bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Hepatitis C Virus Proteins Interferes with the Antiviral Action of Interferon Independently of PKR-Mediated Control of Protein Synthesis
Authors: François C. , Duverlie G. , Rebouillat D. , Khorsi H. , Castelain S. , Blum H. E. , Gatignol A. , Wychowski C. , Moradpour D. , Meurs E. F.
Published: July 2002

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2α (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208–5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262–1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2α after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2α phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.
Interaction of Human Immunodeficiency Virus Type 2 Vpx and Invariant Chain
Authors: Pancio Heather A. , Vander Heyden Nancy , Kosuri Kavitha , Cresswell Peter , Ratner Lee
Published: July 2002

Vpx is a virion-associated protein of human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency viruses. The yeast two-hybrid system was used to identify invariant chain (Ii) as a cellular protein that interacts with HIV-2 Vpx. Vpx-Ii interaction was confirmed in cell-free reactions using bacterially expressed glutathioneS-transferase fusion proteins and by coimmunoprecipitation in transfected and infected cells. In chronically infected cells expressing Vpx, Ii levels were markedly decreased, presumably due to enhanced degradation. These findings suggest that Vpx may disrupt major histocompatibility complex class II antigen presentation.
The Rous Sarcoma Virus Long Terminal Repeat Promoter Is Regulated by TFII-I
Authors: Mobley Constance M. , Sealy Linda
Published: July 2002

Many viral genes contain core promoters with two basal control elements, the TATA box and the pyrimidine-rich initiator (Inr). However, the molecular mechanisms involved in transcription initiation from composite core promoters (TATA
+
Inr
+
) containing Inr elements are unclear. The Rous sarcoma virus (RSV) long terminal repeat (LTR) contains a transcriptionally potent enhancer and core promoter composed of a TATA box and an Inr-like sequence, termed the transcription start site core (TSSC). Previously we demonstrated that the TSSC binds the multifunctional Inr-binding protein YY1. Here we present evidence that the TSSC also binds the multifunctional transcription factor TFII-I and that both TFII-I and YY1 are required for RSV LTR transcriptional activity. Gel shift assays using anti-TFII-I antibody show that TFII-I is present in a protein complex that specifically binds to the TSSC. Mutations in the TSSC that reduce TFII-I binding also reduce RSV LTR enhancer and promoter activity. Transient-transfection assays demonstrate that TFII-I transactivates the RSV LTR from ca. fourfold (basal) to ca. sevenfold (enhanced) in both human and natural host cell lines. Importantly, the activity of the TSSC element can be attributed to the binding activity of TFII-I and the YY1 protein, since mutation of each of these binding sites within the TSSC element abolishes all viral expression as demonstrated by transient-transfection assays. Taken together, these data demonstrate that expression of RSV viral mRNA is dependent on both TFII-I and YY1.
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

Recent 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

In 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

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

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

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

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

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

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

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

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

Infection 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

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

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.
Isolation and Characterization of an Arterivirus Defective Interfering RNA Genome
Authors: Molenkamp Richard , Rozier Babette C. D. , Greve Sophie , Spaan Willy J. M. , Snijder Eric J.
Published: July 2002

Equine arteritis virus
(EAV), the type member of the family
Arteriviridae
, is a single-stranded RNA virus with a positive-stranded genome of approximately 13 kb. EAV uses a discontinuous transcription mechanism to produce a nested set of six subgenomic mRNAs from which its structural genes are expressed. We have generated the first documented arterivirus defective interfering (DI) RNAs by serial undiluted passaging of a wild-type EAV stock in BHK-21 cells. A cDNA copy of the smallest DI RNA (5.6 kb) was cloned. Upon transfection into EAV-infected BHK-21 cells, transcripts derived from this clone (pEDI) were replicated and packaged. Sequencing of pEDI revealed that the DI RNA was composed of three segments of the EAV genome (nucleotides 1 to 1057, 1388 to 1684, and 8530 to 12704) which were fused in frame with respect to the replicase reading frame. Remarkably, this DI RNA has retained all of the sequences encoding the structural proteins. By insertion of the chloramphenicol acetyltransferase reporter gene in the DI RNA genome, we were able to delimitate the sequences required for replication/DI-based transcription and packaging of EAV DI RNAs and to reduce the maximal size of a replication-competent EAV DI RNA to approximately 3 kb.
Uncoating Kinetics of Hepatitis A Virus Virions and Provirions
Authors: Bishop Naomi E. , Anderson David A.
Published: July 2002

