pinecone/core-2020-05-10-deduplication huggingface dataset:
Coronavirus MHV-3-Induced Apoptosis in Macrophages

AbstractInfection with mouse hepatitis virus strain 3 (MHV-3) results in lethal fulminant hepatic necrosis in fully susceptible BALB/c mice compared to the minimal disease observed in resistant strain A/J mice. Macrophages play a central role in the pathogenesis of MHV-3-induced hepatitis. In the present study we have shown that MHV-3 infection of macrophages induces these cells to undergo apoptosis. Three methods to detect apoptosis were applied: flow cytometry analysis of nuclear DNA content, fluorescence microscopic visualization of apoptotic cells labeled by the TUNEL assay, and gel electrophoresis to detect DNA laddering. Apoptosis in A/J and BALB/c macrophages was first detected at 8 h postinfection (p.i.) and reached a maximum by 12 h p.i. The degree of MHV-3-induced apoptosis was much greater in A/J-derived macrophages than in BALB/c-derived cells. Apoptosis was inversely correlated with the development of typical MHV cytopathology, namely syncytia formation. Infected macrophages from A/J mice did not form synctia in contrast to the extensive synctia formation observed in BALB/c-derived macrophages. In MHV-3-infected BALB/c macrophage cultures, apoptotic cells were not incorporated into syncytia. Apoptosis was also inversely correlated with the expression of MHV-3-induced fgl2 prothrombinase in macrophages. These results add the murine coronavirus MHV-3 to the list of RNA-containing viruses capable of inducing apoptosis
Human Papillomavirus Oncoproteins Alter Differentiation-Dependent Cell Cycle Exit on Suspension in Semisolid Medium

AbstractThe suspension of keratinocytes containing episomal forms of the human papillomavirus (HPV)-31 genome in semisolid medium results in the induction of viral late functions. In this study, the suspension in semisolid medium was used to analyze how HPV deregulates the process of cell cycle exit during differentiation. In cells that contain the entire HPV-31 genome, induction of late protein synthesis was found to be linked with the expression of cyclin A. Consistent with analyses in organotypic rafts, the expression of the high-risk E7 oncoprotein alone was sufficient to retain cyclin A expression during suspension-induced differentiation. The cyclin-dependent kinase inhibitors (CKIs) p27 and p57 were found to be up-regulated in normal keratinocytes, as well as in the lines that express the HPV oncoproteins. The up-regulation of these CKIs is coincident with the inhibition of cyclin/cdk activity in normal keratinocytes. Cells expressing E7 were found to retain significant cdk2-associated kinase activity, although it was partially inhibited, coincident with CKI induction. When the phosphorylation state of Rb was examined during differentiation, cells expressing E7 retained phosphorylated forms of Rb, whereas Rb in normal keratinocytes was hypophosphorylated. As previously reported, E7-expressing cells were found to contain less Rb protein than normal keratinocytes. Interestingly, the Rb levels decreased during normal keratinocyte differentiation, and this differentiation-dependent reduction in Rb levels was enhanced by EG and E7 expression. This study identified proteins that may be critical for cell cycle regulation during normal epithelial differentiation and demonstrated that HPV oncoproteins alter their activities
Retrograde Transport of Intact Poliovirus Through the Axon via the Fast Transport System

AbstractIntramuscularly inoculated poliovirus is thought to spread to the central nervous system through neural pathways in humans, monkeys, and the transgenic (Tg) mice carrying the human poliovirus receptor (PVR) gene. To gain insight into molecular mechanisms for the retrograde axonal transport of poliovirus, resulting in the expression of neurovirulence, a poliovirus-sensitive ICR-PVRTg21 mouse line (Tg21) was used as an animal model for poliomyelitis. We detected poliovirus antigens in axons of the sciatic nerve. All of the Tg21 mice, which had been inoculated into the calves with 1 × 106pfu of the Mahoney strain of type 1 poliovirus, showed symptoms of paralysis in the inoculated limbs (initial paralysis) within 48 h after the inoculation. The appearance of this initial paralysis was observed in mice whose sciatic nerves were transected at various times after virus inoculation. The results were indicators of the velocity of poliovirus transportation through the sciatic nerves under analysis. Poliovirus-related materials recovered from the sciatic nerve were mainly composed of intact 160S virion particles. The amount of 160S particle recovered was greatly reduced by coinjection with anti-PVR monoclonal antibody. These results suggest that one of the fast retrograde axonal transport systems is involved in poliovirus dissemination through the sciatic nerve and that IM-inoculated poliovirus is incorporated into the sciatic nerve as intact particles in a PVR-dependent manner, as it is in humans
A Human Liver Cell Line Exhibits Efficient Translation of HCV RNAs Produced by a Recombinant Adenovirus Expressing T7 RNA Polymerase

