bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Hepatitis B Virus Polymerase Interacts with the Molecular Chaperonin Hsp60
Authors: Park Sung Gyoo , Jung Guhung 
Published: July 2002

ABSTRACT
Previous studies showed that hepatitis B virus polymerase (HBV Pol) interacts with host factors such as the Hsp90 complex, which is a critical step in viral genome replication. In this report, we propose that another chaperone, Hsp60, interacts with human HBV Pol and that this is a very important step for maturation of human HBV Pol into the active state. In the immunoprecipitation of recombinant human HBV Pol expressed in insect cells with the recombinant baculovirus expression system, the 60-kDa protein was coimmunoprecipitated with Pol and the protein was identified as Hsp60 through peptide sequencing and immunogenic analysis with an anti-Hsp60 antibody. In vitro experiments showed that Hsp60 strongly affected human HBV Pol activity in that (i) blocking of Hsp60 by the protein-specific antibody reduced human HBV Pol activity, (ii) the activity was increased by addition of Hsp60 in the presence of ATP, and (iii) ATP synergistically activated human HBV Pol with Hsp60. In vivo experiments showed that inhibition of Hsp60 in cells by a mutant Hsp60, CΔ540, resulted in the reduction of human HBV Pol activity. In summary, our results indicate that the interaction is significant for conversion of human HBV Pol into the active state.
Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented intrans
Authors: Grassmann Claus W. , Isken Olaf , Tautz Norbert , Behrens Sven-Erik 
Published: July 2002

ABSTRACTThe functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act incisor intrans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued intrans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to becisdominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.
Interaction of Classical Swine Fever Virus with Membrane-Associated Heparan Sulfate: Role for Virus Replication In Vivo and Virulence
Authors: Hulst M. M. , van Gennip H. G. P. , Vlot A. C. , Schooten E. , de Smit A. J. , Moormann R. J. M. 
Published: July 2002

ABSTRACT

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS). A Ser-to-Arg change in the C terminus of envelope glycoprotein E
rns
(amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553–9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser
476
and an HS-dependent recombinant virus (S-RT virus) with Arg
476
were constructed. Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E
rns
, E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg
476
was mutated back to Ser
476
. Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E
rns
S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by 12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.
Fine Definition of the Epitope on the gp41 Glycoprotein of Human Immunodeficiency Virus Type 1 for the Neutralizing Monoclonal Antibody 2F5
Authors: Parker Carol E. , Deterding Leesa J. , Hager-Braun Christine , Binley James M. , Schülke Norbert , Katinger Hermann , Moore John P. , Tomer Kenneth B. 
Published: July 2002

ABSTRACT
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), in combination with proteolytic protection assays, has been used to identify the functional epitope on human immunodeficiency virus envelope glycoprotein gp41 for the broadly neutralizing anti-gp41 human monoclonal antibody 2F5. In this protection assay-based procedure, a soluble gp140 protein with a stabilizing intermolecular disulfide bond between the gp120 and gp41 subunits (SOS gp140) was affinity bound to immobilized 2F5 under physiological conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Residues of SOS gp140 protected by their binding to 2F5 were then identified based on their molecular weights as determined by direct MALDI-MS of the immobilized antibody beads. The epitope, NEQELLELDKWASLWN, determined by this MALDI-MS protection assay approach consists of 16 amino acid residues near the C terminus of gp41. It is significantly longer than the ELDKWA core epitope previously determined for 2F5 by peptide enzyme-linked immunosorbent assay. This new knowledge of the structure of the 2F5 epitope may facilitate the design of vaccine antigens intended to induce antibodies with the breadth and potency of action of the 2F5 monoclonal antibody.
Gamma Interferon Can Prevent Herpes Simplex Virus Type 1 Reactivation from Latency in Sensory Neurons
Authors: Liu Ting , Khanna Kamal M. , Carriere Brian N. , Hendricks Robert L. 
Published: July 2002

ABSTRACTWe recently demonstrated that CD8+T cells could block herpes simplex virus type 1 (HSV-1) reactivation from latency in ex vivo trigeminal ganglion (TG) cultures without destroying the infected neurons. Here we establish that CD8+T-cell prevention of HSV-1 reactivation from latency is mediated at least in part by gamma interferon (IFN-γ). We demonstrate that IFN-γ was produced in ex vivo cultures of dissociated latently infected TG by CD8+T cells that were present in the TG at the time of excision. Depletion of CD8+T cells or neutralization of IFN-γ significantly enhanced the rate of HSV-1 reactivation from latency in TG cultures. When TG cultures were treated with acyclovir for 4 days to insure uniform latency, supplementation with recombinant IFN-γ blocked HSV-1 reactivation in 80% of cultures when endogenous CD8+T cells were present and significantly reduced and delayed HSV-1 reactivation when CD8+T cells or CD45+cells were depleted from the TG cultures. The effectiveness of recombinant IFN-γ in blocking HSV-1 reactivation was lost when its addition to TG cultures was delayed by more than 24 h after acyclovir removal. We propose that when the intrinsic ability of neurons to inhibit HSV-1 gene expression is compromised, HSV-specific CD8+T cells are rapidly mobilized to produce IFN-γ and perhaps other antiviral cytokines that block the viral replication cycle and maintain the viral genome in a latent state.
Porcine Encephalomyocarditis Virus Persists in Pig Myocardium and Infects Human Myocardial Cells
Authors: Brewer Laurie A. , Lwamba Humphrey C. M. , Murtaugh Michael P. , Palmenberg Ann C. , Brown Corrie , Njenga M. Kariuki 
Published: July 2002

