pinecone/core-2020-05-10-deduplication huggingface dataset:
Gene Expression by Atypical Recombinant Ovine Adenovirus Vectors during Abortive Infection of Human and Animal Cellsin Vitro

AbstractAn ovine adenovirus, which is phylogenetically distinct from the Mastadeno- and Aviadenoviruses, was used to construct recombinants in which reporter genes were expressed from the OAV major late, or human cytomegalovirus promoters. It was demonstrated by transgene expression that OAV could infect bovine nasal turbinate and rabbit kidney cells as well as a range of human cell types, including lung and foreskin fibroblasts as well as liver, prostate, breast, colon, and retinal lines. Some human lines, e.g., 293 and LNCaP were not detectably infected. Infection occurred even though OAV has a fiber protein with a unique cell binding domain and a penton protein that lacks the integrin-binding Arg–Gly–Asp motif which facilitates entry by human adenoviruses. Most cell lines showed little or no ill effect for several days after infection but a prominent cytopathic effect appeared in fibroblasts after 3–4 days. However, no viral DNA synthesis was detected and replication was abortive. Viral promoter activity during infection of nonpermissive cell types was assayed by RT-PCR. Early promoter activity was detectable in some, but not all cell types. In a liver and a colon carcinoma cell line, none of the promoters examined was significantly active, even when a higher multiplicity of infection was used. Major late promoter activity was not detectable in any cell type. The lack of DNA replication and MLP function suggests that a critical transition from early to late gene expression does not occur during abortive infection by OAV
Cleavage of Transcriptional Activator Oct-1 by Poliovirus Encoded Protease 3Cpro

AbstractIn HeLa cells, RNA polymerase II mediated transcription is severely inhibited by poliovirus infection. Both basal and activated transcription are affected to bring about a complete shutoff of host cell transcription. We demonstrate here that the octamer binding transcription factor, Oct-1, is cleaved in HeLa cells infected with poliovirus. Incubation of Oct-1 with the purified, recombinant 3Cproresults in the generation of the cleaved Oct-1 product seen in virus infected cells. Poliovirus infection leads to the formation of altered Oct-1–DNA complexes that can also be generated by incubation of Oct-1 with purified 3Cpro. We also show that Oct-1 cleaved by 3Cproloses its ability to inhibit transcriptional activation by the SV40 B enhancer. These results suggest that cleavage of Oct-1 in poliovirus infected cells leads to the loss of its activity
Heterogeneity and Common Features of Defective Hepatitis B Virus Genomes Derived from Spliced Pregenomic RNA

AbstractDefective hepatitis B virus (HBV) genomes derived from packaging and reverse transcription of spliced RNA pregenomes were reported to be associated with progression to chronic infection. Since only two types with similarly spliced regions were characterized so far we reasoned that additional “spliced” genome variants may exist. Therefore, we isolated a large number of defective HBV genomes from sera of seven chronic carriers by full-length PCR. Forty-eight were found to contain deletions caused by splicing as identified by cloning, subgenomic PCR, and sequencing. In total, 11 types of spliced genomes derived from excision of 10 different introns were present in various combinations in each serum. This diversity resulted from alternative usage of five splice donor and four acceptor sites present in most but not all HBV genotypes. All spliced genomes shared sequence elements essential for replication as well as for transcription of the pre-C and pregenome/C mRNAs and the X mRNA. Moreover, all contained the coding regions for the X protein and for precore/core or precore/core fusion proteins but lacked the pre-S/S gene promoters. These data demonstrate substantial and HBV genotype-dependent diversity of spliced genomes from which a variety of aberrant precore/core fusion proteins and normal X protein but no functional envelope and P proteins could be expressed. These genomes and the encoded proteins may play a role in the viral life cycle, persistence, and pathogenesis
New Satellite RNAs, but No DI RNAs, Are Found in Natural Populations of Tomato Bushy Stunt Tombusvirus

AbstractA collection of 57 field isolates of the tombusvirus tomato bushy stunt virus was obtained from eggplant and tomato during 1994–1997 and was examined for the presence of defective interfering (DI) RNA species by Northern blot hybridization and RT-PCR. No DI RNA species were detected associated with any of the field TBSV isolates. However, serial passaging of two field isolates inNicotiana clevelandiiat high multiplicity of infection resulted in the rapid generation of DI-like RNA species, indicating that the absence of DI RNAs in natural populations of the virus was not due to the inability of the TBSV field isolates to generate them in a suitable host. The results indicate that DI RNAs may not play a role in modulating natural TBSV infections in the hosts examined. In 4 of 57 isolates analyzed we have detected less than full-length RNAs and we show here that they are true satellite RNAs. Two different satellite RNA species were detected, named TBSV sat RNAs B1 (822 nt) and B10 (612 nt). TBSV sat RNAs lack significant open reading frames and do not present sequence homology except in a central box that is also conserved in TBSV-Ch genomic RNA and in all the DI RNAs derived from it. TBSV sat RNA B10 attenuated the symptoms induced by the helper virus inN. clevelandiiwhile sat RNA B1 did not modify the symptoms. This is the first report of sat RNAs associated with TBSV and the first time that sat RNAs are associated with natural tombusvirus infections
Polyomavirus Large T Can Support DNA Replication in Human Cells