AbstractAnin vitrosystem that supports the efficient growth of hepatitis C virus (HCV) and reflects its completein vitroreplication cycle has not yet been established. The establishment of a minigene RNA of HCV in mammalian cells could facilitate the study of virus–cell interactions and the molecular pathogenesis of this virus. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable for at least 11 days after inoculation. Cells infected with AdexCAT7 were then transfected with plasmids carrying the authentic T7 promoter, the 5′ untranslated region (UTR) of encephalomyocarditis virus, a luciferase gene, and a T7 terminator (pT7EMCVLuc) or carrying the modified T7 promoter, the 5′UTR of HCV, a luciferase gene, the coding region of C-terminal of NS5B and the 3′UTR of HCV, a ribozyme of hepatitis D virus and a T7 terminator (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with pT7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma, exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesizedin vitrofrom pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. The T7-adenovirus system for the synthesis of HCV minigenesin vivoprovides useful information on the molecular mechanisms of HCV translation in human liver cells
Cells Expressing the Human Foamy Virus (HFV) Accessory Bet Protein Are Resistant to Productive HFV Superinfection

AbstractBet is a foamy virus (FV) accessory protein not required for virus replication. The function of Bet is not understood. We report on the generation of cell lines stably expressing the HFV Bet protein. In Bet+cells, HFV replication was reduced by ∼3–4 orders of magnitude compared with control cells. The HFV Bet-expressing cells only partially resisted infection by the distantly related feline FV (FFV). Pseudotyping experiments, using murine retroviral vectors with an HFV envelope, revealed that the resistance was not due to downregulation of the unknown HFV receptor. In transfection experiments, using proviral reporter gene constructs and infectious proviruses, no significant differences were detected between Bet+and control cells. In infection experiments, HFV vectors expressing an indicator gene under control of the HFV promoters showed no activity in Bet+cells. The results are best compatible with the hypothesis that the main block to productive superinfection of Bet+cells occurs at an early stage of replication between virus entry and provirus establishment. We suggest that inhibition of provirus integration by Bet protein may serve a distinct function in the unique foamy virus replication cycle
Constitutive Production of a Murine Retrovirus in the Human B-Lymphoblastoid Cell Line, DG-75

AbstractDuring the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission studies, the Epstein–Barr virus (EBV)-negative, B-lymphoblastoid cell line DG-75 was found to be chronically infected with a heretofore unrecognized retrovirus. Two DG-75 sublines obtained from different sources (designated UW and KAR) were found to produce constitutively particles identified as retroviral by electron microscopy and reverse transcriptase activity. The ultrastructure, morphogenesis, and density in sucrose of the particles were typical of C-type retroviruses. Immunoblot analysis of the DG-75(UW) retrovirus proteins showed antigenic similarity to Moloney murine leukemia virus. A third DG-75 subline in early passage, designated HAD, was free of retrovirus. The DG-75(UW) retrovirus was infectious and produced progeny virions that could be passaged to uninfected cells. We have thus demonstrated that DG-75 cells, which have been used extensively in studies of the biological effects of EBV-encoded genes and their promoters, may be chronically infected with a murine retrovirus and that an early passage subline is retrovirus free and available for such studies
Characterization of mRNA for Hepatitis Delta Antigen: Exclusion of the Full-Length Antigenomic RNA as an mRNA

AbstractHepatitis delta virus (HDV) encodes a single protein, the hepatitis delta antigen (HDAg), which is thought to be translated from a 0.8-kb RNA of antigenomic sense. This subgenomic RNA species is present in very small amounts in HDV-infected liver tissues and in cultured cells infected or transfected with HDV, and in some cases it cannot be detected at all. In contrast, HDAg protein is present in large amounts in all natural and experimental models of HDV infection. This study addresses whether other HDV RNA species, such as the antigenomic-sense, genome-size HDV RNA can also serve as the mRNA for HDAg synthesis. Taking advantage of the ability of herpes simplex virus (HSV) to degrade only polyadenylated mRNAs, we examined the effect of HSV coinfection on HDAg synthesis. It was shown that HSV infection did degrade the subgenomic 0.8-kb HDV mRNA but not HDV genome-length RNA. Under such conditions, HDAg synthesis was completely inhibited. Furthermore, the genome-length HDV RNA was found not to be associated with polysomes. Finally,in vitrotranslation studies demonstrated that HDAg could not be translated directly from the genome-length antigenomic-sense HDV RNA. These results suggest that only the subgenomic RNA species of HDV possesses properties characteristic of the mRNA for HDAg and that the genome-length RNA cannot be used for translating HDAg. In addition, we found that HDV RNA replication did not depend onde novoHDAg synthesis
The Vaccinia Virus E3L Gene Product Interacts with both the Regulatory and the Substrate Binding Regions of PKR: Implications for PKR Autoregulation

