pinecone/core-2020-05-10-deduplication huggingface dataset:
The Rep68 Protein of Adeno-Associated Virus Type 2 Increases RNA Levels from the Human Cytomegalovirus Major Immediate Early Promoter

AbstractThe Rep68 and Rep78 proteins of adeno-associated virus type-2 (AAV) are multifunctional DNA binding proteins which are involved in the positive and negative regulation of AAV genes, as well as various cellular and heterologous viral genes. In this study we report that Rep68 enhances expression from the major immediate early promoter (MIEP) of human cytomegalovirus (HCMV). This Rep-mediated enhancement of RNA levels is abrogated by the introduction of a Rep recognition sequence (RRS) at either position −18 or −244 in the HCMV–MIEP. However, a mutant RRS (mRRS), which is not bound by Rep68 is unable to negate the effect of Rep68. Sequence analysis and electrophoretic mobility shift assays showed no Rep68 binding sites within the wild-type HCMV–MIEP. Rep68 may therefore be enhancing expression from the HCMV–MIEP by interacting with other regulatory proteins that have an effect on the expression from this promoter or by altering the expression of a cellular gene whose product influences the HCMV–MIEP. Our results may also help to explain the previous observation that coinfection with AAV enhances the cytopathic effect of HCMV
Mutational Analysis of Transcriptional Activation by the Bovine Papillomavirus Type 1 E6

AbstractWhile the bovine papillomavirus type 1 (BPV-1) E6 induces tumorigenic transformation of murine C127 cells, it does not bind or promote the degradation of p53. We recently showed the cellular protein ERC-55/E6BP binds BPV-1 E6 as well as the cancer-related human papillomavirus (HPV) E6 proteins. BPV-1 E6 also binds E6-AP, a ubiquitin ligase necessary for HPV E6-induced p53 degradation. We previously reported that the transforming activity of a set of BPV-1 E6 mutants correlated with their E6BP-binding ability. Another function of BPV-1 E6 is stimulation of transcription when targeted to a promoter, although cellular promoters responsive to BPV-1 E6 have not been identified. To examine whether its transcriptional function is required for oncogenic activity, or is related to its interactions with E6-AP or E6BP, a series of BPV-1 E6 mutants were analyzed as fusions to a sequence-specific DNA binding domain for activity in yeast and in mammalian cells. We show that some transformation defective mutants retained substantial levels of transcriptional activation activity. These mutants also distinguish transcriptional activation from E6-AP and E6BP binding. These results suggest the transcriptional activation function of BPV-1 E6 is not sufficient for cell transformation
Genetic Characterization of the Rotavirus Nonstructural Protein, NSP4

AbstractThe rotavirus nonstructural protein NSP4 plays a role in viral assembly by acting as an intracellular receptor for single-shelled particles and assisting in the translocation of these particles across the endoplasmic reticulum. Recently, NSP4 has been implicated in rotavirus virulence and is thought to act as an enterotoxin which triggers chloride secretion by a calcium-dependent signal transduction pathway. Limited sequence analysis of NSP4 shows a well-conserved protein. To define the extent of sequence variation in the gene coding for NSP4, we have sequenced this gene from nine human rotavirus strains. These data and the analysis of additional human strains and various animal rotaviruses (bovine, simian, equine, and porcine) by Northern blot hybridization suggested that three NSP4 genotypes were present among rotavirus strains. A correlation between NSP4 genotype and VP6 subgroup was also implied. Two different NSP4 genes (which encoded distinct types of NSP4 proteins) were found among standard human rotaviruses and in strains circulating in the local community and these showed homology to cognate genes in some animal strains
R7, a Spontaneous Mutant of Moloney Murine Sarcoma Virus 124 with Three Direct Repeats and an In-Frame Truncatedgag-mosGene, Induces Brain Lesions

AbstractWe have isolated Recombinant 7 (R7), a spontaneous mutant of SV7, a molecular clone of MoMuSV124. Like SV7, R7 induces subcutaneous fibrosarcomas, spleen tumors, and mesentery tumors infiltrated by proliferating vessels lined by transformed endothelial cells. However, it also induces brain lesions. We have molecularly cloned and sequenced the R7 proviral DNA and shown that the R7 genome consists of 3401 bp. It has three direct repeats in each enhancer. Its coding sequence consists of only 176 bp of p15, 263 bp of p30, a 7-bp insertion, and 853 bp of an N-terminally truncatedmosgene. From the sequence of R7 we have deduced that the truncatedmossequence is in-frame with all of thegagsequence and the 7-bp insertion. The incorporation of the 3′ end of the p15 sequence further suggests that the R7 Gag-Mos is myristylated. We have also shown that the molecularly cloned R7 virus transformed NIH/3T3 fibroblasts about sevenfold better than the parental SV7. We have also confirmed that molecularly cloned R7 induces the same disease phenotype as that induced by the nonmolecularly cloned R7
Poxvirus Genomes Encode a Secreted, Soluble Protein That Preferentially Inhibits β Chemokine Activity yet Lacks Sequence Homology to Known Chemokine Receptors

