bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Herpesvirus 8-Encoded vGPCR Activates Nuclear Factor of Activated T Cells and Collaborates with Human Immunodeficiency Virus Type 1 Tat
Authors: Pati Shibani , Foulke James S. , Barabitskaya Oxana , Kim Jynho , Nair B. C. , Hone David , Smart Jennifer , Feldman Ricardo A. , Reitz Marvin 
Published: April 2003

ABSTRACTHuman herpesvirus 8 (HHV-8), the etiologic agent of Kaposi's sarcoma (KS), encodes a chemokine receptor homologue, the viral G protein-coupled receptor (vGPCR), that has been implicated in KS pathogenesis. Expression of vGPCR constitutively activates several signaling pathways, including NF-κB, and induces the expression of proinflammatory and angiogenic factors, consistent with the inflammatory hyperproliferative nature of KS lesions. Here we show that vGPCR also constitutively activates the nuclear factor of activated T cells (NF-AT), another transcription factor important in regulation of the expression of inflammatory cytokines and related factors. NF-AT activation by vGPCR depended upon signaling through the phosphatidylinositol 3-kinase-Akt-glycogen synthetase kinase 3 (PI3-K/Akt/GSK-3) pathway and resulted in increased expression of NF-AT-dependent cell surface molecules (CD25, CD29, Fas ligand), proinflammatory cytokines (interleukin-2 [IL-2], IL-4), and proangiogenic factors (granulocyte-macrophage colony-stimulating factor GMCSF and TNFα). vGPCR expression also increased endothelial cell-T-cell adhesion. Although infection with HHV-8 is necessary to cause KS, coinfection with human immunodeficiency virus type 1 (HIV-1), in the absence of antiretroviral suppressive therapy, increases the risk of KS by many orders of magnitude. NF-AT and NF-κB activation by vGPCR was greatly increased by the HIV-1 Tat protein, although Tat alone had little effect on NF-AT. The enhancement of NF-AT by Tat appears to be mediated through collaborative stimulation of the PI3-K/Akt/GSK-3 pathway by vGPCR and Tat. Our data further support the idea that vGPCR contributes to the pathogenesis of KS by a paracrine mechanism and, in addition, provide the first evidence of collaboration between an HIV-1 protein and an HHV-8 protein.
Comparison of Adjuvant Efficacy of Herpes Simplex Virus Type 1 Recombinant Viruses Expressing TH1 and TH2 Cytokine Genes
Authors: Osorio Yanira , Ghiasi Homayon 
Published: April 2003

ABSTRACTThe adjuvant effects of cytokines in humoral and cell-mediated immunity to herpes simplex virus type 1 (HSV-1) have been examined in mice using HSV-1 recombinant viruses expressing murine interleukin-2 (IL-2), IL-4, or gamma interferon (IFN-γ) gene. Groups of naive BALB/c mice were immunized intraperitoneally with one or three doses of the HSV-1 recombinant viruses expressing IL-2, IL-4, or IFN-γ or with parental control virus. Despite similar replication kinetics, these three recombinant viruses elicited different immune responses to HSV-1 on immunization. Immunization with the recombinant virus expressing IL-4 elicited a humoral response of greater magnitude than immunization with the recombinant viruses expressing IL-2 or IFN-γ or with parental virus. In contrast, immunization with recombinant virus expressing IL-2 elicited a higher cytotoxic T-cell response than immunization with viruses expressing IL-4 or IFN-γ. Stimulation in vitro of splenocytes obtained from the mice immunized with UV-inactivated HSV-1 McKrae resulted in a TH1 pattern of cytokine expression irrespective of the recombinant virus used in the immunization. As observed for the parental virus, both CD4+and CD8+T cells contributed equally to the production of IL-2 by the splenocytes of mice immunized with any of the three recombinant viruses. However, the pattern of IFN-γ production by CD4+and CD8+T cells differed according to the recombinant virus used. After lethal ocular challenge, all immunized mice were protected completely against death and manifestations of eye disease caused by HSV-1, which are typical responses in unimmunized mice. Mice immunized with IL-4-expressing virus cleared the virus from their eyes more rapidly than mice immunized with IL-2- or IFN-γ-expressing virus. Taken together, our results suggest that, in contrast to IFN-γ which did not exhibit an adjuvant effect, both IL-4 and IL-2 act as adjuvants in immunization with HSV, with IL-4 showing greater efficacy.
Infection-Triggered Regulatory Mechanisms Override the Role of STAT 4 in Control of the Immune Response to Influenza Virus Antigens
Authors: Bot A. , Rodrigo E. , Wolfe T. , Bot S. , Von Herrath M. G. 
Published: April 2003

