bluuebunny/crossref_metadata_2025_split huggingface dataset:
Human Immunodeficiency Virus Tat Modulates the Flk-1/KDR Receptor, Mitogen-Activated Protein Kinases, and Components of Focal Adhesion in Kaposi’s Sarcoma Cells
Authors: Ganju Ramesh K. , Munshi Neru , Nair B. C. , Liu Zhong-Ying , Gill Parkash , Groopman Jerome E. 
Published: December 2019

ABSTRACTKaposi’s sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130cas. Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.
Attenuation of Influenza A Virus mRNA Levels by Promoter Mutations
Authors: Fodor Ervin , Palese Peter , Brownlee George G. , García-Sastre Adolfo 
Published: December 2019

ABSTRACT
We have engineered influenza A/WSN/33 viruses which have viral RNA (vRNA) segments with altered base pairs in the conserved double-stranded region of their vRNA promoters. The mutations were introduced into the segment coding for the neuraminidase (NA) by using a reverse genetics system. Two of the rescued viruses which share a C-G→A-U double mutation at positions 11 and 12′ at the 3′ and 5′ ends of the NA-specific vRNA, respectively, showed approximately a 10-fold reduction of NA levels. The mutations did not dramatically affect the NA-specific vRNA levels found in virions or the NA-specific vRNA and cRNA levels in infected cells. In contrast, there was a significant decrease in the steady-state levels of NA-specific mRNAs in infected cells. Transcription studies in vitro with ribonucleoprotein complexes isolated from the two transfectant viruses indicated that transcription initiation of the NA-specific segment was not affected. However, the majority of NA-specific transcripts lacked poly(A) tails, suggesting that mutations in the double-stranded region of the influenza virus vRNA promoter can attenuate polyadenylation of mRNA molecules. This is the first time that a promoter mutation in an engineered influenza virus has shown a differential effect on influenza virus RNA transcription and replication.
Maintenance of an Intact Human Immunodeficiency Virus Type 1
vpr
Gene following Mother-to-Infant Transmission
Authors: Yedavalli Venkat R. K. , Chappey Colombe , Ahmad Nafees 
Published: December 2019

ABSTRACT

The
vpr
sequences from six human immunodeficiency virus type 1 (HIV-1)-infected mother-infant pairs following perinatal transmission were analyzed. We found that 153 of the 166 clones analyzed from uncultured peripheral blood mononuclear cell DNA samples showed a 92.17% frequency of intact
vpr
open reading frames. There was a low degree of heterogeneity of
vpr
genes within mothers, within infants, and between epidemiologically linked mother-infant pairs. The distances between
vpr
sequences were greater in epidemiologically unlinked individuals than in epidemiologically linked mother-infant pairs. Moreover, the infants’ sequences displayed patterns similar to those seen in their mothers. The functional domains essential for Vpr activity, including virion incorporation, nuclear import, and cell cycle arrest and differentiation were highly conserved in most of the sequences. Phylogenetic analyses of 166 mother-infant pairs and 195 other available
vpr
sequences from HIV databases formed distinct clusters for each mother-infant pair and for other
vpr
sequences and grouped the six mother-infant pairs’ sequences with subtype B sequences. A high degree of conservation of intact and functional
vpr
supports the notion that
vpr
plays an important role in HIV-1 infection and replication in mother-infant isolates that are involved in perinatal transmission.
The Neurovirulence of the DA and GDVII Strains of Theiler’s Virus Correlates with Their Ability To Infect Cultured Neurons
Authors: Jarousse Nadine , Syan Sylvie , Martinat Cécile , Brahic Michel 
Published: December 2019

ABSTRACT
The strains of Theiler’s murine encephalomyelitis virus, a picornavirus, are divided into two groups according to their neurovirulence after intracerebral inoculation. The highly virulent GDVII strain causes an acute, fatal encephalomyelitis, whereas the DA strain causes a mild encephalomyelitis followed by a chronic inflammatory demyelinating disease associated with viral persistence. Studies with recombinant viruses showed that the capsid plays the major role in determining these phenotypes. However, the molecular basis for the effect of the capsid on neurovirulence is still unknown. In this paper, we describe a large difference in the patterns of infection of primary neuron cultures by the GDVII and DA strains. Close to 90% of the neurons were infected 12 h after inoculation with the GDVII strain, and the cytopathic effect was complete 24 h postinoculation. In contrast, with the DA strain, viral antigens were not detected in neurons until 24 h postinoculation. Infected neurons accounted for only 2% of the total number of neurons, even 6 days after inoculation. No cytopathic effect was visible, and the cultures could be kept for the same length of time as the noninfected controls. Because the neurovirulence of the GDVII strain has been mapped to the capsid, we examined the role of the capsid in this difference of phenotype. We showed, using recombinant viruses, that the capsid was indeed responsible for the pattern of infection observed in vitro, most likely through its role in viral entry. Thus, the levels of neurovirulence of the GDVII and DA strains correlate with their abilities to infect cultured neurons, and this ability is controlled by the capsid.
Binding of Sindbis Virus to Cell Surface Heparan Sulfate
Authors: Byrnes Andrew P. , Griffin Diane E. 
Published: December 2019