AbstractHuman cells are generally thought to be nonpermissive for polyomavirus (Py) DNA replication. Using transient transfection, we show that Py large T-antigen (LT) was able to support replication of a Py origin-containing plasmid in two human cell lines. Replication supported by LT in human cells was specific for the Py origin and required its enhancer sequences, as well as the previously reported critical phosphorylation sites within LT. Py replication efficiency was comparable to that of papillomavirus E1- and E2-activated DNA replication in transient assays performed in human 293 and C-33A cells. Previous analysis of DNA replicationin vitrohas pointed to polymerase α–primase as a specificity determinant for polyomavirus. The data presented here imply that in certain cellular environments, Py LT must functionally interact with human polymerase α–primase to permit DNA replication
Hormone Regulation of Bovine Leukemia Virus via the Long Terminal Repeat

AbstractThe hormone regulation of viruses has been of great interest since the discovery of glucocorticoid stimulation of mouse mammary tumor virus via a hormone response element in the viral long terminal repeat (LTR) promoter region. This report describes the investigation of the hormone responsiveness of bovine leukemia virus (BLV), an oncogenic retrovirus that infects dairy and beef cattle worldwide. It is a member of the human T cell leukemia (HTLV)/BLV group of retroviruses, which encode a protein, Tax, that is essential for regulating transcription of their own proviruses and for transforming host cells. We investigated the responsiveness of BLV to the hormones 17β-estradiol, progesterone, prolactin, insulin, and dexamethasone, a potent glucocorticoid. Only dexamethasone, in combination with insulin or insulin/prolactin, consistently stimulated BLV expression, as measured by reverse transcriptase activity, RNA blot hybridization (Northern blots), and CAT (chloramphenicol acetyltransferase) reporter assays of cell lines transiently or stably transfected with the BLV LTR. This effect required the presence of glucocorticoid receptors and Tax. This is the first report of hormone responsiveness in a virus of the HTLV/BLV group
Evidence That a Viral Replicase Protein Is Involved in the Disassembly of Tobacco Mosaic Virus Particlesin Vivo

AbstractTobacco mosaic virus (TMV) particles have been shown to undergo bidirectional disassembly when they are introduced into host cells. Approximately three-quarters of the genomic RNA (i.e., the 126-kDa and 183-kDa protein ORFs) is first uncoated in the 5′-to-3′ direction and the process is then completed by removal of coat protein molecules in the 3′-to-5′ direction. An effort was made to determine whether the 126-kDa protein or the 183-kDa protein, both of which are involved in replication of the viral RNA, is required for the second part of the disassembly reaction. It was shown that progeny negative-strand viral RNA begins to be produced in inoculated cells at about the same time that 3′-to-5′ disassembly is initiated thus suggesting that the two processes may be coupled. Particles containing mutant forms of the viral RNA in which large sections of the 126-kDa and 183-kDa protein ORFs were missing were not disassembled in the 3′-to-5′ direction when they were introduced into cells. However, they were disassembled when the inoculum contained purified TMV RNA from which, presumably, the two functional proteins could be translated. Particles containing mutants of the RNA from which a few codons had been deleted in or near conserved regions in the 126-kDa protein ORF also did not undergo 3′-to-5′ disassembly unless mixed with wild type viral RNA prior to inoculation. These results suggest that the 126-kDa and/or 183-kDa protein plays a role in the completion of disassembly of TMV particles at the onset of the infection process
Megalomicin Inhibits HIV-1 Replication and Interferes with gp160 Processing

AbstractThe inhibitory effects on HIV replication of megalomicin (MGM), an inhibitor of intra-Golgi vesicle transport, have been studied. In experiments at low multiplicity of infection on Jurkat and MT2 cell lines, MGM inhibited the production of p24 antigen, the formation of syncytia, and the induction of apoptosis at concentrations below 5 μM. Furthermore, PCR analysis of genomic DNA showed that, in the presence of MGM, HIV-1 had been eradicated from the culture. MGM also inhibited replication of primary isolates of HIV-1 in blood lymphoblasts and more importantly, at 1 μM, MGM inhibited depletion of CD4+T cells in cultures of blood lymphocytes from seropositive patients. Finally, MGM inhibited the generation of infectious virions and the processing of the envelope protein precursor gp160 to its mature forms, resulting in the rapid degradation of gp160. These data suggest that MGM induces a powerful inhibitory effect on HIV-1 replication at nontoxic concentrations by preventing the processing of HIV-1 gp160 envelope protein and the subsequent formation of infectious viral particles
Increasing the Ratio of PP2A Core Enzyme to Holoenzyme Inhibits Tat-Stimulated HIV-1 Transcription and Virus Production