AbstractPoxvirus genomes encode several proteins which inhibit specific elements of the host immune response. We show the “35K” virulence gene in variola and cowpox viruses, whose vaccinia and Shope fibroma virus equivalents are strongly conserved in sequence, actually encodes a secreted soluble protein with high-affinity binding to virtually all known β chemokines, but only weak or no affinity to the α and γ classes. The viral protein completely inhibits the biological activity of monocyte chemotactic protein-1 (MCP-1) by competitive inhibition of chemokine binding to cellular receptors. As all β chemokines are also shown to cross-compete with MCP1 binding to the viral protein, we conclude that this viral chemokine inhibitor (vCCI) not only interacts through a common binding site, but is likely a potent general inhibitor of β chemokine activity. Unlike many poxvirus virulence genes to date, which are clearly altered forms of acquired cellular genes of the vertebrate immune system, this viral chemokine inhibitor (vCCI) shares no sequence homology with known proteins, including known cellular chemokine receptors, all of which are multiple membrane-spanning proteins. Thus, vCCI presumably has no cellular analogue and instead may be the product of unrelenting sequence variations which gave rise to a completely new protein with similar binding properties to native chemokine receptors. The proposed function of vCCI is inhibition of the proinflammatory (antiviral) activities of β chemokines
Structural Analysis of a Necrogenic Strain of Cucumber Mosaic Cucumovirus Satellite RNAin Planta

AbstractStructural studies of plant viral RNA molecules have been based onin vitrochemical and enzymatic modification. That approach, along with mutational analysis, has proven valuable in predicting structural models for some plant viruses such as tobacco mosaic tobamovirus and brome mosaic bromovirus. However,in plantaconditions may be dramatically different from those foundin vitro.In this study we analyzed the structure of cucumber mosaic cucumovirus satellite RNA (sat RNA) strain D4in vivoand compared it to the structures foundin vitroand in purified virions. Following a methodology developed to determine the structure of 18S rRNA within intact plant tissues, different patterns of adenosine and cytosine modification were found for D4-sat RNA moleculesin vivo, in vitro,and in virions. This chemical probing procedure identifies adenosine and cytosine residues located in unpaired regions of the RNA molecules. Methylation data, a genetic algorithm in the STAR RNA folding program, and sequence alignment comparisons of 78 satellite CMV RNA sequences were used to identify several helical regions located at the 5′ and 3′ ends of the RNA molecule. Data from previous mutational and sequence comparison studies between satellite RNA strains inducing necrosis in tomato plants and those strains not inducing necrosis allowed us to identify one helix and two tetraloop regions correlating with the necrogenicity syndrome
Concurrent Suppression of Virus Replication and Rescue of Movement-Defective Virus in Transgenic Plants Expressing the Coat Protein of Potato Virus X

AbstractA line of transgenic tobacco expressing the coat protein (CP) of potato virus X (PVX) was resistant against a broad spectrum of PVX strains. Inoculation of leaves and protoplasts with PVX expressing the jellyfish green fluorescent protein reporter gene revealed that this resistance mechanism suppressed PVX replication in the initially infected cell and systemic spread of the virus. Cell-to-cell movement was also slower in the resistant plants. The resistance at the level of replication was effective against wild-type PVX and also against movement-defective isolates with a frameshift mutation or deletion in the CP ORF. However, the cell-to-cell movement defect of the mutant viruses was rescued on the resistant plants. Based on these results it is proposed that the primary resistance mechanism is at the level of replication
Increased Expression of the N Protein of Respiratory Syncytial Virus Stimulates Minigenome Replication but Does Not Alter the Balance between the Synthesis of mRNA and Antigenome

