bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiple Effector Functions Mediated by Human Immunodeficiency Virus-Specific CD4+T-Cell Clones
Authors: Norris Philip J. , Sumaroka Marina , Brander Christian , Moffett Howell F. , Boswell Steven L. , Nguyen Tam , Sykulev Yuri , Walker Bruce D. , Rosenberg Eric S. 
Published: July 2002

ABSTRACTMounting evidence suggests that human immunodeficiency virus type 1 (HIV-1) Gag-specific T helper cells contribute to effective antiviral control, but their functional characteristics and the precise epitopes targeted by this response remain to be defined. In this study, we generated CD4+T-cell clones specific for Gag from HIV-1-infected persons with vigorous Gag-specific responses detectable in peripheral blood mononuclear cells. Multiple peptides containing T helper epitopes were identified, including a minimal peptide, VHAGPIAG (amino acids 218 to 226), in the cyclophilin binding domain of Gag. Peptide recognition by all clones examined induced cell proliferation, gamma interferon (IFN-γ) secretion, and cytolytic activity. Cytolysis was abrogated by concanamycin A and EGTA but not brefeldin A or anti-Fas antibody, implying a perforin-mediated mechanism of cell lysis. Additionally, serine esterase release into the extracellular medium, a marker for cytolytic granules, was demonstrated in an antigen-specific, dose-dependent fashion. These data indicate that T helper cells can target multiple regions of the p24 Gag protein and suggest that cytolytic activity may be a component of the antiviral effect of these cells.
Rhabdoviruses and the Cellular Ubiquitin-Proteasome System: a Budding Interaction
Authors: Harty Ronald N. , Brown Melissa E. , McGettigan James P. , Wang Guangli , Jayakar Himangi R. , Huibregtse Jon M. , Whitt Michael A. , Schnell Matthias J. 
Published: July 2002

ABSTRACT
The matrix (M) proteins of vesicular stomatitis virus (VSV) and rabies virus (RV) play a key role in both assembly and budding of progeny virions. A PPPY motif (PY motif or late-budding domain) is conserved in the M proteins of VSV and RV. These PY motifs are important for virus budding and for mediating interactions with specific cellular proteins containing WW domains. The PY motif and flanking sequences of the M protein of VSV were used as bait to screen a mouse embryo cDNA library for cellular interactors. The mouse Nedd4 protein, a membrane-localized ubiquitin ligase containing multiple WW domains, was identified from this screen. Ubiquitin ligase Rsp5, the yeast homolog of Nedd4, was able to interact both physically and functionally with full-length VSV M protein in a PY-dependent manner. Indeed, the VSV M protein was multiubiquitinated by Rsp5 in an in vitro ubiquitination assay. To demonstrate further that ubiquitin may be involved in the budding process of rhabdoviruses, proteasome inhibitors (e.g., MG132) were used to decrease the level of free ubiquitin in VSV- and RV-infected cells. Viral titers measured from MG132-treated cells were reproducibly 10- to 20-fold lower than those measured from untreated control cells, suggesting that free ubiquitin is important for efficient virus budding. Last, release of a VSV PY mutant was not inhibited in the presence of MG132, signifying that the functional L domain of VSV is required for the inhibitory effect exhibited by MG132. These data suggest that the cellular ubiquitin-proteasome machinery is involved in the budding process of VSV and RV.
Selective Loss of Natural Killer T Cells by Apoptosis following Infection with Lymphocytic Choriomeningitis Virus
Authors: Hobbs Jacqueline A. , Cho Sungyoo , Roberts Tonya J. , Sriram Venkataraman , Zhang Jianhua , Xu Ming , Brutkiewicz Randy R. 
Published: July 2002

