bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Kinetic Analyses of the Protease from an Amprenavir-Resistant Human Immunodeficiency Virus Type 1 Mutant Rendered Resistant to Saquinavir and Resensitized to Amprenavir
Authors: Markland W. , Rao B. G. , Parsons J. D. , Black J. , Zuchowski L. , Tisdale M. , Tung R. 
Published: July 2002

ABSTRACTRecent drug regimens have had much success in the treatment of human immunodeficiency virus (HIV)-infected individuals; however, the incidence of resistance to such drugs has become a problem that is likely to increase in importance with long-term therapy of this chronic illness. An analysis and understanding of the molecular interactions between the drug(s) and the mutated viral target(s) is crucial for further progress in the field of AIDS therapy. The protease inhibitor amprenavir (APV) generates a signature set of HIV type 1 (HIV-1) protease mutations associated with in vitro resistance (M46I/L, I47V, and I50V [triple mutant]). Passage of the triple-mutant APV-resistant HIV-1 strain in MT4 cells, in the presence of increasing concentrations of saquinavir (SQV), gave rise to a new variant containing M46I, G48V, I50V, and I84L mutations in the protease and a resulting phenotype that was resistant to SQV and, unexpectedly, resensitized to APV. This phenotype was consistent with a subsequent kinetic analysis of the mutant protease, together with X-ray crystallographic analysis and computational modeling which elucidated the structural basis of these observations. The switch in protease inhibitor sensitivities resulted from (i) the I50V mutation, which reduced the area of contact with APV and SQV; (ii) the compensating I84L mutation, which improved hydrophobic packing with APV; and (iii) the G-to-V mutation at residue 48, which introduced steric repulsion with the P3 group of SQV. This analysis establishes the fine detail necessary for understanding the loss of protease binding for SQV in the quadruple mutant and gain in binding for APV, demonstrating the powerful combination of virology, molecular biology, enzymology, and protein structural and modeling studies in the elucidation and understanding of viral drug resistance.
Characterization and Epitope Mapping of Neutralizing Monoclonal Antibodies Produced by Immunization with Oligomeric Simian Immunodeficiency Virus Envelope Protein
Authors: Edinger Aimee L. , Ahuja Ména , Sung Tina , Baxter Kelly C. , Haggarty Beth , Doms Robert W. , Hoxie James A. 
Published: July 2002

ABSTRACTIn an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmΔB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmΔB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.
ATP Is Required for Correct Folding and Disulfide Bond Formation of Rotavirus VP7
Authors: Mirazimi Ali , Svensson Lennart 
Published: July 2002

ABSTRACT
Rotavirus is one of very few viruses that utilize the endoplasmic reticulum (ER) for assembly, and therefore it has been used as an attractive model to study ER-associated protein folding. In this study, we have examined the requirements for metabolic energy (ATP) for correct folding of the luminal and ER-associated VP7 of rotavirus. We found that VP7 rapidly misfolds in an energy-depleted milieu and is not degraded within 60 min. We also found that VP7 attained a stable minimum-energy state soon after translation in the ER. Most surprisingly, energy-misfolded VP7 could be recovered and establish correct disulfide bonds and antigenicity following a shift to an ATP-rich milieu. Using a Semliki Forest virus expression system, we observed that VP7 requires ATP and cellular, but not viral, factors for correct disulfide bond formation. Our results show for the first time that the disulfide bond formation of rotavirus VP7 is an ATP-dependent process. It has previously been shown that chaperones hydrolyze ATP during interaction with newly synthesized polypeptides and prevent nonproductive intra- and intermolecular interactions. The most reasonable explanation for the energy requirement of VP7 is thus a close interaction during folding with an ATP-dependent chaperone, such as BiP (Grp78), and possibly with protein disulfide isomerase. Taken together, our observations provide new information about folding of ER-associated proteins in general and rotavirus VP7 in particular.
Intercellular Transfer of a Soluble Viral Superantigen
Authors: Reilly Melissa , Mix Denise , Reilly Andrew A. , Yang Ye Xiang , Winslow Gary M. 
Published: July 2002

ABSTRACT

Mouse mammary tumor virus (MMTV) superantigens (vSAgs) can undergo intercellular transfer in vivo and in vitro such that a vSAg can be presented to T cells by major histocompatibility complex (MHC) class II proteins on antigen-presenting cells (APCs) that do not express the superantigen. This process may allow T-cell activation to occur prior to viral infection. Consistent with these findings, vSAg produced by Chinese hamster ovary (CHO) cells was readily transferred to class II IE and IA (