When the growth kinetics of immature hepatitis A virus provirions and mature virions were monitored, distinct eclipse phases were noted for both types of particles. Strikingly, uncoating of virions occurred around 4 h postinfection, while uncoating of provirions occurred predominantly between 8 and 10 h postinfection. It is proposed that the heterogenous mixture of infectious hepatitis A virus particles (virions and provirions) typically present in inocula is responsible for the normally asynchronous nature of hepatitis A virus uncoating kinetics.
A Murine Leukemia Virus (MuLV) Long Terminal Repeat Derived from Rhesus Macaques in the Context of a Lentivirus Vector and MuLV
gag
Sequence Results in High-Level Gene Expression in Human T Lymphocytes
Authors: Kung Sam K. P. , An Dong Sung , Chen Irvin S. Y.
Published: July 2002

We constructed human immunodeficiency virus type 1 (HIV-1) vectors that will allow higher levels of gene expression in T cells. Gene expression under the control of an internal cytomegalovirus (CMV) immediate-early promoter in a self-inactivating lentiviral vector (CSCG) is 4- to 15-fold lower in T-cell lines (SUPT1 and CEMX174) than in non-lymphoid-cell lines (HeLa and 293T). This is in contrast to a Moloney murine leukemia virus (MoMLV)-based retrovirus vector (SRαLEGFP). We therefore replaced the internal CMV promoter of CSCG with three different murine oncoretroviral long terminal repeat (LTR) promoters—murine sarcoma virus (MSV), MoMLV (MLV), and the LTR (termed Rh-MLV) that is derived from the ampho-mink cell focus-forming (AMP/MCF) retrovirus in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation of enriched bone marrow stem cells transduced with a retrovirus vector preparation containing replication-competent viruses (E. F. Vanin, M. Kaloss, C. Broscius, and A. W. Nienhuis, J. Virol. 68:4241–4250, 1994). We found that the combination of Rh-MLV LTR and a partial
gag
sequence of MoMLV (Δ
gag
871–1612
) in CS-Rh-MLV-E gave the highest level of enhanced green fluorescent protein (EGFP) gene expression compared with MLV, MSV LTR, phosphoglycerate kinase, and CMV promoters in T-cell lines, as well as activated primary T cells. Interestingly, there was a further two- to threefold increase in EGFP expression (thus, 10-fold-higher expression than with CMV) when the Rh-MLV promoter and Δ
gag
871–1612
were used in a self-inactivating-vector setting that has a further deletion in the U3 region of the HIV-1 LTR. These hybrid vectors should prove useful in gene therapy applications for T cells.
Simian Immunodeficiency Virus Containing Mutations in N-Terminal Tyrosine Residues and in the PxxP Motif in Nef Replicates Efficiently in Rhesus Macaques
Authors: Carl Silke , Iafrate A. John , Lang Sabine M. , Stolte Nicole , Stahl-Hennig Christiane , Mätz-Rensing Kerstin , Fuchs Dietmar , Skowronski Jacek , Kirchhoff Frank
Published: July 2002

SIVmac Nef contains two N-terminal tyrosines that were proposed to be part of an SH2-ligand domain and/or a tyrosine-based endocytosis signal and a putative SH3-ligand domain (P104xxP107). In the present study, we investigated the effects of combined mutations in these tyrosine and proline residues on simian immunodeficiency virus (SIV) Nef interactions with the cellular signal transduction and endocytic machinery. We found that mutation of Y28F, Y39F, P104A, and P107A (FFAA-Nef) had little effect on Nef functions such as the association with the cellular tyrosine kinase Src, downregulation of cell surface expression of CD4 and class I major histocompatibility complex, and enhancement of virion infectivity. However, mutations in the PxxP sequence reduced the ability of Nef to stimulate viral replication in primary lymphocytes. Three macaques infected with the SIVmac239 FFAA-Nef variant showed high viral loads during the acute phase of infection. Reversions in the mutated prolines were observed between 12 and 20 weeks postinfection. Importantly, reversion of A107→P, which restored the ability of Nef to coprecipitate a 62-kDa phosphoprotein in in vitro kinase assays, did not precede the development of a high viral load. The Y28/Y39→F28/F39substitutions did not revert. In conclusion, mutations in both the tyrosine residues and the putative SH3 ligand domain apparently do not disrupt major aspects of SIV Nef function in vivo.
Genetic Evidence of a Role for ATM in Functional Interaction between Human T-Cell Leukemia Virus Type 1 Tax and p53
Authors: Van Phillip L. , Yim Kyong-Wook , Jin Dong-Yan , Dapolito George , Kurimasa Akihiro , Jeang Kuan-Teh
Published: July 2002

