bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activation and Inhibition of Cellular Calcium and Tyrosine Kinase Signaling Pathways Identify Targets of the HBx Protein Involved in Hepatitis B Virus Replication
Authors: Bouchard Michael J. , Puro Robyn J. , Wang Lihua , Schneider Robert J. 
Published: June 2003

ABSTRACT
Human hepatitis B virus (HBV) HBx protein is a multifunctional protein that activates cellular signaling pathways and is thought to be essential for viral infection. Woodchuck HBV mutants that lack HBx are unable to replicate in vivo or are severely impaired. HBV replication in HepG2 cells, a human hepatoblastoma cell line, is stimulated 5- to 10-fold by HBx protein. We have utilized the HepG2, HBx-dependent HBV replication system to study the effects of activators and inhibitors of cytosolic calcium and tyrosine kinase signaling pathways on viral replication. By transfecting either a wild-type HBV genome or an HBV genome that does not express HBx and then treating transfected cells with activators or inhibitors of signaling pathways, we identified compounds that either impair wild-type HBV replication or rescue HBx-deficient HBV replication. Geldanamycin or herbimycin A, tyrosine kinase inhibitors, blocked HBV replication. Derivatives of cyclosporine, i.e., cyclosporine A, cyclosporine H, and SDZ NIM811, which block cytosolic calcium signaling and specifically the mitochondrial permeability transition pore (SDZ NIM811), also impaired HBV replication. Treatment of cells with compounds that increase cytosolic calcium levels by a variety of mechanisms rescued replication of an HBx-deficient HBV mutant. Transcription of viral RNA and production of viral capsids were only minimally affected by these treatments. These results define a functional signaling circuit for HBV replication that includes calcium signaling and activation of cytosolic signaling pathways involving Src kinases, and they suggest that these pathways are stimulated by HBx acting on the mitochondrial transition pore.
Tax and Overlapping Rex Sequences Do Not Confer the Distinct Transformation Tropisms of Human T-Cell Leukemia Virus Types 1 and 2
Authors: Ye Jianxin , Xie Li , Green Patrick L. 
Published: June 2003

ABSTRACT

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are distinct oncogenic retroviruses that infect several cell types but display their biological and pathogenic activity only in T cells. Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4
+
T cells, whereas HTLV-2 in vivo tropism is less clear but appears to favor CD8
+
T cells. Both CD4
+
and CD8
+
T cells are susceptible to HTLV-1 and HTLV-2 infection in vitro, and HTLV-1 has a preferential immortalization and transformation tropism of CD4
+
T cells, whereas HTLV-2 immortalizes and transforms primarily CD8
+
T cells. The molecular mechanism that determines this tropism of HTLV-1 and HTLV-2 has not been determined. HTLV-1 and HTLV-2 carry the
tax
and
rex
transregulatory genes in separate but partially overlapping reading frames. Since Tax has been shown to be critical for cellular transformation in vitro and interacts with numerous cellular processes, we hypothesized that the viral determinant of transformation tropism is encoded by
tax
. Using molecular clones of HTLV-1 (Ach) and HTLV-2 (pH6neo), we constructed recombinants in which
tax
and overlapping
rex
genes of the two viruses were exchanged. p19 Gag expression from proviral clones transfected into 293T cells indicated that both recombinants contained functional Tax and Rex but with significantly altered activity compared to the wild-type clones. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells. Both recombinants were competent to transform T lymphocytes with an efficiency similar to that of the parental viruses. Flow cytometry analysis indicated that HTLV-1 and HTLV-1/TR2 had a preferential tropism for CD4
+
T cells and that HTLV-2 and HTLV-2/TR1 had a preferential tropism for CD8
+
T cells. Our results indicate that
tax/rex
in different genetic backgrounds display altered functional activity but ultimately do not contribute to the different in vitro transformation tropisms. This first study with recombinants between HTLV-1 and HTLV-2 is the initial step in elucidating the different pathobiologies of HTLV-1 and HTLV-2.
Infectivity and Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1 Variants Isolated during Primary Infection
Authors: Simon Viviana , Padte Neal , Murray Deya , Vanderhoeven Jeroen , Wrin Terri , Parkin Neil , Di Mascio Michele , Markowitz Martin 
Published: June 2003