H-2
k

and

H-2
d

) proteins on a B-cell lymphoma or mouse splenocytes. Fixed class II-expressing acceptor cells were used to demonstrate that the vSAg, but not the class II proteins, underwent intercellular transfer, indicating that vSAg binding to class II MHC could occur directly at the cell surface. Intercellular transfer also occurred efficiently to splenocytes from endogenous retrovirus-free mice, indicating that other proviral proteins were not involved. Presentation of vSAg7 produced by a class II-negative, furin protease-deficient CHO variant (FD11) was unsuccessful, indicating that proteolytic processing was a requisite event and that proteolytic activity could not be provided by an endoprotease on the acceptor APC. Furthermore, vSAg presentation was effected using cell-free supernatant from class II-negative, vSAg-positive cells, indicating that a soluble molecule, most likely produced by proteolytic processing, was sufficient to stimulate T cells. Because the membrane-proximal endoproteolytic cleavage site in the vSAg (residues 68 to 71) was not necessary for intercellular transfer, the data support the notion that the carboxy-terminal endoproteolytic cleavage product is an active vSAg moiety.
Activation of the Interferon-Inducible 2′-5′-Oligoadenylate Synthetase Gene by Hepatitis C Virus Core Protein
Authors: Naganuma Atsushi , Nozaki Akito , Tanaka Torahiko , Sugiyama Kazuo , Takagi Hitoshi , Mori Masatomo , Shimotohno Kunitada , Kato Nobuyuki 
Published: July 2002

ABSTRACT
The effects of hepatitis C virus (HCV) proteins on several signal transduction pathways in human nonneoplastic hepatocyte PH5CH8 cells were investigated using expression vectors encoding HCV proteins derived from HCV-infected human nonneoplastic cultured T-lymphocyte and hepatocyte cells (MT-2C and PH5CH7), which could support HCV replication. The amino acid sequences of HCV proteins obtained from HCV-infected human cells were identical or very close to the consensus sequences of the proteins derived from the original inoculum used for HCV infection. During the course of the study, we found that HCV core protein specifically activated the 40/46-kDa 2′-5′-oligoadenylate synthetase (2′-5′-OAS) gene promoter in a dose-dependent manner in different human hepatocyte cell lines (PH5CH8, HepG2, and PLC/PRF/5). We also found that the activation by core protein was further enhanced in the cells treated with alpha interferon. The expression of E1 or E2 envelope protein or nonstructural NS5A protein did not activate the 2′-5′-OAS gene promoter. We demonstrated that the activation by core protein in the hepatocyte cells was suppressed by antisense RNA complementary to core-encoding RNA. Deletion mutant analysis of core protein and deletion analysis of the 2′-5′-OAS gene promoter have been performed. Finally, we demonstrated that the activation of the 2′-5′-OAS gene occurred at the transcriptional level and furthermore demonstrated that the endogenous 2′-5′-OAS gene was also activated by core protein. This is the first report to show that a viral protein activated the 2′-5′-OAS gene.
Herpes Simplex Virus 1 Open Reading Frames O and P Are Not Necessary for Establishment of Latent Infection in Mice
Authors: Randall Glenn , Lagunoff Michael , Roizman Bernard 
Published: July 2002

ABSTRACT

Open reading frame (ORF) O and ORF P partially overlap and are located antisense to the γ
1
34.5 gene within the domain transcribed during latency. In wild-type virus-infected cells, ORF O and ORF P are completely repressed during productive infection by ICP4, the major viral transcriptional activator/repressor. In cells infected with a mutant in which ORF P was derepressed there was a significant delay in the appearance of the viral α-regulatory proteins ICP0 and ICP22. The ORF O protein binds to and inhibits ICP4 binding to its cognate DNA site in vitro. These characteristics suggested a role for ORF O and ORF P in the establishment of latency. To test this hypothesis, two recombinant viruses were constructed. In the first, R7538(P−/O−), the ORF P initiator methionine codon, which also serves as the initiator methionine codon for ORF O, was replaced and a diagnostic restriction endonuclease was introduced upstream. In the second, R7543(P−/O−)R, the mutations were repaired to restore the wild-type virus sequences. We report the following. (i) The R7538(P−/O−) mutant failed to express ORF O and ORF P proteins but expressed a wild-type γ
1
34.5 protein. (ii) R7538(P−/O−) yields were similar to that of the wild type following infection of cell culture or following infection of mice by intracerebral or ocular routes. (iii) R7538(P−/O−) virus reactivated from latency following explanation and cocultivation of murine trigeminal ganglia with Vero cells at a frequency similar to that of the wild type, herpes simplex virus 1(F). (iv) The amount of latent R7538(P−/O−) virus as assayed by quantitative PCR is eightfold less than that of the repair virus. The repaired virus could not be differentiated from the wild-type parent in any of the assays done in this study. We conclude that ORF O and ORF P are not essential for the establishment of latency in mice but may play a role in determining the quantity of latent virus maintained in sensory neurons.
Adeno-Associated Virus Type 2 Rep Protein Inhibits Human Papillomavirus Type 16 E2 Recruitment of the Transcriptional Coactivator p300
Authors: Marcello Alessandro , Massimi Paola , Banks Lawrence , Giacca Mauro 
Published: July 2002