Recent evidence from several investigators suggest that the human T-cell leukemia virus type 1 Tax oncoprotein represses the transcriptional activity of the tumor suppressor protein, p53. An examination of published findings reveals serious controversy as to the mechanism(s) utilized by Tax to inhibit p53 activity and whether the same mechanism is used by Tax in adherent and suspension cells. Here, we have investigated Tax-p53 interaction simultaneously in adherent epithelial (HeLa and Saos) and suspension T-lymphocyte (Jurkat) cells. Our results indicate that Tax activity through the CREB/CREB-binding protein (CBP), but not NF-κB, pathway is needed to repress the transcriptional activity of p53 in all tested cell lines. However, we did find that while CBP binding by Tax is necessary, it is not sufficient for inhibiting p53 function. Based on knockout cell studies, we correlated a strong genetic requirement for the ATM, but not protein kinase-dependent DNA, protein in conferring a Tax-p53-repressive phenotype.
Transport of Human Immunodeficiency Virus Type 1 Pseudoviruses across the Blood-Brain Barrier: Role of Envelope Proteins and Adsorptive Endocytosis
Authors: Banks William A. , Freed Eric O. , Wolf Kathleen M. , Robinson Sandra M. , Franko Mark , Kumar Vijaya B.
Published: July 2002

Blood-borne human immunodeficiency virus type 1 (HIV-1) crosses the blood-brain barrier (BBB) to induce brain dysfunction. How HIV-1 crosses the BBB is unclear. Most work has focused on the ability of infected immune cells to cross the BBB, with less attention devoted to the study of free virus. Since the HIV-1 coat glycoprotein gp120 can cross the BBB, we postulated that gp120 might be key in determining whether free virus can cross the BBB. We used radioactive virions which do (Env+) or do not (Env−) bear the envelope proteins to characterize the ability of HIV-1 to be taken up by the murine BBB. In vivo and in vitro studies showed that the envelope proteins are key to the uptake of free virus and that uptake was enhanced by wheat germ agglutinin, strongly suggesting that the envelope proteins induce viral adsorptive endocytosis and transcytosis in brain endothelia. Capillary depletion showed that Env+virus completely crossed the vascular BBB to enter the parenchyma of the brain. Virus also entered the cerebrospinal fluid, suggesting passage across the choroid plexus as well. About 0.22% of the intravenously injected dose was taken up per g of brain. In vitro studies showed that postinternalization membrane cohesion (membrane binding not reversed with acid wash or cell lysis) was a regulated event. Intact virus was recovered from the brain endothelial cytosol and was effluxed from the endothelial cells. These results show that free HIV-1 can cross the BBB by an event related to adsorptive endocytosis and mediated by the envelope proteins.
Requirement of Interaction of Nectin-1α/HveC with Afadin for Efficient Cell-Cell Spread of Herpes Simplex Virus Type 1
Authors: Sakisaka Toshiaki , Taniguchi Tomokuni , Nakanishi Hiroyuki , Takahashi Kenichi , Miyahara Masako , Ikeda Wataru , Yokoyama Shigekazu , Peng Ying-Feng , Yamanishi Koichi , Takai Yoshimi
Published: July 2002