AbstractThe vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2α, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to functionin vitroby sequestration of dsRNA. We have utilizedin vitrobinding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1–99) located in the regulatory domain and the carboxyl-terminal (amino acids 367–523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2α and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR–PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2α and its subsequent phosphorylation
Asymmetric Infectivity of Pseudorecombinants of Cabbage Leaf Curl Virus and Squash Leaf Curl Virus: Implications for Bipartite Geminivirus Evolution and Movement

AbstractThe bipartite geminiviruses squash leaf curl virus (SqLCV) and cabbage leaf curl virus (CLCV) have distinct host ranges. SqLCV infects a broad range of plants within the Cucurbitaceae, including pumpkin and squash, and CLCV has a broad host range within Brassicaceae that includes cabbage andArabidopsis thaliana.Despite this, the genomic A components of these viruses share a high degree of sequence identity, particularly in the gene encoding the replication protein AL1, and their common regions are 77% identical. However, there is unexpected sequence diversity in the common regions of the two CLCV genomic A and B components, these being only 80% identical. Based on these sequence similarities, we investigated the host range properties of pseudorecombinants of SqLCV and CLCV. We found that in a pseudorecombinant virus consisting of the A component of CLCV and the B component of SqLCV, both components replicated in tobacco protoplasts, and this pseudorecombinant was infectious and caused systemic disease inNicotiana benthamiana, a common host to all bipartite geminiviruses. However, this pseudorecombinant did not move systemically in pumpkin orArabidopsis, despite the demonstrated replication compatibility of the genome components. As a result of the greater sequence differences between the common regions, the pseudorecombinant of SqLCV A and CLCV B components neither replicated the CLCV B component nor systemically infected any of the hosts tested. These findings demonstrate that for different geminiviruses with distinct host ranges, the replication origins and AL1 proteins can be sufficiently similar to permit infectious pseudorecombinants, but replication alone is not sufficient to cause systemic disease, and host range may ultimately be limited at the level of movement. The results of this study further suggest that CLCV is an evolving virus that can provide insights into how new bipartite geminiviruses arise from mixed infections
Marburg Virus Vaccines Based upon Alphavirus Replicons Protect Guinea Pigs and Nonhuman Primates

AbstractMarburg virus (MBGV), for which no vaccines or treatments currently exist, causes an acute hemorrhagic fever with a high mortality rate in humans. We previously showed that immunization with either killed MBGV or a glycoprotein (GP) subunit prevented lethal infection in guinea pigs. In the studies reported here, an RNA replicon, based upon Venezuelan equine encephalitis (VEE) virus, was used as a vaccine vector; the VEE structural genes were replaced by genes for MBGV GP, nucleoprotein (NP), VP40, VP35, VP30, or VP24. Guinea pigs were vaccinated with recombinant VEE replicons (packaged into VEE-like particles), inoculated with MBGV, and evaluated for viremia and survival. Results indicated that either GP or NP were protective antigens while VP35 afforded incomplete protection. As a more definitive test of vaccine efficacy, nonhuman primates (cynomolgus macaques) were inoculated with VEE replicons expressing MBGV GP and/or NP. Three monkeys received packaged control replicons (influenza HA); these died 9 or 10 days after challenge, with typical MBGV disease. MBGV NP afforded incomplete protection, sufficient to prevent death but not disease in two of three macaques. Three monkeys vaccinated with replicons which expressed MBGV GP, and three others vaccinated with both replicons that expressed GP or NP, remained aviremic and were completely protected from disease
Structural and Functional Analysis of the Membrane-Spanning Domain of the Human Immunodeficiency Virus Type 1 Vpu Protein