ABSTRACTRecent advances toward using pig tissues in human transplantation have made it necessary to determine the risk of transmitting zoonotic viruses from pigs to humans or vice versa. We investigated the suitability of the porcine encephalomyocarditis virus (EMCV) model for such studies by determining its ability to persist in pigs, escape detection by routine serological methods, and infect human cells. Intraperitoneal inoculation of 5-week-old pigs with EMCV-30, a strain isolated from commercial pigs, resulted in acute cellular degeneration, infiltration of lymphocytes, and apoptosis in myocardium in 13 of 15 (86.7%) pigs during the acute phase of disease (3 to 21 days postinfection), followed by less-severe lymphocytic infiltration and apoptosis in 5 of 10 (50%) pigs during the chronic phase of the disease (day 45 to 90 postinfection). In the brain, lymphocytic infiltration, neuronal degeneration, and gliosis were observed in 26 to 33% of pigs in the acute phase of disease whereas perivascular cuffing was the predominant feature during chronic disease. EMCV antigens and RNA were demonstrated in the myocardium and brain during the chronic phase of disease. Analysis of 100 commercial pigs that were negative for EMCV antibodies identified two pig hearts positive for EMCV RNA. Porcine EMCV productively infected primary human cardiomyocytes as demonstrated by immunostaining using a monoclonal antibody specific for EMCV RNA polymerase, which is expressed only in productively infected cells, and by a one-step growth curve that showed production of 100 to 1,000 PFU of virus per cell within 6 h. The findings that porcine EMCV can persist in pig myocardium and can infect human myocardial cells make it an important infectious agent to screen for in pig-to-human cardiac transplants and a good model for xenozoonosis.
CD154 Costimulated Ovine Primary B Cells, a Cell Culture System That Supports Productive Infection by Bovine Leukemia Virus
Authors: Van den Broeke A. , Cleuter Y. , Beskorwayne T. , Kerkhofs P. , Szynal M. , Bagnis C. , Burny A. , Griebel P. 
Published: July 2002

ABSTRACTBovine leukemia virus (BLV) is closely associated with the development of B-cell leukemia and lymphoma in cattle. BLV infection has also been studied extensively in an in vivo ovine model that provides a unique system for studying B-cell leukemogenesis. There is no evidence that BLV can directly infect ovine B cells in vitro, and there are no direct data regarding the oncogenic potential of the viral Tax transactivator in B cells. Therefore, we developed ovine B-cell culture systems to study the interaction between BLV and its natural target, the B cell. In this study, we used murine CD154 (CD40 ligand) and γ-chain-common cytokines to support the growth of B cells isolated from ovine lymphoid tissues. Integrated provirus, extrachromosomal forms, and viral transcripts were detected in BLV-exposed populations of immature, rapidly dividing surface immunoglobulin M-positive B cells from sheep ileal Peyer's patches and also in activated mature B cells isolated from blood. Conclusive evidence of direct B-cell infection by BLV was obtained through the use of cloned B cells derived from sheep jejunal Peyer's patches. Finally, inoculation of sheep with BLV-infected cultures proved that infectious virus was shed from in vitro-infected B cells. Collectively, these data confirm that a variety of ovine B-cell populations can support productive infection by BLV. The development of ovine B-cell cultures permissive for BLV infection provides a controlled system for investigating B-cell leukemogenic processes and the pathogenesis of BLV infection.
Characterization of Herpes Simplex Viruses Selected in Culture for Resistance to Penciclovir or Acyclovir
Authors: Sarisky Robert T. , Quail Matthew R. , Clark Philip E. , Nguyen Tammy T. , Halsey Wendy S. , Wittrock Robert J. , Bartus Joan O'Leary , Van Horn Marion M. , Sathe Ganesh M. , Van Horn Stephanie , Kelly Michael D. , Bacon Teresa H. , Leary Jeffry J. 
Published: July 2002

ABSTRACT

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv
r
clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv
r
mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.
DNase Protection Analysis of Retrovirus Integrase at the Viral DNA Ends for Full-Site Integration In Vitro
Authors: Vora Ajaykumar , Grandgenett Duane P. 
Published: July 2002