AbstractA popular model for RNA synthesis by nonsegmented negative-strand RNA viruses is that transcription and RNA replication are executed by the same polymerase complex and that there is a dynamic balance between the two processes that is mediated by the nucleocapsid N protein. According to this model, transcription occurs until sufficient soluble N protein accumulates to initiate encapsidation of the nascent RNA product, which somehow switches the polymerase into a readthrough replicative mode. This model was examined for respiratory syncytial virus (RSV) using a reconstituted transcription and RNA replication system that involves a minireplicon and viral proteins that are expressed intracellularly from transfected plasmids. Preliminary experiments showed that reconstituted RNA replication was highly productive, such that on average each molecule of plasmid-supplied minigenome that became encapsidated was amplified 10- to 50-fold. N protein was increased on its own or in concert with the phosphoprotein P and in the presence or absence of the M2 ORF1 transcription elongation factor. The maximum level of N and P protein expression achieved from plasmids equalled or exceeded that obtained in RSV-infected cells. Increased levels of N protein stimulated RNA replication. This is consistent with the idea that RNA replication is dependent on the availability of N protein for encapsidation, which is one postulate of the model. The M2 ORF1 protein had no detectable effect on RNA replication under the various conditions of expression of N and P, which confirmed and extended previous results. However, there was no evidence of a significant switch in positive-sense RNA synthesis from transcription (synthesis of mRNAs) to RNA replication (synthesis of antigenome). The synthesis of positive-sense antigenome and mRNA appeared to occur at a fixed ratio, with mRNA being by far the more abundant product
Inhibition of Vaccinia Virus Replication byN-(Phosphonoacetyl)-l-aspartate: Differential Effects on Viral Gene Expression Result from a Reduced Pyrimidine Nucleotide Pool

AbstractThe replication of vaccinia virus was reduced by 3 logs in cells that had been treated before and during infection with a concentration ofN-(phosphonoacetyl)-l-aspartate (PALA) which lowered the UTP and CTP to 5 and 20% of controls, respectively, without affecting cell viability. The antiviral activity of PALA was reversed with uridine, indicating that it was entirely due to the diminution in pyrimidine nucleotides. Analysis of viral proteins revealed prolonged synthesis of some early stage species but a drastic reduction in late stage species, even though the nucleotide concentrations remained relatively constant throughout the infection. Although the gene expression pattern resembled that caused by a potent inhibitor of DNA synthesis, viral DNA accumulation was reduced by only 60%. Very little of the DNA made in the presence of PALA was converted to genome length molecules. The effect of PALA on transcription of early genes was complex: there was a twofold increase in the amount of a relatively short mRNA of 500 nucleotides but a two- to threefold decrease in the amount of a 4300-nucleotide mRNA encoding the largest subunit of RNA polymerase. In contrast, PALA severely inhibited the accumulation of viral intermediate and late stage mRNAs. The extreme sensitivity of vaccinia virus to PALA and the differential effects of the drug on viral gene expression result from the cascade mechanism of viral gene regulation
The Coat and Cylindrical Inclusion Proteins of a Potyvirus Are Associated with Connections between Plant Cells

AbstractThe subcellular locations of two potyviral proteins, the coat (CP) and nonstructural cylindrical inclusion (CI) proteins of tobacco vein mottling virus (TVMV), during early stages in the development of systemic infections in plants, have been investigated. Ultrathin sections of newly emerged leaves in infected plants were treated with antibodies specific to these proteins and then with gold-labeled secondary antibodies and examined by electron microscopy. CI was detected near plasmodesmatal connections between mesophyll cells prior to the appearance of CP or any virus-induced features or effects. Further accumulation of CI was evident in the form of conical structures, many of which appeared to penetrate the cell wall and to be connected to cones in neighboring cells. Prior to its appearance in other parts of the cells, the viral CP was detected, often in linear arrays, near the vertices or inside the cones and in plasmodesmata.In situhybridization analysis of similar tissue sections with a TVMV RNA-specific oligoribonucleotide probe revealed the presence of the viral RNA in plasmodesmata. These results lend support to the notion that the formation of specific structures by potyviral CI proteins is required for and plays a direct role in the intercellular passage of viral genetic material, in the form of virus particles or complexes containing viral CP and RNA, in infected plants
Early Region 4 Modulates Adenovirus DNA Replication by Two Genetically Separable Mechanisms

AbstractThree viral proteins, all products of early region 2 (E2), participate directly in adenovirus DNA replication. Three products of early region 4 (E4) also affect viral DNA synthesis: the product of E4 ORF4 inhibits viral DNA accumulation, while the products of E4 ORFs 3 and 6 antagonize that effect of ORF4 expression. Because no E4 products are required for DNA synthesis, these proteins probably act indirectly. The E4 ORF3, 4, and 6 proteins all participate in aspects of the regulation of viral gene expression. To determine whether they modulate DNA replication by effects on expression of viral replication proteins, we examined E2 expression in E4 mutant-infected cells. In cells infected by ORF3−, 6−mutants, expression of ORF4 substantially depressed the steady-state levels of replication proteins and E2 mRNAs, reduced E2 transcription rates, and profoundly inhibited viral DNA replication. Thus, in the absence of E4 ORFs 3 and 6, ORF4 acts as a transcriptional regulator of E2 expression, and reduced replication protein levels largely account for the inhibition of DNA replication by ORF4. Cells infected by viruses that express ORFs 3 and 6 in addition to ORF4 accumulated much larger quantities of viral DNA than did cells infected by the ORF3−, 6−, 4+mutant. Increased DNA accumulation was not accompanied by a comparable increase in E2 expression. Therefore, the ORF3 and 6 products counteract the ORF4-induced reduction of DNA replication by a mechanism other than reversing the inhibitory effect of ORF4 on E2 expression. The effect of ORF4 on E2 expression is consistent with its ability to regulate levels of the transcription factor AP-1 (Mülleret al.,1992,J. Virol.66, 5867–5878); the mechanism by which ORFs 3 and 6 enhance replication is unknown
Innate and Acquired Immunity to Herpes Simplex Virus Type 1

