rapidly and more efficiently than WT virus. As a consequence, delNS1 virus was also more lethal for chicken embryos than WT virus. In IFN-deficient Vero cells, however, apoptosis was delayed and developed with similar intensity after infection with both viruses. Taken together, these data indicate that the IFN antagonistic NS1 protein of influenza A viruses has IFN-dependent antiapoptotic potential.
Virion Disassembly Is Required for Apoptosis Induced by Reovirus
Authors: Connolly Jodi L. , Dermody Terence S.
Published: July 2002

Reovirus infection leads to apoptosis in cultured cells and in vivo. Binding of viral attachment protein ς1 to both sialic acid and junction adhesion molecule is required for induction of apoptosis. However, it is not known whether viral engagement of receptors is sufficient to elicit this cellular response. To determine whether steps in reovirus replication subsequent to viral attachment are required for reovirus-induced apoptosis, we used inhibitors of viral disassembly and RNA synthesis, viral disassembly intermediates, temperature-sensitive (ts) reovirus mutants, and reovirus particles deficient in genomic double-stranded RNA (dsRNA). We found that reovirus-induced apoptosis is abolished in the presence of the viral disassembly inhibitors ammonium chloride and E64. Infectious subvirion particles (ISVPs), which are intermediates in reovirus disassembly that can be generated in vitro by protease treatment, are capable of inducing apoptosis in the presence or absence of these inhibitors. Treatment of cells with the viral RNA synthesis inhibitor ribavirin does not diminish the capacity of reovirus to induce apoptosis, and reovirustsmutants arrested at defined steps in viral replication produce apoptosis with efficiency similar to that of wild-type virus. Furthermore, reovirus particles lacking dsRNA are capable of inducing apoptosis. Finally, we found that viral attachment and disassembly must occur within the same cellular compartment for reovirus to elicit an apoptotic response. These results demonstrate that disassembly of reovirus virions to form ISVPs, but not viral transcription or subsequent steps in viral replication, is required for reovirus to induce apoptosis.
Positively Charged Amino Acid Substitutions in the E2 Envelope Glycoprotein Are Associated with the Emergence of Venezuelan Equine Encephalitis Virus
Authors: Brault Aaron C. , Powers Ann M. , Holmes Edward C. , Woelk C. H. , Weaver Scott C.
Published: July 2002

Epidemic-epizootic Venezuelan equine encephalitis (VEE) viruses (VEEV) have emerged repeatedly via convergent evolution from enzootic predecessors. However, previous sequence analyses have failed to identify common sets of nucleotide or amino acid substitutions associated with all emaergence events. During 1993 and 1996, VEEV subtype IE epizootics occurred on the Pacific Coast of the states of Chiapas and Oaxaca in southern Mexico. Like other epizootic VEEV strains, when inoculated into guinea pigs and mice, the Mexican isolates were no more virulent than closely related enzootic strains, complicating genetic studies of VEE emergence. Complete genomic sequences of 4 of the Mexican strains were determined and compared to those of closely related enzootic subtype IE isolates from Guatemala. The epizootic viruses were less than 2% different at the nucleotide sequence level, and phylogenetic relationships confirmed that the equine-virulent Mexican strains probably evolved from enzootic progenitors on the Pacific Coast of Mexico or Guatemala. Of 35 amino acids that varied among the Guatemalan and Mexican isolates, only 8 were predicted phylogenetically to have accompanied the phenotypic change. One mutation at position 117 of the E2 envelope glycoprotein, involving replacement of Glu by Lys, resulted in a small-plaque phenotype characteristic of epizootic VEEV strains. Analysis of additional E2 sequences from representative enzootic and epizootic VEEV isolates implicated similar surface charge changes in the emergence of previous South American epizootic phenotypes, indicating that E2 mutations are probably important determinants of the equine-virulent phenotype and of VEE emergence. Maximum-likelihood analysis indicated that one change at E2 position 213 has been influenced by positive selection and convergent evolution of the epizootic phenotype.
Expression and Self-Assembly of Norwalk Virus Capsid Protein from Venezuelan Equine Encephalitis Virus Replicons
Authors: Baric Ralph S. , Yount Boyd , Lindesmith Lisa , Harrington Patrick R. , Greene Shermalyn R. , Tseng Fan-Chen , Davis Nancy , Johnston Robert E. , Klapper David G. , Moe Christine L.
Published: July 2002