ABSTRACT
Alphaviruses are arthropod-borne viruses with wide species ranges and diverse tissue tropisms. The cell surface receptors which allow infection of so many different species and cell types are still incompletely characterized. We show here that the widely expressed glycosaminoglycan heparan sulfate can participate in the binding of Sindbis virus to cells. Enzymatic removal of heparan sulfate or the use of heparan sulfate-deficient cells led to a large reduction in virus binding. Sindbis virus bound to immobilized heparin, and this interaction was blocked by neutralizing antibodies against the viral E2 glycoprotein. Further experiments showed that a high degree of sulfation was critical for the ability of heparin to bind Sindbis virus. However, Sindbis virus was still able to infect and replicate on cells which were completely deficient in heparan sulfate, indicating that additional receptors must be involved. Cell surface binding of another alphavirus, Ross River virus, was found to be independent of heparan sulfate.
Caspase Activation and Specific Cleavage of Substrates after Coxsackievirus B3-Induced Cytopathic Effect in HeLa Cells
Authors: Carthy Christopher M. , Granville David J. , Watson Kathleen A. , Anderson Daniel R. , Wilson Janet E. , Yang Decheng , Hunt David W. C. , McManus Bruce M. 
Published: December 2019

ABSTRACTCoxsackievirus B3 (CVB3), an enterovirus in the familyPicornaviridae, induces cytopathic changes in cell culture systems and directly injures multiple susceptible organs and tissues in vivo, including the myocardium, early after infection. Biochemical analysis of the cell death pathway in CVB3-infected HeLa cells demonstrated that the 32-kDa proform of caspase 3 is cleaved subsequent to the degenerative morphological changes seen in infected HeLa cells. Caspase activation assays confirm that the cleaved caspase 3 is proteolytically active. The caspase 3 substrates poly(ADP-ribose) polymerase, a DNA repair enzyme, and DNA fragmentation factor, a cytoplasmic inhibitor of an endonuclease responsible for DNA fragmentation, were degraded at 9 h following infection, yielding their characteristic cleavage fragments. Inhibition of caspase activation by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) did not inhibit the virus-induced cytopathic effect, while inhibition of caspase activation by ZVAD.fmk in control apoptotic cells induced by treatment with the porphyrin photosensitizer benzoporphyrin derivative monoacid ring A and visible light inhibited the apoptotic phenotype. Caspase activation and cleavage of substrates may not be responsible for the characteristic cytopathic effect produced by picornavirus infection yet may be related to late-stage alterations of cellular homeostatic processes and structural integrity.
Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro
Authors: Joachims Michelle , Van Breugel Pieter C. , Lloyd Richard E. 
Published: December 2019

ABSTRACT

Many enteroviruses, members of the family
Picornaviridae
, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.
Lack of Effect of Cytoplasmic Tail Truncations on Human Immunodeficiency Virus Type 2 ROD Env Particle Release Activity
Authors: Bour Stephan P. , Aberham Claudia , Perrin Christèle , Strebel Klaus 
Published: December 2019

ABSTRACT
In addition to its role in receptor binding, the envelope glycoprotein of certain human immunodeficiency virus type 2 (HIV-2) isolates, including ROD10, exhibits a biological activity that enhances the release of HIV-2, HIV-1, and simian immunodeficiency virus particles from infected cells. The present study aims at better defining the functional domains involved in this biological activity. To this end, we have characterized the envelope protein of the ROD14 isolate of HIV-2, which, despite 95% homology with the ROD10 envelope at the amino acid level, is unable to enhance viral particle release. Site-directed mutagenesis showed that the presence of a truncation in the cytoplasmic tail of the ROD14 envelope was not responsible for the lack of activity, as previously reported for the HIV-2 ST isolate (G. D. Ritter, Jr., G. Yamshchikov, S. J. Cohen, and M. J. Mulligan, J. Virol. 70:2669–2673, 1996). Similarly, several modifications of the length of the ROD10 envelope cytoplasmic tail did not impair its ability to enhance particle release, suggesting that, in the case of the HIV-2 ROD isolate, particle release activity is not regulated by the length of the cytoplasmic tail.
Effect of Distance between Homologous Sequences and 3′ Homology on the Frequency of Retroviral Reverse Transcriptase Template Switching
Authors: Delviks Krista A. , Pathak Vinay K. 
Published: December 2019