ABSTRACT

It is believed that replication capacity is an important determinant of human immunodeficiency virus type 1 (HIV-1) pathogenicity and transmissibility. To explore this, we conducted a comprehensive analysis of the replication properties of nine drug-resistant and nine drug-susceptible viral isolates derived from patients with primary HIV-1 infection. Viral isolates were tested for single-cycle infectivity in the GHOST cell line. The infectivity of isolates carrying resistance-associated mutations was significantly higher than that of drug-susceptible isolates. Additionally, the growth kinetics of these isolates were determined in CD4
+
T lymphocytes. Drug-resistant isolates replicated as well as drug-susceptible viruses. Insertion of the resistance-conferring regions into an NL4-3-based molecular background resulted in chimeras that displayed a modest but significant reduction in replication capacity compared to the drug-susceptible chimeric viruses. Of note, two multidrug-resistant isolates and one protease inhibitor-resistant isolate displayed higher rates of infectivity and growth kinetics than the other drug-resistant or drug-susceptible isolates. These distinct replicative features, however, were not seen in the corresponding chimeras, indicating that changes within the C-terminal region of Gag as well as within the protease and reverse transcriptase genes contribute to but are not sufficient for the level of compensatory adaptation observed. These findings suggest that some drug-resistant viruses isolated during primary infection possess unique adaptive changes that allow for both high viral replication capacity and resistance to one or more classes of antiretroviral drugs. Further studies are needed to elucidate the precise regions that are essential for these characteristics.
Quantitative Analysis of Long-Term Virus-Specific CD8+-T-Cell Memory in Mice Challenged with Unrelated Pathogens
Authors: Liu Haiyan , Andreansky Samita , Diaz Gabriela , Turner Stephen J. , Wodarz Dominik , Doherty Peter C. 
Published: June 2003

ABSTRACTThe consequences for the long-term maintenance of virus-specific CD8+-T-cell memory have been analyzed experimentally for sequential respiratory infections with readily eliminated (influenza virus) and persistent (gammaherpesvirus 68 [γHV68]) pathogens. Sampling a broad range of tissue sites established that the numbers of CD8+T cells specific for the prominent influenza virus DbNP366epitope were reduced by about half in mice that had been challenged 100 days previously with γHV68, though the prior presence of a large CD8+DbNP366+population caused no selective defect in the γHV68-specific CD8+Kbp79+response. Conversely, mice that had been primed and boosted to generate substantial γHV68-specific CD8+Dbp56+populations did not show any decrease in prevalence for this set of CD8+memory cytotoxic T lymphocytes (CTL) at 200 days after respiratory exposure to an influenza A virus. However, in both experiments, the total magnitude of the CD8+-T-cell pool was significantly diminished in those that had been infected with γHV68 and the influenza A virus. The broader implications of these findings, especially under conditions of repeated exposure to unrelated pathogens, are explored with a mathematical model which emphasizes that the immune effector and memory “phenome” is a function of the overall infection experience of the individual.
Mutations within the ADP (E3-11.6K) Protein Alter Processing and Localization of ADP and the Kinetics of Cell Lysis of Adenovirus-Infected Cells
Authors: Tollefson Ann E. , Scaria Abraham , Ying Baoling , Wold William S. M. 
Published: June 2003

ABSTRACTADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic NendoCexonuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2adpgene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.
Kunjin Virus Replicon Vectors for Human Immunodeficiency Virus Vaccine Development
Authors: Harvey Tracey J. , Anraku Itaru , Linedale Richard , Harrich David , Mackenzie Jason , Suhrbier Andreas , Khromykh Alexander A. 
Published: June 2003