The Norwalk virus (NV) capsid protein was expressed using Venezuelan equine encephalitis virus replicon particles (VRP-NV1). VRP-NV1 infection resulted in large numbers of recombinant NV-like particles that were primarily cell associated and were indistinguishable from NV particles produced from baculoviruses. Mutations located in the N-terminal and P1 domains of the NV capsid protein ablated capsid self-assembly in mammalian cells.
Mutation of the Dominant Endocytosis Motif in Human Immunodeficiency Virus Type 1 gp41 Can Complement Matrix Mutations without Increasing Env Incorporation
Authors: West John T. , Weldon Sally K. , Wyss Stephanie , Lin Xiaoxu , Yu Qin , Thali Markus , Hunter Eric
Published: July 2002

The human immunodeficiency virus type 1 transmembrane glycoprotein (TM) is efficiently endocytosed in a clathrin-dependent manner. Internalization is mediated by a tyrosine-containing motif within the cytoplasmic domain, and replacement of the cytoplasmic tyrosine by cysteine or phenylalanine increased expression of mutant glycoprotein on the surface of transfected cells by as much as 2.5-fold. Because interactions between the cytoplasmic domain of Env and the matrix protein (MA) have been suggested to mediate incorporation of Env in virus particles, we examined whether perturbation of endocytosis would alter incorporation. Proviruses were constructed to contain the wild-type or mutant Env in conjunction with point mutations in MA that had previously been shown to block Env incorporation. These constructs were used to evaluate the effect of glycoprotein endocytosis on incorporation into virus particles and to test the necessity for a specific interaction between Env and MA to mediate incorporation. Viruses produced from transfected 293T cells were used to infect various cell lines, including MAGI, H9, and CEMx174. Viruses encoding both a disrupted endocytosis motif signal and mutations within MA were significantly more infectious in MAGI cells than their counterparts encoding a mutant MA and wild-type Env. This complementation of infectivity for the MA incorporation mutant viruses was not due to increased glycoprotein incorporation into particles but instead reflected an enhanced fusogenicity of the mutated Env proteins. Our findings further support the concept that a specific interaction between the long cytoplasmic domain of TM and MA is required for efficient incorporation of Env into assembling virions. Alteration of the endocytosis signal of Env, and the resulting increase in cell surface glycoprotein, has no effect on incorporation despite demonstrable effects on fusion, virus entry, and infectivity.
Intrinsic Stability of Episomal Circles Formed during Human Immunodeficiency Virus Type 1 Replication
Authors: Pierson Theodore C. , Kieffer Tara L. , Ruff Christian T. , Buck Christopher , Gange Stephen J. , Siliciano Robert F.
Published: July 2002

The development of surrogate markers capable of detecting residual ongoing human immunodeficiency virus type 1 (HIV-1) replication in patients receiving highly active antiretroviral therapy is an important step in understanding viral dynamics and in developing new treatment strategies. In this study, we evaluated the utility of circular forms of the viral genome for the detection of recent infection of cells by HIV-1. We measured the fate of both one-long terminal repeat (1-LTR) and 2-LTR circles following in vitro infection of logarithmically growing CD4
+
T cells under conditions in which cell death was not a significant contributing factor. Circular forms of the viral genome were found to be highly stable and to decrease in concentration only as a function of dilution resulting from cell division. We conclude that these DNA circles are not intrinsically unstable in all cell types and suggest that the utility of 2-LTR circle assays in measuring recent HIV-1 infection of susceptible cells in vivo needs to be reevaluated.
Calorimetric Study of the Interaction of Local Anesthetics [2-(Alkyloxy)phenyl]-2-(1-piperidinyl)ethyl Carbamates with Model Dipalmitoylphosphatidylcholine Membranes
Authors: Gallová Jana , Bágeľová Jaroslava , Čižmárik Jozef , Balgavý Pavol
Published: July 2002