AbstractThe human immunodeficiency virus type 1 (HIV-1)vpugene product is a class I integral membrane phosphoprotein that is capable of oligomerization. Two distinct biological activities have been attributed to Vpu: induction of CD4 degradation in the endoplasmic reticulum and enhancement of viral particle release from the plasma membrane of infected cells. These two biological activities were shown to involve two separable structural domains: the N-terminal transmembrane (TM) domain and the C-terminal cytoplasmic domain. The TM domain mediates enhancement of viral particle release, whereas phosphorylation of the cytoplasmic domain is essential for Vpu-induced CD4 degradation. In this study, we performed a mutational analysis of the TM domain of Vpu to delineate amino acids that are important in the process of viral particle release or in Vpu-induced CD4 degradation. Substitution of conserved amino acids from the N-terminal, middle, or C-terminal parts of the native VpuTM domain generated proteins that integrated normally into canine pancreatic microsomal membranes, exhibited subcellular localization similar to those of wild-type Vpu, but partially lost their ability to enhance viral particle release, strongly suggesting that the VpuTM domain contains determinants responsible for Vpu-mediated enhancement of viral particle release. Interestingly, the C-terminal TM mutant VpuIVW, in contrast to the other mutants, also lost its ability to bind and consequently degrade the CD4 molecule, indicating that the alteration of the C-terminal part of the TM did interfere with this function of Vpu. Taken together, our study supports the notion that both structural elements of Vpu (TM and cytoplasmic) contribute to the biological activities of Vpu
Nuclear Localization of the Functional Bel 1 Transactivator but Not of the Gag Proteins of the Feline Foamy Virus

AbstractInteractions between host cells and foamy or spumaretroviruses are different from those of other known retroviruses. Previous work has suggested that the Gag and high-affinity DNA-binding Bel 1 transactivator of human foamy virus are localized in the nuclei of infected cells. Using two independent detection methods, we show here that the functionally active Bel 1 transactivator protein of feline foamy virus is of nuclear localization. In contrast to that reported for the human foamy virus Gag protein, the cat foamy virus Gag proteins exclusively localized in the cytoplasm close to perinuclear regions
Particle-Bombardment-Mediated DNA Vaccination with Rotavirus VP4 or VP7 Induces High Levels of Serum Rotavirus IgG but Fails to Protect Mice against Challenge

AbstractWe recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997)Virology232, 129–138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4.In vitroassays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus.In vitroexpression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Δ1–162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge
Characterization of Two Avian Reoviruses That Exhibit Strain-Specific Quantitative Differences in Their Syncytium-Inducing and Pathogenic Capabilities

AbstractWe previously proposed that the conservation of the nonessential syncytium-inducing phenotype among all reported avian reovirus (ARV) isolates may reflect a mechanism for enhanced virus disseminationin vivo, which in turn could contribute to the natural pathogenicity of ARV. Direct testing of this hypothesis has been hampered by the lack of available virus strains with defined differences in their fusion-inducing capability. We now report on the characterization of two ARV strains, ARV-176 and ARV-138, that exhibited strain-specific differences in their fusogenic properties, which correlated with their pathogenic potential in embryonated eggs. Moreover, both virus strains possessed similar replicative abilities in cell culture, suggesting that the weakly fusogenic ARV-138 virus is specifically inhibited in its syncytium-inducing ability. To test the use of these viruses for reassortant studies aimed at assessing the role of cell fusion in viral pathogenesis, a preliminary genetic analysis was undertaken using a monoreassortant that contained nine genome segments from the parental ARV-138 virus and the S1 genome segment from the highly fusogenic and pathogenic ARV-176 parental virus. The monoreassortant possessed the full fusogenic potential of the ARV-176 parental virus and displayed enhanced embryo pathogenicity, providing the first genetic evidence implicating the ARV S1 genome segment in both syncytium formation and viral pathogenesis
L1-Specific Protection from Tumor Challenge Elicited by HPV16 Virus-like Particles

AbstractA single injection of HPV16 L1 virus-like particles induced potent CD8-mediated protection from tumor challenge by C3 cells, a line derived from embryonic mouse cells transfected with the HPV16 genome. L1 RNA, but not protein, was detected biochemically in C3 cells. These results indicate that low-level expression of HPV16 L1 can occur in proliferating cells and serve as a tumor vaccine target. Although L1 expression is generally thought to be restricted to terminally differentiated epithelial cells, these results suggest that additional analysis for low-level L1 expression in proliferating cells of HPV-induced lesions is warranted and might help in predicting the clinical potential of HPV L1 virus-like particle-based vaccines