ABSTRACT
Deletion of direct repeats in retroviral genomes provides an in vivo system for analysis of reverse transcriptase (RT) template switching. The effect of distance between direct repeats on the rate of deletion was determined for 16 murine leukemia virus (MLV)-based vectors containing a 701-bp direct repeat of overlapping fragments of the herpes simplex virus thymidine kinase gene (HTK). The direct repeats were separated by spacer fragments of various lengths (0.1 to 3.5 kb). Southern analysis of infected cells after one replication cycle indicated that all vectors in which the distance between homologous sequences was >1,500 bp deleted at very high rates (>90%). In contrast, vectors containing <1,500 bp between homologous sequences exhibited lower frequencies of deletion (37 to 82%). To analyze the pattern of locations at which RT switched templates, restriction site markers were introduced to divide the downstream direct repeat into five regions. RT switched templates within all five regions of the 701-bp direct repeat and the frequency of template switching was greater within the 5′ regions in comparison to the 3′ regions. The probability of RT switching templates within the 5′ regions doubled when the MLV packaging sequence (Ψ) was placed between the 701-bp direct repeats. However, Ψ did not increase the rate of template switching for shorter direct repeats. These results indicate that linear distance between homologous sequences increases the rate of template switching and suggest that duplex formation between nascent DNA and homologous template sequences 3′ of RT promote template switching.
Regulation of Bovine Leukemia VirustaxandpolmRNA Levels by Interleukin-2 and -10
Authors: Pyeon Dohun , Splitter Gary A. 
Published: December 2019

ABSTRACTRecently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLVtaxandpolmRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLVtaxandpolmRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLVtaxandpolmRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLVtaxandpolmRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLVtaxandpolmRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.
Macrophage Infiltration, but Not Apoptosis, Is Correlated with Immune-Mediated Demyelination following Murine Infection with a Neurotropic Coronavirus
Authors: Wu Gregory F. , Perlman Stanley 
Published: December 2019

ABSTRACTMice infected with mouse hepatitis virus strain JHM (MHV-JHM) develop a chronic demyelinating encephalomyelitis that is in large part immune mediated. Potential mechanisms of immune activity were assessed using an adoptive transfer system. Mice deficient in recombinase-activating gene function (RAG1−/−), defective in B- and T-cell maturation, become persistently infected with MHV but do not develop demyelination. Adoptive transfer of splenocytes from mice immunized to MHV into RAG1−/−mice infected with an attenuated strain of the virus results in the rapid and progressive development of demyelination. Most striking, adoptive transfer resulted, within 5 to 6 days, in extensive recruitment of activated macrophages/microglia to sites of demyelination within the spinal cord. Clearance of virus antigen occurred preferentially from the gray matter of the spinal cord. Apoptotic cells were identified in both the gray and white matter of the central nervous system (CNS) from RAG1−/−mice before and after adoptive transfer, with a moderate increase in number, but not distribution, of apoptotic cells following the development of demyelination. These results suggest that apoptosis following MHV-JHM infection of the murine CNS is not sufficient to cause demyelination. These results, showing that macrophage recruitment and myelin destruction occur rapidly after immune reconstitution of RAG−/−mice, suggest that this will be a useful system for investigating MHV-induced demyelination.
Replication-Defective Bovine Adenovirus Type 3 as an Expression Vector
Authors: Reddy P. Seshidhar , Idamakanti Neeraja , Chen Yan , Whale Tyler , Babiuk Lorne A. , Mehtali Majid , Tikoo Suresh Kumar 
Published: December 2019

ABSTRACT
Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans.
Induction of Syncytia by Neuropathogenic Murine Leukemia Viruses Depends on Receptor Density, Host Cell Determinants, and the Intrinsic Fusion Potential of Envelope Protein
Authors: Chung Maeran , Kizhatil Krishnakumar , Albritton Lorraine M. , Gaulton Glen N. 
Published: December 2019

ABSTRACT
Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.