ABSTRACT
Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300.
Characterization of an Internal Ribosomal Entry Segment in the 5′ Leader of Murine Leukemia Virus
env
RNA
Authors: Deffaud Clarence , Darlix Jean-Luc 
Published: July 2002

ABSTRACT

The 5′ untranslated region, also called the leader, of oncoretroviruses and lentiviruses is long and is formed of several structured domains critically important in virus replication. The 5′ leader of murine leukemia virus (MLV) RNA contains an internal ribosomal entry segment (IRES) which promotes synthesis of Gag and glyco-Gag polyprotein precursors. In the present study we investigated the translational features of the 5′ leader of MLV subgenomic RNA (
env
RNA) encoding the Env polyprotein precursor. When the
env
leader was inserted between two genes, such as
lacZ
and the neomycin resistance cassette, in a dicistronic vector, it allowed IRES-dependent translation of the 3′ cistron in the rabbit reticulocyte lysate system and in murine cells. The drug rapamycin and the foot-and-mouth disease virus L protease, known to inhibit cap-dependent translation, caused an enhancement of the translation driven by the
env
leader sequence, consistent with an IRES activity promoting Env expression. Analysis of several deletion mutants led us to localize the minimal
env
IRES between the splice junction and the
env
AUG start codon.
High-Mobility-Group Protein I Can Modulate Binding of Transcription Factors to the U5 Region of the Human Immunodeficiency Virus Type 1 Proviral Promoter
Authors: Henderson Angus , Bunce Michael , Siddon Nicole , Reeves Raymond , Tremethick David John 
Published: July 2002

ABSTRACT
HMG I/Y appears to be a multifunctional protein that relies on in its ability to interact with DNA in a structure-specific manner and with DNA, binding transcriptional activators via distinct protein-protein interaction surfaces. To investigate the hypothesis that HMG I/Y may have a role in human immunodeficiency virus type 1 (HIV-1) expression, we have analyzed whether HMG I/Y interacts with the 5′ long terminal repeat and whether this interaction can modulate transcription factor binding. Using purified recombinant HMG I, we have identified several high-affinity binding sites which overlap important transcription factor binding sites. One of these HMG I binding sites coincides with an important binding site for AP-1 located downstream of the transcriptional start site, in the 5′ untranslated region at the boundary of a positioned nucleosome. HMG I binding to this composite site inhibits the binding of recombinant AP-1. Consistent with this observation, using nuclear extracts prepared from Jurkat T cells, we show that HMG I (but not HMG Y) is strongly induced upon phorbol myristate acetate stimulation and this induced HMG I appears to both selectively inhibit the binding of basal DNA-binding proteins and enhance the binding of an inducible AP-1 transcription factor to this AP-1 binding site. We also report the novel finding that a component present in this inducible AP-1 complex is ATF-3. Taken together, these results argue that HMG I may play a fundamental role in HIV-1 expression by determining the nature of transcription factor-promoter interactions.
A Human Nuclear Shuttling Protein That Interacts with Human Immunodeficiency Virus Type 1 Matrix Is Packaged into Virions
Authors: Gupta Kalpana , Ott David , Hope Thomas J. , Siliciano Robert F. , Boeke Jef D. 
Published: July 2002

ABSTRACT

Active nuclear import of the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC) is essential for the productive infection of nondividing cells. Nuclear import of the PIC is mediated by the HIV-1 matrix protein, which also plays several critical roles during viral entry and possibly during virion production facilitating the export of Pr55
Gag
and genomic RNA. Using a yeast two-hybrid screen, we identified a novel human virion-associated matrix-interacting protein (VAN) that is highly conserved in vertebrates and expressed in most human tissues. Its expression is upregulated upon activation of CD4
+
T cells. VAN is efficiently incorporated into HIV-1 virions and, like matrix, shuttles between the nucleus and cytoplasm. Furthermore, overexpression of VAN significantly inhibits HIV-1 replication in tissue culture. We propose that VAN regulates matrix nuclear localization and, by extension, both nuclear import of the PIC and export of Pr55
Gag
and viral genomic RNA during virion production. Our data suggest that this regulatory mechanism reflects a more global process for regulation of nucleocytoplasmic transport.
A Central Role for CD4+T Cells and RANTES in Virus-Induced Central Nervous System Inflammation and Demyelination
Authors: Lane Thomas E. , Liu Michael T. , Chen Benjamin P. , Asensio Valérie C. , Samawi Roger M. , Paoletti Alyssa D. , Campbell Iain L. , Kunkel Stephen L. , Fox Howard S. , Buchmeier Michael J. 
Published: July 2002