We recently found a novel cell-cell adhesion system at cadherin-based adherens junctions (AJs), consisting at least of nectin, a Ca
2+
-independent homophilic immunoglobulin-like adhesion molecule, and afadin, an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin is associated with cadherin through afadin and α-catenin. The cadherin-catenin system increases the concentration of nectin at AJs in an afadin-dependent manner. Nectin constitutes a family consisting of three members: nectin-1, -2, and -3. Nectin-1 serves as an entry and cell-cell spread mediator of herpes simplex virus type 1 (HSV-1). We studied here a role of the interaction of nectin-1α with afadin in entry and/or cell-cell spread of HSV-1. By the use of cadherin-deficient L cells overexpressing the full length of nectin-1α capable of interacting with afadin and L cells overexpressing a truncated form of nectin-1α incapable of interacting with afadin, we found that the interaction of nectin-1α with afadin increased the efficiency of cell-cell spread, but not entry, of HSV-1. This interaction did not affect the binding to nectin-1α of glycoprotein D, a viral component mediating entry of HSV-1 into host cells. Furthermore, the cadherin-catenin system increased the efficiency of cell-cell spread of HSV-1, although it also increased the efficiency of entry of HSV-1. It is likely that efficient cell-cell spread of HSV-1 is caused by afadin-dependent concentrated localization of nectin-1α at cadherin-based AJs.
Genotypic Correlates of Phenotypic Resistance to Efavirenz in Virus Isolates from Patients Failing Nonnucleoside Reverse Transcriptase Inhibitor Therapy
Authors: Bacheler Lee , Jeffrey Susan , Hanna George , D'Aquila Richard , Wallace Lany , Logue Kelly , Cordova Beverly , Hertogs Kurt , Larder Brendan , Buckery Renay , Baker David , Gallagher Karen , Scarnati Helen , Tritch Radonna , Rizzo Chris
Published: July 2002

Efavirenz (also known as DMP 266 or SUSTIVA) is a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity and of HIV-1 replication in vitro and in vivo. Most patients on efavirenz-containing regimens have sustained antiviral responses; however, rebounds in plasma viral load have been observed in some patients in association with the emergence of mutant strains of HIV-1. Virus isolates from the peripheral blood mononuclear cells (PBMCs) of patients with such treatment failures, as well as recombinant viruses incorporating viral sequences derived from patient plasma, show reduced in vitro susceptibility to efavirenz in association with mutations in the RT gene encoding K103N, Y188L, or G190S/E substitutions. Patterns of RT gene mutations and in vitro susceptibility were similar in plasma virus and in viruses isolated from PBMCs. Variant strains of HIV-1 constructed by site-directed mutagenesis confirmed the role of K103N, G190S, and Y188L substitutions in reduced susceptibility to efavirenz. Further, certain secondary mutations (V106I, V108I, Y181C, Y188H, P225H, and F227L) conferred little resistance to efavirenz as single mutations but enhanced the level of resistance of viruses carrying these mutations in combination with K103N or Y188L. Viruses with K103N or Y188L mutations, regardless of the initial selecting nonnucleoside RT inhibitor (NNRTI), exhibited cross-resistance to all of the presently available NNRTIs (efavirenz, nevirapine, and delavirdine). Some virus isolates from nevirapine or delavirdine treatment failures that lacked K103N or Y188L mutations remained susceptible to efavirenz in vitro, although the clinical significance of this finding is presently unclear.
The NH
2
-Terminal Domain of the Human T-Cell Leukemia Virus Type 1 Capsid Protein Is Involved in Particle Formation
Authors: Rayne Fabienne , Bouamr Fadila , Lalanne Jacqueline , Mamoun Robert Z.
Published: July 2002

The human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) capsid proteins (CA) display similar structures formed by two independently folded N-terminal (NTD) and C-terminal (CTD) domains. To characterize the functions harbored by the HTLV-1 CA domains in particle formation, 12 sites scattered throughout the protein were mutated. The effects of the mutations on Gag membrane binding, proteolytic processing, and virus-like particle secretion were analyzed. It appears that the NTD is the major partner of indirect or direct Gag-Gag interactions. In particular, most of the NTD mutations impaired virion morphogenesis, and no mutation located in the NTD could be fully rescued by coexpression of wild-type Gag. In contrast, the CTD seems not to be involved in Gag-Gag interactions. Nevertheless, an unknown function required for particle formation is located in the CTD. Thus, despite an overall structural similarity between the HIV-1 and HTLV-1 CA proteins, their NTDs and CTDs exhibit different functions.
Nasal-Associated Lymphoid Tissue Is a Site of Long-Term Virus-Specific Antibody Production following Respiratory Virus Infection of Mice
Authors: Liang Bin , Hyland Lisa , Hou Sam
Published: July 2002