ABSTRACTRetrovirus intasomes purified from virus-infected cells contain the linear viral DNA genome and integrase (IN). Intasomes are capable of integrating the DNA termini in a concerted fashion into exogenous target DNA (full site), mimicking integration in vivo. Molecular insights into the organization of avian myeloblastosis virus IN at the viral DNA ends were gained by reconstituting nucleoprotein complexes possessing intasome characteristics. Assembly of IN–4.5-kbp donor complexes capable of efficient full-site integration appears cooperative and is dependent on time, temperature, and protein concentration. DNase I footprint analysis of assembled IN-donor complexes capable of full-site integration shows that wild-type U3 and other donors containing gain-of-function attachment site sequences are specifically protected by IN at low concentrations (<20 nM) with a defined outer boundary mapping ∼20 nucleotides from the ends. A donor containing mutations in the attachment site simultaneously eliminated full-site integration and DNase I protection by IN. Coupling of wild-type U5 ends with wild-type U3 ends for full-site integration shows binding by IN at low concentrations probably occurs only at the very terminal nucleotides (<10 bp) on U5. The results suggest that assembly requires a defined number of avian IN subunits at each viral DNA end. Among several possibilities, IN may bind asymmetrically to the U3 and U5 ends for full-site integration in vitro.
Herpes Simplex Virus Type 1 Entry Is Inhibited by the Cobalt Chelate Complex CTC-96
Authors: Schwartz Jennifer A. , Lium Erik K. , Silverstein Saul J. 
Published: July 2002

ABSTRACT
The CTC series of cobalt chelates display in vitro and in vivo activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). The experiments described here identify the stage in the virus life cycle where CTC-96 acts and demonstrate that the drug inhibits infection of susceptible cells. CTC-96 at 50 μg/ml has no effect on adsorption of virions to Vero cell monolayers. Penetration assays reveal that CTC-96 inhibits entry of the virus independent of gC and cellular entry receptors. This observation was supported by the failure to detect the accumulation of virus-specified proteins and α mRNA transcripts when CTC-96 is present at the onset of infection. Moreover, virion-associated αTIF does not accumulate in the nucleus of cells infected in the presence of CTC-96. CTC-96 targets the initial fusion event between the virus and the cell and also inhibits cell-to-cell spread and syncytium formation. Furthermore, CTC-96 inhibits plaque formation by varicella-zoster virus and vesicular stomatitis virus as efficiently as by HSV-1. Collectively, these experiments suggest that CTC-96 is a broad-spectrum inhibitor of infection by enveloped viruses and that it inhibits HSV-1 infection at the point of membrane fusion independent of the type of virus and cellular receptors present.
Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture
Authors: Neumann Gabriele , Feldmann Heinz , Watanabe Shinji , Lukashevich Igor , Kawaoka Yoshihiro 
Published: July 2002

ABSTRACT
Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.
Early Pathogenesis of Transmucosal Feline Immunodeficiency Virus Infection
Authors: Obert Leslie A. , Hoover Edward A. 
Published: July 2002

ABSTRACTTo identify the early target cells and tissues in transmucosal feline immunodeficiency virus (FIV) infection, cats were exposed to a clade C FIV isolate via the oral-nasal or vaginal mucosa and multiple tissues were examined by virus isolation coculture (VI), DNA PCR, catalyzed tyramide signal-amplified in situ hybridization (TSA-ISH), and immunohistochemistry between days 1 and 12 postinoculation (p.i.). FIV RNA was detected in tonsil and oral or vaginal mucosa as early as 1 day p.i. by TSA-ISH and in retropharyngeal, tracheobronchial, or external iliac lymph nodes and sometimes in spleen or blood mononuclear cells by day 2, indicating that regional and distant spread of virus-infected cells occurred rapidly after mucosal exposure. By day 8, viral RNA, DNA, and culturable virus were uniformly detected in regional and distant tissues, connoting systemic infection. TSA-ISH proved more sensitive than DNA PCR in detecting early FIV-infected cells. In mucosal tissues, the earliest demonstrable FIV-bearing cells were either within or subjacent to the mucosal epithelium or were in germinal centers of regional lymph nodes. The FIV+cells were of either of two morphological types, large stellate or small round. Those FIV RNA+cells which could be colabeled for a phenotype marker, were labeled for either dendritic-cell-associated protein p55 or T-lymphocyte receptor antigen CD3. These studies indicate that FIV crosses mucous membranes within hours after exposure and rapidly traffics via dendritic and T cells to systemic lymphoid tissues, a pathway similar to that thought to occur in the initial phase of infection by the human and simian immunodeficiency viruses.
Identification and Characterization of the UL56 Gene Product of Herpes Simplex Virus Type 2
Authors: Koshizuka Tetsuo , Goshima Fumi , Takakuwa Hiroki , Nozawa Naoki , Daikoku Tohru , Koiwai Osamu , Nishiyama Yukihiro 
Published: July 2002

ABSTRACT
The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.