ABSTRACTAccurate control of the balance of the T1 and T2 cells during antiviral immunity is essential for optimizing immune effector functions and for avoiding potentially severe immunopathology. We examined the in vivo role of the signal transducer and activator of transcription (STAT) 4 in regulating the T1/T2 balance during the response to live influenza virus and isolated viral proteins. We found that the differentiation of gamma interferon (IFN-γ)-producing Th1 and Tc1 cells after inoculation of live virus occurred independently of STAT 4 expression. Influenza virus-specific T2 and Tc2 responses were well controlled in such STAT 4-deficient mice unless IFN-γ was eliminated as well. In contrast, the STAT 4-dependent signaling pathway played a more essential role in regulating the T1/T2 balance after immunization with viral proteins and, in particular, inactivated nonreplicating virus. Pulmonary infection was cleared even in the absence of both functional STAT 4 genes and functional IFN-γ genes because virus-neutralizing antibodies were still generated, consistent with a substantial redundancy in different antiviral effector pathways. Thus, replicating agents such as live influenza virus can elicit IFN-γ and control T2 immunity independently of STAT 4, whereas the profile of immunity to isolated proteins is more reliant on an intact STAT 4 signaling pathway.
Comprehensive Investigation of the Molecular Defect in
vif
-Deficient Human Immunodeficiency Virus Type 1 Virions
Authors: Gaddis Nathan C. , Chertova Elena , Sheehy Ann M. , Henderson Louis E. , Malim Michael H. 
Published: April 2003

ABSTRACT

Replication of human immunodeficiency virus type 1 (HIV-1) in primary blood lymphocytes, certain T-cell lines (nonpermissive cells), and most likely in vivo is highly dependent on the virally encoded Vif protein. Evidence suggests that Vif acts late in the viral life cycle during assembly, budding, and/or maturation to counteract the antiviral activity of the CEM15 protein and possibly other antiviral factors. Because HIV-1 virions produced in the absence of Vif are severely restricted at a postentry, preintegration step of infection, it is presumed that such virions differ from wild-type virions in some way. In the present study, we established a protocol for producing large quantities of
vif
-deficient HIV-1 (HIV-1/Δ
vif
) from an acute infection of nonpermissive T cells and performed a thorough examination of the defect in these virions. Aside from the expected lack of Vif, we observed no apparent abnormalities in the packaging, modification, processing, or function of proteins in Δ
vif
virions. In addition, we found no consistent defect in the ability of Δ
vif
virions to perform intravirion reverse transcription under a variety of assay conditions, suggesting that the reverse transcription complexes in these particles can behave normally under cell-free conditions. Consistent with this finding, neither the placement of the primer tRNA3Lys nor its ability to promote reverse transcription in an in vitro assay was affected by a lack of Vif. Based on the inability of this comprehensive analysis to uncover molecular defects in Δ
vif
virions, we speculate that such defects are likely to be subtle and/or rare.
In Vivo Evolution of Human Immunodeficiency Virus Type 1 toward Increased Pathogenicity through CXCR4-Mediated Killing of Uninfected CD4 T Cells
Authors: Jekle Andreas , Keppler Oliver T. , De Clercq Erik , Schols Dominique , Weinstein Mark , Goldsmith Mark A. 
Published: April 2003

ABSTRACTThe destruction of the immune system by progressive loss of CD4 T cells is the hallmark of AIDS. CCR5-dependent (R5) human immunodeficiency virus type 1 (HIV-1) isolates predominate in the early, asymptomatic stages of HIV-1 infection, while CXCR4-dependent (X4) isolates typically emerge at later stages, frequently coinciding with a rapid decline in CD4 T cells. Lymphocyte killing in vivo primarily occurs through apoptosis, but the importance of apoptosis of HIV-1-infected cells relative to apoptosis of uninfected bystander cells is controversial. Here we show that in human lymphoid tissues ex vivo, apoptosis of uninfected bystander CD4 T cells is a major mechanism of lymphocyte depletion caused by X4 HIV-1 strains but is only a minor mechanism of depletion by R5 strains. Further, X4 HIV-1-induced bystander apoptosis requires the interaction of the viral envelope glycoprotein gp120 with the CXCR4 coreceptor on CD4 T cells. These results emphasize the contribution of bystander apoptosis to HIV-1 cytotoxicity and suggest that in association with a coreceptor switch in HIV disease, T-cell killing evolves from an infection-restricted stage to generalized toxicity that involves a high degree of bystander apoptosis.
Molecular Features of the Broadly Neutralizing Immunoglobulin G1 b12 Required for Recognition of Human Immunodeficiency Virus Type 1 gp120
Authors: Zwick Michael B. , Parren Paul W. H. I. , Saphire Erica O. , Church Sarah , Wang Meng , Scott Jamie K. , Dawson Philip E. , Wilson Ian A. , Burton Dennis R. 
Published: April 2003