AbstractWe demonstrated previously that PP2A exists in many cell types as two abundant forms: (1) holoenzyme composed of two regulatory subunits, A and B, and a catalytic subunit C; and (2) core enzyme consisting of the A and C subunits. These two forms have different substrate specificities. Since published data suggested that HIV-1 transcription may be regulated by a cellular protein phosphatase, it was of interest to determine whether changing the ratio between PP2A core and holoenzyme affects HIV-1 gene expression. This question was addressed by expression in COS cells of an N-terminal mutant of the A subunit, AΔ5, which binds the C but not the B subunit. This resulted in an increase in the amount of core enzyme and a decrease in the amount of holoenzyme concomitant with the expected change in phosphatase activity. Tat-stimulated transcription from the HIV-1 LTR was inhibited 5-fold by mutant AΔ5, whereas mRNA synthesis directed by the actin promoter was not affected. Furthermore, virus production in COS, HeLa, and Jurkat T cells was inhibited 45-, 5-, and 3-fold, respectively, by mutant AΔ5. These results demonstrate that the balance between PP2A holoenzyme and core enzyme is important for HIV-1 gene expression and virus production
An Imperfect Correlation between DNA Replication Activity of Epstein–Barr Virus Nuclear Antigen 1 (EBNA1) and Binding to the Nuclear Import Receptor, Rch1/importin α

AbstractEpstein–Barr virus (EBV) replicates as a stable multicopy episome in latently infected mammalian cells. Latent cycle DNA replication requires only two viral elements, the cis-acting origin of plasmid replication (oriP) and the trans-acting origin binding protein (EBNA1). EBNA1 binds multiple recognition sites in oriP, but has no other enzymatic activities associated with replication functions. To identify human cellular proteins that mediate EBNA1 function, we designed a one-hybrid assay in yeast to select for proteins that bind to EBNA1 when bound to oriPin vivo.A human cDNA encoding the Rch1/hSRP1α/importinα protein was isolated and shown to bind to full-length EBNA1, but not to an amino terminal deletion mutant of EBNA1 when bound to oriP in yeast. The interaction of EBNA1 with Rch1 was confirmed biochemically by coimmunoprecipitation from nuclear extracts and by direct binding of recombinant proteinsin vitro.Internal deletion mutations in EBNA1 which compromised DNA replication activity were similarly reduced for binding to Rch1. Mutations with no effect on DNA replication activity were similarly unaffected for Rch1 binding. Rch1/importin α has been shown to bind to the nuclear localization sequence (NLS) of several proteins and stimulate nuclear import. A substitution mutation in the EBNA1 nuclear localization sequence reduced Rch1 binding, but had no effect on DNA replication function, indicating that Rch1 binding affinity does not correspond precisely with replication activity. Nevertheless, the identification of a stable interaction between Rch1 and EBNA1 at the origin of viral DNA replication raises the intriguing possibility that Rch1 contributes to the nuclear functions of EBNA1
Reassessing the Organization of the UL42–UL43 Region of the Human Cytomegalovirus Strain AD169 Genome

AbstractA polymorphism in the UL42–UL43 region of the human cytomegalovirus genome has been characterized by nucleotide sequence analysis, revealing a 929-bp insertion following nt 54,612 relative to the published strain AD169-UK genome sequence (M. S. Cheeet al.,1990,Curr. Top. Microbiol. Immunol.154, 125–170). Although AD169-UK exhibited polymorphism in this genomic region, other CMV strains (Towne, Toledo, and AD169-ATCC) carried only the newly characterized longer form. The additional sequence altered the assignment of UL42 and UL43 open reading frames. UL42 decreased in size from 157 to 125 codons, retaining 76 of the previously reported carboxyl terminal codons, and UL43 increased in size from 187 to 423 codons, retaining 185 of the previously reported amino terminal codons. This additional sequence makes UL43 a more conserved betaherpesvirus US22 family member. Only AD169-UK exhibited restriction fragment length polymorphism in this region, suggesting that a deletion occurred during the propagation of this strain in cell culture. The additional sequence should be considered a bona fide part of the cytomegalovirus genome and the AD169 genome size should be corrected to 230,283 bp
Replicon-Helper Systems from Attenuated Venezuelan Equine Encephalitis Virus: Expression of Heterologous Genesin Vitroand Immunization against Heterologous Pathogensin Vivo

AbstractA replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of thecis-acting 5′ and 3′ ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were suppliedin transby expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains. Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen
Characterization of Torovirus from Human Fecal Specimens