AbstractImmunization with heat-inactivated herpes simplex virus type 1 (HSV-1) 2–5 days before ocular infection reduced the frequency of establishment of latent HSV-1 infection in the trigeminal ganglion (TG); this induction of resistence coincided with reduced expression of IFN-γ mRNA in the TG. Immunization with unrelated antigens was not protective. In part, this resistance to nervous system invasion correlated with the appearance of serum antibody to HSV-1. Immunization reduced viral replication in the eye and trigeminal ganglion, and prevented HSV-1 spread to the cerebellum. IFN-γ was detected in immunized mice 4 days postocular infection as determined by plaque reduction using neutralizing Ab to IFN-α/β and IFN-γ. Injection of antibody (Ab) to IFN-α/β and IFN-γ administered at the time of immunization did not affect survival. Anti-IFN-γ-treated mice had significantly reduced levels of IFN in their serum. Treatment with anti-IFN-α/β Ab resulted in an elevation in viral replication as determined by the expression of latency associated transcripts in the TG of mice. Likewise, there was a significant increase in the CD8, IL-12 (p40), and TNF-α mRNA levels in the TG of the anti-IFN-α/β-treated mice. TG explant cultures demonstrated that viral load was significantly increased in the TG of anti-IFN-α/β-treated mice relative to TG of control mice 7 days after infection. The results suggest that exposure to viral antigens 2–5 days before infection is an important determinant of the extent of HSV-1 spread to the nervous system. Moreover, the data suggest that both an antibody response and IFN-α/β play a role in limiting the progress of infection from the peripheral tissues to the central nervous system
P32/TAP, a Cellular Protein That Interacts with EBNA-1 of Epstein–Barr Virus

AbstractThe Epstein–Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1–282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40–60 and aa 325–376, each of which contains arginine–glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244–282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation
Detection of Proviral DNA of Bovine Immunodeficiency Virus in Bovine Tissues by Polymerase Chain Reaction (PCR) and PCRin SituHybridization

AbstractIn this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCRin situhybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conservedpolgene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIVpolgene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed byin situamplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected toin situhybridization with an internal biotinylated probe and detected with streptavidin–gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissuesin vivoand has a broad cell tropism
Characterization of RNA Products Associated with or Aborted by a Viral RNA-Dependent RNA Polymerase

AbstractTernary RNA-dependent RNA polymerase complexes were arrested at various stages of synthesisin vitroby limiting one or more NTPs. The RNAs synthesized prior to/during the arrest of the complex were characterized and the ability of the arrested complexes to continue elongation of these nascent RNAs in the presence of all four NTPs was analyzed. Nascent RNAs of 10 nt and longer remained associated with the RdRp complex and could be extended into full-length RNAs while shorter nascent RNAs were released by the complex. These results establish that previously observed RdRp-synthesized oligoribonucleotides of 8 nt or less are abortive initiation products and confirms that RdRp undergoes a transition from initiation to elongation after the synthesis of 8 nt
Assembly of Hepatitis Delta Virus-like Empty Particles in Yeast

AbstractLarge delta antigen (L-HDAg) of hepatitis delta virus (HDV) and small-form hepatitis B surface antigen (HBsAg) of helper hepatitis B virus have previously been shown to be the minimum components for the assembly of HDV-like particles in mammalian cells. Extending from this finding, we coexpressed L-HDAg and small HBsAg inSaccharomyces cerevisiaeto study their assembly in yeast cells. The assembly of virus particles from L-HDAg and HBsAg in yeast was demonstrated by their coexistence in the same isopycnic fractions and by the coimmunoprecipitation of L-HDAg with HBsAg using an antibody against HBsAg (anti-HBs). Furthermore, after purification by affinity chromatography with anti-HBs, HDV-like particles with size and morphology similar to those derived from mammalian cells could be visualized by electron microscopy. Mice immunized with yeast-derived HDV-like particles simultaneously acquired antibodies against HBsAg and HDAg, indicating that both viral proteins are antigenic. The results indicated thatS. cerevisiaecould serve as a host for the assembly of HDV-like empty particles. This system may be useful in investigating cellular processes involved in HDV assembly and in producing ample amount of HDV-like particles for structural and immunological studies