Local anesthetics of the homologous series of monohydrochlorides of [2-(alkoxy)phenyl]-2-(1-piperidinyl)ethyl esters of carbamic acid decrease the temperature of phase transitions Lβ' → Pβ (pretransition) and Pβ → Lα (main transition) of 1,2-dipalmitoyl-sn-phosphatidylcholine in aqueous phase at pH 6.2. The efficiency of the anesthetics in decreasing the pretransition temperature increases with the number of carbon atoms n of the alkoxy substituent up to n = 6. For n > 6 no pretransition could be detected. The efficiency in decreasing the temperature of the main transition increases up to n = 8 - 9 while it starts decreasing at n = 10. Computer simulation of the thermograms has shown that the decrease of efficiency at n = 10 is caused by the dependence of the partition coefficients of the anesthetics KP,G and KP,LC between the aqueous phase and the Pβ and Lα phases, respectively, on the length of the alkoxy substituent, thus: log KP,LC = ALC + BLC n and log KP,G = AG + BG n, where BLC < BG.
Study of the Conversion of CaSO4 to CaCO3 within the CaSO4 + Na2CO3 = CaCO3 + Na2SO4 Four-Component Water-Salt System
Authors: Trendafelov Dimitar , Christov Christomir , Balarew Christo , Karapetkova Aneta
Published: July 2002

The Pitzer ion-interaction model was employed to predict the conversion in the title quaternary water-salt system. The thermodynamic solubility products (Ks0) were determined and the conversion of CaSO4 to CaCO3 in the CaSO4 + Na2CO3 = CaCO3 + Na2SO4 system was predicted. The standard molar Gibbs free energy ∆rG0 of the conversion reaction was calculated to be -23.62 kJ mol-1. A 98% degree of conversion of CaSO4 was achieved by using calcium sulfate isolated from sea salt production waste brines. Suitable conditions were found for obtaining CaCO3 exhibiting a high dispersity and whiteness.
Cyclohexylation of Naphthalene Over USY Zeolites
Authors: Michvocík Miroslav , Mravec Dušan , Hronec Milan , Smiešková Agáta , Hudec Pavol
Published: July 2002

The influence of thermal stabilization of NH4-Y zeolite and modification of USY zeolites with solutions of hydrochloric acid on the cyclohexylation of naphthalene in the liquid phase was studied. Removal of the part of extra-framework aluminium from zeolite structure has a positive effect on both conversion of naphthalene and amount of dicyclohexylnaphthalenes formed. Modification of zeolites leads to an increase in conversion and selectivity of β-substitution in the naphthalene cyclohexylation.
Incidence, site, and nature of injuries in amateur rugby league over three consecutive seasons
Authors: Gabbett Tim J
Published: July 2002

Objectives—To report the incidence, site, and nature of injuries in amateur rugby league over three consecutive seasons.

Methods—Six hundred players registered with an amateur rugby league organisation were studied over three consecutive seasons. All injuries sustained during the amateur rugby league matches were recorded. Information recorded included the date and time of injury, name of injured player, anatomical site and nature of injury, and position of the player.

Results—The incidence of injury was 160.6 per 1000 player-position game hours, with forwards having a significantly higher incidence of injury than backs (182.3 per 1000v142.0 per 1000, χ2= 14.60, df = 1, p<0.001). Over 25% of the total injuries (40.6 per 1000) sustained during the three year period were to the head and neck, while injuries to the face (21.3 per 1000, 13.3%), abdomen and thorax (21.3 per 1000, 13.3%), and knee (17.8 per 1000, 11.1%) were less common (χ2= 21.83, df = 8, p<0.01). Muscular injuries (haematomas and strains) were the most common type of injury (45.7 per 1000, 28.5%, χ2= 17.98, df = 7, p<0.05). Significantly more injuries occurred in the latter stages of the season (χ2= 22.94, df = 1, p<0.001), with most injuries (70.8%, χ2= 162.29, df = 1, p<0.001) sustained in the second half of matches.

Conclusions—The results show that muscular injuries and injuries to the head and neck are the most commonly sustained injuries in amateur rugby league. Furthermore, injuries are more often sustained in the latter stages of the season and during the second half of matches. These findings suggest that fatigue or accumulative microtrauma, or both, may contribute to injuries in amateur rugby league players.