ABSTRACT

We have previously demonstrated the ability of the vaccine vectors based on replicon RNA of the Australian flavivirus Kunjin (KUN) to induce protective antiviral and anticancer CD8
+
T-cell responses using murine polyepitope as a model immunogen (I. Anraku, T. J. Harvey, R. Linedale, J. Gardner, D. Harrich, A. Suhrbier, and A. A. Khromykh, J. Virol. 76:3791-3799, 2002). Here we showed that immunization of BALB/c mice with KUN replicons encoding HIV-1 Gag antigen resulted in induction of both Gag-specific antibody and protective Gag-specific CD8
+
T-cell responses. Two immunizations with KUN
gag
replicons in the form of virus-like particles (VLPs) induced anti-Gag antibodies with titers of ≥1:10,000. Immunization with KUN
gag
replicons delivered as plasmid DNA, naked RNA, or VLPs induced potent Gag-specific CD8
+
T-cell responses, with one immunization of KUN
gag
VLPs inducing 4.5-fold-more CD8
+
T cells than the number induced after immunization with recombinant vaccinia virus carrying the
gag
gene (rVV
gag
). Two immunizations with KUN
gag
VLPs also provided significant protection against challenge with rVV
gag
. Importantly, KUN replicon VLP vaccinations induced long-lasting immune responses with CD8
+
T cells able to secrete gamma interferon and to mediate protection 6 to 10 months after immunization. These results illustrate the potential value of the KUN replicon vectors for human immunodeficiency virus vaccine design.
Molecular and Functional Analyses of Kunjin Virus Infectious cDNA Clones Demonstrate the Essential Roles for NS2A in Virus Assembly and for a Nonconservative Residue in NS3 in RNA Replication
Authors: Liu Wen Jun , Chen Hua Bo , Khromykh Alexander A. 
Published: June 2003

ABSTRACT

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by ∼100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an ∼5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type Ile residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in
trans
by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in
trans
by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.
An Infectious Clone of Woolly Monkey Hepatitis B Virus
Authors: Lanford Robert E. , Chavez Deborah , Barrera Azeneth , Brasky Kathleen M. 
Published: June 2003

ABSTRACT

Members of the
Hepadnaviridae
family have been isolated from birds, rodents, and primates. A new hepadnavirus isolated from the woolly monkey, a New World primate, is phylogenetically distinct from other primate isolates. An animal model has been established for woolly monkey hepatitis B virus (WMHBV) by using spider monkeys, since woolly monkeys are endangered. In this study, a greater-than-genome length construct was prepared without amplification by using covalently closed circular DNA extracted from the liver of an infected woolly monkey. Transfection of the human liver cell line Huh7 with WMHBV DNA resulted in the production of viral transcripts, DNA replicative intermediates, and secreted virions at levels similar to those obtained with an infectious human HBV clone, demonstrating that the host range restriction of WMHBV is not at the level of genome replication. WMHBV particles from the medium of transfected cultures initiated an infection in a spider monkey similar to that obtained with virions derived from woolly monkey serum. In an attempt to adapt the virus for higher levels of replication in spider monkeys, immunosuppressed and newborn animals were inoculated. Neither procedure produced persistent infections, and the level of viral replication remained several logs lower than that observed in persistently infected woolly monkeys. These data demonstrate the production of an infectious clone for WMHBV and extend the characterization of the spider monkey animal model.
Drug Resistance Patterns of Recombinant Herpes Simplex Virus DNA Polymerase Mutants Generated with a Set of Overlapping Cosmids and Plasmids
Authors: Bestman-Smith Julie , Boivin Guy 
Published: June 2003