ABSTRACTNatural killer T (NKT) cells, a unique subpopulation of T cells, coexpress markers also present on NK cells and recognize the major histocompatibility complex class I-like CD1d1 molecule. We studied the effect of an acute virus infection on NKT cells. Mice were infected with the nonhepatotropic Armstrong strain of lymphocytic choriomeningitis virus (LCMV), and at various times postinfection, mononuclear cells from the liver, peritoneum, and spleen were isolated. It was found that within 2 to 3 days, there was a selective loss of NKT cells from the liver with an apparent rapid recovery within 8 to 14 days. There was no increase in peritoneal or splenic NKT cells, indicating that NKT cells did not traffic to these tissues. This loss of NKT cells was independent of gamma interferon (IFN-γ) and interleukin 12 (IL-12) production, but did occur in mice treated with poly(I-C), a classical inducer of IFN-α/β. The reduction in NKT cells was CD28 andfas/fasLindependent and occurred via apoptosis. It was not observed in LCMV-infected DNA fragmentation factor 45-deficient mice, and an increase in active caspase 3-specific staining was found in liver NKT cells from LCMV-infected and poly(I-C)-treated mice compared to uninfected wild-type mice. Interestingly, it was also found that liver NKT cells from LCMV-infected mice were themselves infected. These results suggest that the loss of NKT cells following an acute LCMV infection could be due to the induction of IFN-α/β resulting in NKT-cell apoptosis and is important for the host's immune response to LCMV.
Packaging of Genomic and Amplicon DNA by the Herpes Simplex Virus Type 1 UL25-Null Mutant KUL25NS
Authors: Stow Nigel D. 
Published: July 2002

ABSTRACT
The herpes simplex virus type 1 (HSV-1) mutant KUL25NS, containing a null mutation within the UL25 gene, was isolated and characterized by McNab and coworkers (A. R. McNab, P. Desai, S. Person, L. L. Roof, D. R. Thomsen, W. W. Newcomb, J. C. Brown, and F. L. Homa, J. Virol. 72:1060–1070, 1998). This mutant was able to cleave the concatemeric products of viral DNA replication into monomeric units, but in contrast to wild-type (wt) HSV-1, they were degraded by DNase treatment, indicating that they were not stably packaged into virus capsids. I have examined the packaging of the KUL25NS genome and an HSV-1 amplicon in cells infected with the mutant virus. In contrast to the previous results, a low level of KUL25NS DNA was resistant to DNase digestion, indicating that it was retained in capsids. The proportion of this packaged DNA present as full-length genomes was much lower than in cells infected by wt HSV-1, and there was a significant overrepresentation of the long terminus and underrepresentation of the short terminus. KUL25NS was less impaired in stably packaging amplicon DNA than in packaging its own genome, and the packaged molecules contained approximately equimolar amounts of the two terminal fragments. Below about 100 kbp, the packaged amplicon molecules exhibited an abundance and size distribution similar to those generated using wt HSV-1 as a helper, but the mutant was relatively impaired in packaging longer amplicon molecules. Both packaged genomic and amplicon DNAs were retained in the nuclei of KUL25NS-infected cells. These results suggest that the UL25 protein may play an important role during the later stages of the head-filling process, prior to release of capsids into the cytoplasm.
Role of Polypyrimidine Tract Binding Protein in the Function of the Hepatitis B Virus Posttranscriptional Regulatory Element
Authors: Zang Wei-Qing , Li Bin , Huang Pei-Yong , Lai Michael M. C. , Yen T. S. Benedict 
Published: July 2002

ABSTRACT
The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.
Antigenic Properties of the Human Immunodeficiency Virus Envelope during Cell-Cell Fusion
Authors: Finnegan Catherine M. , Berg Werner , Lewis George K. , DeVico Anthony L. 
Published: July 2002