ABSTRACTInfection of C57BL/6 mice with mouse hepatitis virus (MHV) results in a demyelinating encephalomyelitis characterized by mononuclear cell infiltration and white matter destruction similar to the pathology of the human demyelinating disease multiple sclerosis. The contributions of CD4+and CD8+T cells in the pathogenesis of the disease were investigated. Significantly less severe inflammation and demyelination were observed in CD4−/−mice than in CD8−/−and C57BL/6 mice (P≤ 0.002 andP≤ 0.001, respectively). Immunophenotyping of central nervous system (CNS) infiltrates revealed that CD4−/−mice had a significant reduction in numbers of activated macrophages/microglial cells in the brain compared to the numbers in CD8−/−and C57BL/6 mice, indicating a role for these cells in myelin destruction. Furthermore, CD4−/−mice displayed lower levels of RANTES (a C-C chemokine) mRNA transcripts and protein, suggesting a role for this molecule in the pathogenesis of MHV-induced neurologic disease. Administration of RANTES antisera to MHV-infected C57BL/6 mice resulted in a significant reduction in macrophage infiltration and demyelination (P≤ 0.001) compared to those in control mice. These data indicate that CD4+T cells have a pivotal role in accelerating CNS inflammation and demyelination within infected mice, possibly by regulating RANTES expression, which in turn coordinates the trafficking of macrophages into the CNS, leading to myelin destruction.
Novel Intertypic Recombinants of Epstein-Barr Virus in the Chinese Population
Authors: Midgley R. S. , Blake N. W. , Yao Q. Y. , Croom-Carter D. , Cheung S. T. , Leung S. F. , Chan A. T. C. , Johnson P. J. , Huang D. , Rickinson A. B. , Lee S. P. 
Published: July 2002

ABSTRACT
Among 34 Epstein-Barr virus isolates from nonimmunocompromised Chinese donors, we identified three intertypic recombinants with type 1 sequences at the EBNA2 locus and type 2 sequences at some or all of the EBNA3A, -3B, and -3C loci. These appear to have arisen from independent, evolutionarily recent recombination events; such events may be commoner in nonimmunocompromised populations than hitherto imagined.
Cellular Uptake and Infection by Canine Parvovirus Involves Rapid Dynamin-Regulated Clathrin-Mediated Endocytosis, Followed by Slower Intracellular Trafficking
Authors: Parker John S. L. , Parrish Colin R. 
Published: July 2002

ABSTRACT
Canine parvovirus (CPV) is a small, nonenveloped virus that is a host range variant of a virus which infected cats and changes in the capsid protein control the ability of the virus to infect canine cells. We used a variety of approaches to define the early stages of cell entry by CPV. Electron microscopy showed that virus particles concentrated within clathrin-coated pits and vesicles early in the uptake process and that the infecting particles were rapidly removed from the cell surface. Overexpression of a dominant interfering mutant of dynamin in the cells altered the trafficking of capsid-containing vesicles. There was a 40% decrease in the number of CPV-infected cells in mutant dynamin-expressing cells, as well as a ∼40% decrease in the number of cells in S phase of the cell cycle, which is required for virus replication. However, there was also up to 10-fold more binding of CPV to the surface of mutant dynamin-expressing cells than there was to uninduced cells, suggesting an increased receptor retention on the cell surface. In contrast, there was little difference in virus binding, virus infection rate, or cell cycle distribution between induced and uninduced cells expressing wild-type dynamin. CPV particles colocalized with transferrin in perinuclear endosomes but not with fluorescein isothiocyanate-dextran, a marker for fluid-phase endocytosis. Cells treated with nanomolar concentrations of bafilomycin A1 were largely resistant to infection when the drug was added either 30 min before or 90 min after inoculation, suggesting that there was a lag between virus entering the cell by clathrin-mediated endocytosis and escape of the virus from the endosome. High concentrations of CPV particles did not permeabilize canine A72 or mink lung cells to α-sarcin, but canine adenovirus type 1 particles permeabilized both cell lines. These data suggest that the CPV entry and infection pathway is complex and involves multiple vesicular components.