ABSTRACT

Herpes simplex virus (HSV) DNA polymerase (Pol) mutations can confer resistance to all currently available antiherpetic drugs. However, discrimination between mutations responsible for drug resistance and those that are part of viral polymorphism can be difficult with current methodologies. A new system is reported for rapid generation of recombinant HSV type 1 (HSV-1) DNA Pol mutants based on transfection of a set of overlapping viral cosmids and plasmids. With this approach, twenty HSV-1 recombinants with single or dual mutations within the DNA
pol
gene were successfully generated and subsequently evaluated for their susceptibilities to acyclovir (ACV), foscarnet (FOS), cidofovir (CDV), and adefovir (ADV). Mutations within DNA Pol conserved regions II (A719T and S724N), VI (L778M, D780N, and L782I), and I (F891C) were shown to induce cross-resistance to ACV, FOS, and ADV, with two of these mutations (S724N and L778M) also conferring significant reduction in CDV susceptibility. Mutant F891C was associated with the highest levels of resistance towards ACV and FOS and was strongly impaired in its replication capacity. One mutation (D907V) lying outside of the conserved regions was also associated with this ACV-, FOS-, and ADV-resistant phenotype. Some mutations (K522E and Y577H) within the δ-region C were lethal, whereas others (P561S and V573M) induced no resistance to any of the drugs tested. Recombinants harboring mutations within conserved regions V (N961K) and VII (Y941H) were resistant to ACV but susceptible to FOS. Finally, mutations within conserved region III were associated with various susceptibility profiles. This new system allows a rapid and accurate evaluation of the functional role of various DNA Pol mutations, which should translate into improved management of drug-resistant HSV infections.
Involvement of Lipids in Different Steps of the Flavivirus Fusion Mechanism
Authors: Stiasny Karin , Koessl Christian , Heinz Franz X. 
Published: June 2003

ABSTRACT
Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and—as shown by the use of cholesterol analogs—the underlying interactions involve the 3β-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses.
The Tailless Icosahedral Membrane Virus PRD1 Localizes the Proteins Involved in Genome Packaging and Injection at a Unique Vertex
Authors: Gowen Brent , Bamford Jaana K. H. , Bamford Dennis H. , Fuller Stephen D. 
Published: June 2003

ABSTRACT
The double-stranded DNA (dsDNA) virus PRD1 carries its genome in a membrane surrounded by an icosahedral protein shell. The shell contains 240 copies of the trimeric P3 protein arranged with a pseudo T = 25 triangulation that is reminiscent of the mammalian adenovirus. DNA packaging and infection are believed to occur through the vertices of the particle. We have used immunolabeling to define the distribution of proteins on the virion surface. Antibodies to protein P3 labeled the entire surface of the virus. Most of the 12 vertices labeled with antibodies directed against proteins P5, P2, and P31. These proteins are known to function in virus binding to the cell surface. Proteins P6, P11, and P20 were found on a single vertex per virion. The P6 and P20 proteins are believed to function in DNA packaging. Protein P11 is a pilot protein that is involved in a complex that mediates the early stages of DNA entry to the host cell. Labeling with antibodies to P5 or P2 did not affect the labeling of P6, the unique vertex protein. Labeling with antibodies to the unique vertex protein P6 interfered with the labeling by antibodies to the unique vertex protein P20. We conclude that PRD1 utilizes 11 of its vertices for initial receptor binding. It utilizes a single, unique vertex for both DNA packing during assembly and DNA delivery during infection.
Increased Thymic Output during Acute Measles Virus Infection
Authors: Permar Sallie R. , Moss William J. , Ryon Judith J. , Douek Daniel C. , Monze Mwaka , Griffin Diane E. 
Published: June 2003

ABSTRACTMeasles virus infects thymic epithelia, induces a transient lymphopenia, and impairs cell-mediated immunity, but thymic function during measles has not been well characterized. Thirty Zambian children hospitalized with measles were studied at entry, hospital discharge, and at 1-month follow-up and compared to 17 healthy children. During hospitalization, percentages of naïve (CD62L+, CD45RA+) CD4+and CD8+T lymphocytes decreased (P= 0.01 for both), and activated (HLA-DR+, CD25+, or CD69+) CD4+and CD8+T lymphocytes increased (P= 0.02 and 0.03, respectively). T-cell receptor rearrangement excision circles (TRECs) in measles patients were increased in CD8+T cells at entry compared to levels at hospital discharge (P =0.02) and follow-up (P =0.04). In CD4+T cells, the increase in TRECS occurred later but was more sustained. At discharge, TRECs in CD4+T cells (P= 0.05) and circulating levels of interleukin-7 (P= 0.007) were increased compared to control values and remained elevated for 1 month, similar to observations in two measles virus-infected rhesus monkeys. These findings suggest that a decrease in thymic output is not the cause of the lymphopenia and depressed cellular immunity associated with measles.