ABSTRACT

Human immunodeficiency virus (HIV) fusion and entry involves sequential interactions between the viral envelope protein, gp120, cell surface CD4, and a G-protein-coupled coreceptor. Each interaction creates an intermediate gp120 structure predicted to display distinct antigenic features, including key functional domains for viral entry. In this study, we examined the disposition of these features during the fusion of HeLa cells expressing either HIV
HXB2
envelope (Env cells) or CXCR4 and CD4 (target cells). Cell-cell fusion, indicated by cytoplasmic dye transfer, was allowed to progress for various times and then arrested. The cells were then examined for reactivity with antibodies directed against receptor-induced epitopes on gp120. Analyses of cells arrested by cooling to 4
°
C revealed that antibodies against the CD4-induced coreceptor-binding domain, i.e., 17b, 48d, and CG10, faintly react with Env cells even in the absence of target cell or soluble CD4 (sCD4) interactions. Such reactivity increased after exposure to sCD4 but remained unchanged during fusion with target cells and was not intensified at the Env-target cell interface. Notably, the antibodies did not react with Env cells when treated with a covalent cross-linker either alone or during fusion with target cells. Immunoreactivity could not be promoted or otherwise altered on either temperature arrested or cross-linked cells by preventing coreceptor interactions or by using a 17b Fab. In comparison, two other gp120-CD4 complex-dependent antibodies against epitopes outside the coreceptor domain, 8F101 and A32, exhibited a different pattern of reactivity. These antibodies reacted with the Env-target cell interface only after 30 min of cocultivation, concurrent with the first visible transfer of cytoplasmic dye from Env to target cells. At later times, the staining surrounded entire syncytia. Such binding was entirely dependent on the formation of gp120-CD4-CXCR4 tricomplexes since staining was absent with SDF-treated or coreceptor-negative target cells. Overall, these studies show that access to the CD4-induced coreceptor-binding domain on gp120 is largely blocked at the fusing cell interface and is unlikely to represent a target for neutralizing antibodies. However, new epitopes are presented on intermediate gp120 structures formed as a result of coreceptor interactions. Such findings have important implications for HIV vaccine approaches based on conformational alterations in envelope structures.
Structural and Functional Analysis of Interhelical Interactions in the Human Immunodeficiency Virus Type 1 gp41 Envelope Glycoprotein by Alanine-Scanning Mutagenesis
Authors: Lu Min , Stoller Marisa O. , Wang Shilong , Liu Jie , Fagan Melinda B. , Nunberg Jack H. 
Published: July 2002

ABSTRACT
Membrane fusion by human immunodeficiency virus type 1 (HIV-1) is promoted by the refolding of the viral envelope glycoprotein into a fusion-active conformation. The structure of the gp41 ectodomain core in its fusion-active state is a trimer of hairpins in which three antiparallel carboxyl-terminal helices pack into hydrophobic grooves on the surface of an amino-terminal trimeric coiled coil. In an effort to identify amino acid residues in these grooves that are critical for gp41 activation, we have used alanine-scanning mutagenesis to investigate the importance of individual side chains in determining the biophysical properties of the gp41 core and the membrane fusion activity of the gp120-gp41 complex. Alanine substitutions at Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 positions severely impaired membrane fusion activity in envelope glycoproteins that were for the most part normally expressed. Whereas alanine mutations at Leu-565 and Val-570 destabilized the trimer-of-hairpins structure, mutations at Gly-572 and Arg-579 led to the formation of a stable gp41 core. Our results suggest that the Leu-565 and Val-570 residues are important determinants of conserved packing interactions between the amino- and carboxyl-terminal helices of gp41. We propose that the high degree of sequence conservation at Gly-572 and Arg-579 may result from selective pressures imposed by prefusogenic conformations of the HIV-1 envelope glycoprotein. Further analysis of the gp41 activation process may elucidate targets for antiviral intervention.
Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spread in Epidermal Cells after Axonal Transmission
Authors: Mikloska Zorka , Cunningham Anthony L. 
Published: July 2002

ABSTRACTThe ability of alpha interferon (IFN-α) and IFN-γ to inhibit transmission of herpes simplex virus type 1 (HSV-1) from neuronal axon to epidermal cells (ECs), and subsequent spread in these cells was investigated in an in vitro dual-chamber model consisting of human fetal dorsal root ganglia (DRG) innervating autologous skin explants and compared with direct HSV-1 infection of epidermal explants. After axonal transmission from HSV-1-infected DRG neurons, both the number and size of viral cytopathic plaques in ECs was significantly reduced by addition of recombinant IFN-γ and IFN-α to ECs in the outer chamber in a concentration-dependent fashion. Inhibition was maximal when IFNs were added at the same time as the DRG were infected with HSV-1. The mean numbers of plaques were reduced by 52% by IFN-α, 36% by IFN-γ, and by 62% when IFN-α and IFN-γ were combined, and the mean plaque size was reduced by 64, 43, and 72%, respectively. Similar but less-inhibitory effects of both IFNs were observed after direct infection of EC explants, being maximal when IFNs were added simultaneously or 6 h before HSV-1 infection. These results show that both IFN-α and IFN-γ can interfere with HSV-1 infection after axonal transmission and subsequent spread of HSV-1 in ECs by a direct antiviral effect. Therefore, both IFN-α and -γ could contribute to the control of HSV-1 spread and shedding in a similar fashion in recurrent herpetic lesions.
Mechanisms Governing Expression of the v-FLIP Gene of Kaposi's Sarcoma-Associated Herpesvirus
Authors: Grundhoff Adam , Ganem Don 
Published: July 2002