AbstractThe toroviruses, Berne virus (BEV) and Breda virus (BRV), are recognized pathogens of horses and cattle, respectively. Torovirus-like particles (TVLPs) that are immunologically related to BRV have been reported as etiological agents of gastroenteritis in humans. Of the toroviruses, only BEV has been shown to replicate in cell culture. Hence, these agents can be routinely detected only by electron microscopy (EM), although serological testing has been used as well. Our studies have provided supporting evidence that the TVLPs detected in the stool specimens of pediatric patients with gastroenteritis are human toroviruses. By EM, these particles are morphologically similar to BEV and BRV. Thin-section electron microscopy revealed that TVLPs contain toroidal-shaped nucleocapsids. Viruses purified from human fecal specimens agglutinate rabbit erythrocytes. BRV antiserum as well as convalescent sera from patients with gastroenteritis whose stools contain TVLPs were shown to contain antibodies that react with purified TVLPs as demonstrated by hemagglutination inhibition, immunoelectron microscopy, and immunoblotting. RNA extracted from partially purified TVLP preparations is amplifiable by RT-PCR using primers bracketing a 219-base region at the 3′ end of the Berne virus genome. Sequence analysis of amplicons from five isolates showed a high degree of identity with the corresponding BEV sequence
Incomplete dsRNA Genomes in Purified Infectious Pancreatic Necrosis Virus

AbstractThe dsRNA containing birnavirus, infectious pancreatic necrosis virus, possesses a virion-associated RNA-dependent RNA polymerase which acts both as primer and as polymerase duringin vitroRNA synthesis (P. Dobos, 1995,Virology208, 19–25). Using [α32P]GTP, we have radiolabeled virion RNAin vitroand found that after deproteinization most of the labeled product comigrated in agarose gels with the 3-kbp viral genome, while the remainder migrated faster than the dsRNA and as a heterogeneous smear. Agarose gel electrophoresis (AGE) of denatured labeled virion RNA showed a radioactive smear ranging from approximately 100 nucleotides to up to 3000 nucleotides, the size of genome-length single stranded RNA. Hybridization experiments using strand-specific and end-specific oligonucleotides on Northern blots revealed that the radioactivity which migrates with the dsRNA during AGE represents small, 5′ end plus RNA molecules of 100–500 nucleotides. The radioactivity in the faster migrating smear denotes incomplete dsRNAs where full-length, unlabeled minus strands are base-paired with labeled plus strands that are 3′ truncated to different extents. This was confirmed by reverse transcription–polymerase chain reaction (RT-PCR) using end- and strand-specific oligonucleotide primers. The results indicated that 95% of incomplete dsRNA molecules consisted of full-length minus strands and 3′ truncated plus strands. The implications of these findings are discussed in light of RNA replication mechanisms of dsRNA viruses belonging to other families
Complementation of Vaccinia Virus Deleted of the E3L Gene by Mutants of E3L

AbstractVaccinia virus devoid of its E3L gene is sensitive to treatment of RK-13 cells with interferon-α and fails to replicate or form plaques in HeLa cells. In order to determine function of the E3L gene, vaccinia virus recombinants were constructed by inserting mutant E3L genes or a gene coding for an alternative dsRNA-binding protein into virus deleted of its wild type E3L gene. Those viruses that expressed proteins that retained dsRNA binding activity were resistant to the effects of interferon in RK-13 cells and could replicate in HeLa cells. Recombinant viruses that expressed E3L mutant proteins which were unable to bind to dsRNA were interferon sensitive in RK-13 cells and could not replicate in HeLa cells. In addition, a virus that expressed a mutant E3L protein previously characterized as having a low binding affinity for dsRNA exhibited an intermediate phenotype: it was interferon resistant in RK-13 cells but could not replicate in HeLa cells. This work suggests that the E3L gene of vaccinia virus functions primarily as a dsRNA-binding protein in allowing resistance to interferon and in promoting replication in HeLa cells
CD4+T Cells Are Ineffective in Clearing a Pulmonary Infection with Influenza Type A Virus in the Absence of B Cells

AbstractRecovery from influenza virus infection is dependent on T cell functions which can be provided either by CD8 or CD4 T cells. To identify the functions involved in recovery promoted by CD4 T cells, we have studied the course of the infection in B-cell deficient μMT mice which had been depleted of CD8 T cells by antibody treatment. Upon infection with PR8 [A/PR/8/34(H1N1)], such B- and CD8 T cell-deficient mice mounted strong CD4 T cell responses that were comparable in size and cytokine secretion to those seen in intact mice. Yet, these B- and CD8 T cell-deficient mice could not clear the infection, in contrast to (CD8-depleted) mice containing both B- and CD4 T cells. These findings indicate that the promotion of the T-dependent antibody response is an indispensable component in the CD4 T cell-dependent recovery process