ABSTRACTIgG1 b12 is a broadly neutralizing antibody against human immunodeficiency virus type 1 (HIV-1). The epitope recognized by b12 overlaps the CD4 receptor-binding site (CD4bs) on gp120 and has been a target for vaccine design. Determination of the three-dimensional structure of immunoglobulin G1 (IgG1) b12 allowed modeling of the b12-gp120 interaction in which the protruding third complementarity-determining region (CDR) of the heavy chain (H3) was crucial for antibody binding. In the present study, extensive mutational analysis of the antigen-binding site of Fab b12 was carried out to investigate the validity of the model and to identify residues important for gp120 recognition and, by inference, key to the anti-HIV-1 activity of IgG1 b12. In all, 50 mutations were tested: 40 in H3, 4 each in H2 and L1, and 2 in L3. The results suggest that the interaction of gp120 with H3 of b12 is crucially dependent not only on a Trp residue at the apex of the H3 loop but also on a number of residues at the base of the loop. The arrangement of these residues, including aromatic side chains and side chains that hydrogen bond across the base of the loop, may rigidify H3 for penetration of the recessed CD4-binding cavity. The results further emphasize the importance to gp120 binding of a Tyr residue at the apex of the H2 loop that forms a second finger-like structure and a number of Arg residues in L1 that form a positively charged, shelf-like structure. In general, the data are consistent with the b12-gp120 interaction model previously proposed. At the gene level, somatic mutation is seen to be crucial for the generation of many of the structural features described. The Fab b12 mutants were also tested against the b12 epitope-mimic peptide B2.1, and the reactivity profile had many similarities but also significant differences from that observed for gp120. The paratope map of b12 may facilitate the design of molecules that are able to elicit b12-like activities.
Hyperglycosylated Mutants of Human Immunodeficiency Virus (HIV) Type 1 Monomeric gp120 as Novel Antigens for HIV Vaccine Design
Authors: Pantophlet Ralph , Wilson Ian A. , Burton Dennis R. 
Published: April 2003

ABSTRACTThe ability to induce broadly neutralizing antibodies should be a key component of any forthcoming vaccine against human immunodeficiency virus type 1. One potential vaccine candidate, monomeric gp120, has generally failed to elicit such antibodies. We postulated that gp120 might be a better immunogen if it could be engineered to preferentially bind known broadly neutralizing antibodies. In a first study, we found that four alanine substitutions on the perimeter of the so-called Phe-43 cavity of gp120 could reduce binding of weakly neutralizing CD4-binding site antibodies (R. Pantophlet, E. O. Saphire, P. Poignard, P. W. H. I. Parren, I. A. Wilson, and D. R. Burton, J. Virol. 77:642-658, 2003), while slightly enhancing binding of the potent, broadly neutralizing antibody b12. In the present study, we sought to reduce or abolish the binding of a wider range of nonneutralizing antibodies, by incorporating extra N-glycosylation motifs at select positions into the hypervariable loops and the gp120 core. A hyperglycosylated mutant containing seven extra glycosylation sequons (consensus sequences) and the four alanine substitutions described above did not bind an extensive panel of nonneutralizing and weakly neutralizing antibodies, including a polyclonal immunoglobulin preparation (HIVIG) of low neutralizing potency. Binding of b12, at lowered affinity, and of four antibodies to the C1 and C5 regions was maintained. Removal of N- and C-terminal residues in the C1 and C5 regions, respectively, reduced or abolished binding of the four antibodies, but this also adversely affected b12 binding. The hyperglycosylated mutant and its analogues described here are novel antigens that may provide a new approach to eliciting antibodies with b12-like neutralizing properties.
The Latency-Associated Nuclear Antigen Homolog of Herpesvirus Saimiri Inhibits Lytic Virus Replication
Authors: Schäfer Alexandra , Lengenfelder Doris , Grillhösl Christian , Wieser Carsten , Fleckenstein Bernhard , Ensser Armin 
Published: April 2003