ABSTRACT

Open reading frame 71 (ORF 71) of Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a death effector domain-containing protein that is homologous to cellular FLIPs (FLICE-inhibitory proteins) and is proposed to inhibit Fas-mediated apoptosis. Transcripts bearing ORF 71 (v-FLIP) sequences are present in all latently infected cells. However, mapping studies reveal these to be bi- or tricistronic mRNAs with ORF 71 located 3′ to ORFs 72 (v-cyclin) and 73 (latency-associated nuclear antigen), raising the question of how efficient expression of v-FLIP is achieved. We explored this question by examining the expression of model bicistronic (v-cyclin/LUC) transcripts in which a luciferase (LUC) reporter replaced v-FLIP coding sequences. SLK spindle cells transfected with such constructs efficiently expressed luciferase from the 3′ position, and this expression was independent of the expression of the 5′ v-cyclin gene. Surprisingly, transcript mapping showed that in these cultures, efficient splicing occurred to remove v-cyclin sequences and generate monocistronic LUC transcripts. Similar splicing events produced monocistronic v-FLIP transcripts in KSHV-infected primary effusion lymphoma cells. However, these RNAs were of low abundance and were inducible by treatment with 12-
O
-tetradecanoylphorbol-13-acetate. Examination of the more abundant bicistronic latent RNAs revealed the presence of an efficient internal ribosome entry site (IRES) overlapping ORF 72 coding sequences. Thus, two potential mechanisms exist for v-FLIP expression, but the evidence suggests that IRES-mediated internal translational initiation on latent polycistronic mRNAs is the principal source of v-FLIP in latency.
Cytoplasmic Domain of Herpes Simplex Virus gE Causes Accumulation in the
trans
-Golgi Network, a Site of Virus Envelopment and Sorting of Virions to Cell Junctions
Authors: McMillan Tom N. , Johnson David C. 
Published: July 2002

ABSTRACT

Alphaherpesviruses express a heterodimeric glycoprotein, gE/gI, that facilitates cell-to-cell spread between epithelial cells and neurons. Herpes simplex virus (HSV) gE/gI accumulates at junctions formed between polarized epithelial cells at late times of infection. However, at earlier times after HSV infection, or when gE/gI is expressed using virus vectors, the glycoprotein localizes to the
trans
-Golgi network (TGN). The cytoplasmic (CT) domains of gE and gI contain numerous TGN and endosomal sorting motifs and are essential for epithelial cell-to-cell spread. Here, we swapped the CT domains of HSV gE and gI onto another HSV glycoprotein, gD. When the gD-gI
CT
chimeric protein was expressed using a replication-defective adenovirus (Ad) vector, the protein was found on both the apical and basolateral surfaces of epithelial cells, as was gD. By contrast, the gD-gE
CT
chimeric protein, gE/gI, and gE, when expressed by using Ad vectors, localized exclusively to the TGN. However, gD-gE
CT
, gE/gI, and TGN46, a cellular TGN protein, became redistributed largely to lateral surfaces and cell junctions during intermediate to late stages of HSV infection. Strikingly, gE and TGN46 remained sequestered in the TGN when cells were infected with a gI
−
HSV mutant. The redistribution of gE/gI to lateral cell surfaces did not involve widespread HSV inhibition of endocytosis because the transferrin receptor and gE were both internalized from the cell surface. Thus, gE/gI accumulates in the TGN in early phases of HSV infection then moves to lateral surfaces, to cell junctions, at late stages of infection, coincident with the redistribution of a TGN marker. These results are related to recent observations that gE/gI participates in the envelopment of nucleocapsids into cytoplasmic vesicles (A. R. Brack, B. G. Klupp, H. Granzow, R. Tirabassi, L. W. Enquist, and T. C. Mettenleiter, J. Virol. 74:4004–4016, 2000) and that gE/gI can sort nascent virions from cytoplasmic vesicles specifically to the lateral surfaces of epithelial cells (D. C. Johnson, M. Webb, T. W. Wisner, and C. Brunetti, J. Virol. 75:821–833, 2000). Therefore, gE/gI localizes to the TGN, through interactions between the CT domain of gE and cellular sorting machinery, and then participates in envelopment of cytosolic nucleocapsids there. Nascent virions are then sorted from the TGN to cell junctions.
cis
-Acting Signals in Encapsidation of Hantaan Virus S-Segment Viral Genomic RNA by Its N Protein
Authors: Severson William E. , Xu Xiaolin , Jonsson Colleen B. 
Published: July 2002