Nasal immunoglobulin A provides an initial defense against inhaled respiratory pathogens. However, it is not known whether the nasal-associated lymphoid tissues (NALT) are able to mount an effective long-lasting pathogen-specific immune response, nor is it known whether functional differences exist between the organized NALT (O-NALT) and the diffuse NALT lining the nasal passages (D-NALT). Here we show that although both the O-NALT and the D-NALT are capable of producing virus-specific antibody in response to influenza virus infection, the frequency of specific antibody-forming cells in the D-NALT is much greater than the frequency observed in the O-NALT. Furthermore, we show that the D-NALT but not the O-NALT is the site of long-term virus-specific humoral immunity which lasts for the life of the animal. These results indicate that the D-NALT is not only the major effector site of the NALT but also the site of local long-term specific antibody production.
Characterization of Nuclear RNases That Cleave Hepatitis B Virus RNA near the La Protein Binding Site
Authors: Heise Tilman , Guidotti Luca G. , Chisari Francis V.
Published: July 2002

Hepatitis B virus (HBV) RNA is downregulated by inflammatory cytokines induced in the liver by adoptively transferred HBV-specific cytotoxic T lymphocytes (CTLs) and during murine cytomegalovirus (MCMV) infections of the livers of HBV transgenic mice. The disappearance of HBV RNA is tightly associated with the cytokine-induced proteolytic cleavage of a previously defined HBV RNA-binding protein known as La autoantigen. La binds to a predicted stem-loop structure at the 5′ end of the posttranscriptional regulatory element of HBV RNA between nucleotides 1243 and 1333. In the present study, we searched for nuclear RNase activities that might be involved in HBV RNA decay. Nuclear extracts derived from control livers and CTL-injected and MCMV-infected livers were analyzed for the ability to cleave HBV RNA. Endonucleolytic activity that cleaved HBV RNA at positions 1269 to 1270 and 1271 to 1272, immediately 5′ of the stem-loop bound by the La protein (positions 1272 to 1293), was detected. Furthermore, we provide evidence that the cytokine-dependent downregulation of HBV RNA following MCMV infection is temporally associated with the upregulation of the endonucleolytic activity herein described. Collectively, these results suggest a model in which the steady-state HBV RNA content is controlled by the stabilizing influence of La and the destabilizing influence of nuclear RNase activities.
Endoplasmic Reticulum andcis-Golgi Localization of Human T-Lymphotropic Virus Type 1 p12I: Association with Calreticulin and Calnexin
Authors: Ding Wei , Albrecht Björn , Luo Rushu , Zhang Weiqing , Stanley James R. L. , Newbound Garret C. , Lairmore Michael D.
Published: July 2002

Human T-lymphotropic virus type 1 (HTLV-1) is a complex retrovirus encoding regulatory and accessory genes in four open reading frames (ORF I to IV) of the pX region. We have demonstrated an important role of pX ORF I expression, which encodes p12I, in establishment of HTLV-1 infection in a rabbit model and for optimal viral infectivity in quiescent primary lymphocytes. These data indicated that p12Imay enhance lymphocyte activation and thereby promote virus infection. To further define the role of p12Iin cell activation, we characterized the subcellular localization of p12Iin transfected 293T cells and HeLa-Tat cells by multiple methods, including immunofluorescence confocal microscopy, electron microscopy, and subcellular fractionation. Herein, we demonstrate that p12Iaccumulates in the endoplasmic reticulum (ER) andcis-Golgi apparatus. The location of p12Iwas unchanged following treatments with both cycloheximide (blocking de novo protein synthesis) and brefeldin A (disrupting ER-to-Golgi protein transport), indicating that the protein is retained in the ER andcis-Golgi. Moreover, using coimmunoprecipitation assays, we identify the direct binding of p12Iwith both calreticulin and calnexin, resident ER proteins which regulate calcium storage. Our results indicate that p12Idirectly binds key regulatory proteins involved in calcium-mediated cell signaling and suggest a role of p12Iin the establishment of HTLV-1 infection by activation of host cells.
Identification of a Putative Coreceptor on Vero Cells That Participates in Dengue 4 Virus Infection
Authors: Martı́nez-Barragán José de Jesús , del Angel Rosa M.
Published: July 2002

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (α-mannose orN-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.