ABSTRACT

Herpesvirus saimiri (HVS), a T-lymphotropic tumor virus of neotropical primates, and the Kaposi's sarcoma-associated human herpesvirus 8 (KSHV) belong to the gamma-
2
-herpesvirus (
Rhadinovirus
) subfamily and share numerous features of genome structure and organization. The KSHV latency-associated nuclear antigen (LANA) protein appears to be relevant for viral persistence, latency, and transformation. It binds to DNA, colocalizes with viral episomal DNA, and presumably mediates efficient persistence of viral genomes. LANA further represses the transcriptional and proapoptotic activities of the p53 tumor suppressor protein. Here we report on the ORF73 gene of HVS strain C488, which is the positional and structural homolog of KSHV LANA. The ORF73 gene in OMK cells can encode a 62-kDa protein that localizes to the nucleus in a pattern similar to that of LANA. We show that the ORF73 gene product can regulate viral gene expression by acting as a transcriptional modulator of latent and lytic viral promoters. To define the HVS ORF73 function in the background of a replication-competent virus, we constructed a viral mutant that expresses ORF73 under the transcriptional control of a mifepristone (RU-486)-inducible promoter. The HVS ORF73 gene product efficiently suppresses lytic viral replication in permissive cells, indicating that it defines a critical control point between viral persistence and lytic replication.
Sodium-Dependent
myo
-Inositol Transporter 1 Is a Cellular Receptor for
Mus cervicolor
M813 Murine Leukemia Virus
Authors: Hein Sibyll , Prassolov Vladimir , Zhang Yuanming , Ivanov Dmitry , Löhler Jürgen , Ross Susan R. , Stocking Carol 
Published: April 2003

ABSTRACT

Retrovirus infection is initiated by binding of the surface (SU) portion of the viral envelope glycoprotein (Env) to specific receptors on cells. This binding triggers conformational changes in the transmembrane portion of Env, leading to membrane fusion and cell entry, and is thus a major determinant of retrovirus tissue and species tropism. The M813 murine leukemia virus (MuLV) is a highly fusogenic gammaretrovirus, isolated from
Mus cervicolor
, whose host range is limited to mouse cells. To delineate the molecular mechanisms of its restricted host range and its high fusogenic potential, we initiated studies to characterize the cell surface protein that mediates M813 infection. Screening of the T31 mouse-hamster radiation hybrid panel for M813 infectivity localized the receptor gene to the distal end of mouse chromosome 16. Expression of one of the likely candidate genes (
slc5a3
) within this region in human cells conferred susceptibility to both M813 infection and M813-induced fusogenicity.
slc5a3
encodes sodium
myo
-inositol transporter 1 (SMIT1), thus adding another sodium-dependent transporter to the growing list of proteins used by MuLVs for cell entry. Characterization of SMIT1 orthologues in different species identified several amino acid variations within two extracellular loops that may restrict susceptibility to M813 infection.
Reovirus σNS and μNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection
Authors: Becker Michelle M. , Peters Timothy R. , Dermody Terence S. 
Published: April 2003

ABSTRACTReovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, σNS and μNS, and structural protein σ3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed σNS, μNS, and σ3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, σNS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed σNS is functional as it complements the defect in temperature-sensitive, σNS-mutant virustsE320. In both transfected and infected cells, μNS is found in punctate cytoplasmic structures and σ3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of μNS and σ3 is not altered when the proteins are expressed together or with σNS. However, when expressed with μNS, σNS colocalizes with μNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both σNS and μNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, σNS interacts with μNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that σNS and μNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.
Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus
Authors: Flick Ramon , Flick Kirsten , Feldmann Heinz , Elgh Fredrik 
Published: April 2003

ABSTRACT

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus
Nairovirus
, family
Bunyaviridae
. The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family
Bunyaviridae
. This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.
Threonine 157 of Influenza Virus PA Polymerase Subunit Modulates RNA Replication in Infectious Viruses
Authors: Huarte Maite , Falcón Ana , Nakaya Yuri , Ortín Juan , García-Sastre Adolfo , Nieto Amelia 
Published: April 2003

ABSTRACT

Previous results have shown a correlation between the decrease in protease activity of several influenza A virus PA protein mutants and the capacity to replicate of the corresponding mutant ribonucleoproteins (RNPs) reconstituted in vivo
.
In this work we studied the phenotype of mutant viruses containing these mutations. Viruses with a T162A mutation, which showed a very moderate decrease both in protease and replication activities of reconstituted RNPs, showed a wild-type phenotype. Viruses with a T157A mutation, which presented a severe decrease in protease activity and replication of RNPs, showed a complex phenotype: (i) transport to the nucleus of PAT157A protein was delayed, (ii) virus multiplication was reduced at both low and high multiplicities, (iii) transcriptive synthesis was unaltered while replicative synthesis, especially cRNA, was diminished, and (iv) viral pathogenesis in mice was reduced, as measured by loss of body weight and virus titers in lungs. Finally, recombinant viruses with a T157E mutation in PA protein, which resulted in a drastic reduction of protease and replication activities of RNPs, were not viable. These results indicate that residue T157 in PA protein is important for the capacity of viral polymerase to synthesize cRNA.