ABSTRACT

The nucleocapsid (N) protein encapsidates both viral genomic RNA (vRNA) and the antigenomic RNA (cRNA), but not viral mRNA. Previous work has shown that the N protein has preference for vRNA, and this suggested the possibility of a
cis
-acting signal that could be used to initiate encapsidation for the S segment. To map the
cis
-acting determinants, several deletion RNA derivatives and synthetic oligoribonucleotides were constructed from the S segment of the Hantaan virus (HTNV) vRNA. N protein-RNA interactions were examined by UV cross-linking studies, filter-binding assays, and gel electrophoresis mobility shift assays to define the ability of each to bind HTNV N protein. The 5′ end of the S-segment vRNA was observed to be necessary and sufficient for the binding reaction. Modeling of the 5′ end of the vRNA revealed a possible stem-loop structure (SL) with a large single-stranded loop. We suggest that a specific interaction occurs between the N protein and sequences within this region to initiate encapsidation of the vRNAs.
Ross River Virus Glycoprotein-Pseudotyped Retroviruses and Stable Cell Lines for Their Production
Authors: Sharkey C. Matthew , North Cynthia L. , Kuhn Richard J. , Sanders David Avram 
Published: July 2002

ABSTRACT
Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)-based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.
Mouse-Human Heterokaryons Support Efficient Human Immunodeficiency Virus Type 1 Assembly
Authors: Mariani Roberto , Rasala Beth A. , Rutter Gabriel , Wiegers Klaus , Brandt Stephanie M. , Kräusslich Hans-Georg , Landau Nathaniel R. 
Published: July 2002

ABSTRACTMurine cells do not support human immunodeficiency virus type 1 (HIV-1) replication because of blocks to virus entry, proviral expression, and virion assembly. In murine 3T3 fibroblasts, the block to HIV-1 entry is relieved by the introduction of human CD4 and CCR5 or CXCR4, and proviral expression is increased by the introduction of the Tat cofactor, human cyclin T1; however, because of the assembly block, virus fails to spread. A panel of rodent cell lines expressing human CD4, CCR5, and cyclin T1 was established and studied for the ability to support virus replication.Mus musculuslymphoid cell lines EL4 and L1-2 andMus dunnifibroblasts supported only low levels of virus assembly and released small amounts of infectious virus. CHO and Rat2 cell lines produced more infectious virus, but this production was still 40-fold lower than production in human cells. Only CHO cells expressing the three human cofactors were partially permissive for HIV-1 replication. To investigate the basis of the block to HIV-1 assembly, mouse-human heterokaryons were tested for ability to assemble and release virus. Fusion of human cells to HIV-1-infected mouse cells expressing CD4, CCR5, and cyclin T1 caused a 12-fold increase in virion release and a 700-fold increase in infectious virus production. Fusion of HIV-1-infectedM. dunnitail fibroblasts to uninfected human cells caused a similar increase in virus release. More efficient virus release was not caused by increased proviral transcription or increased synthesis of virion components. Analysis of reciprocal heterokaryons suggested the absence of an inhibitor of virus assembly. Taken together, the results suggested that murine fibroblasts lack a cofactor that is required for efficient virus assembly and release.