pinecone/core-2020-05-10-deduplication huggingface dataset:
The Positive Transcription Elongation Factor Activity of the Vaccinia Virus J3 Protein Is Independent from Its (Nucleoside-2′-O-) Methyltransferase and Poly(A) Polymerase Stimulatory Functions

AbstractPrevious genetic and biochemical experiments have shown that the vaccinia virus J3 protein has three different roles in mRNA synthesis and modification. First, J3 is a (nucleoside-2′-O-)methyltransferase which methylates the 2′ position of the first transcribed nucleotide, thus converting a cap-0 to a cap-1 structure at the 5′ ends of mRNAs. Second, J3 is a processivity factor for the virus coded poly(A) polymerase. Third, J3 has recently been shown to have intermediate and late gene positive transcription elongation factor activity in vivo. Previous experiments have shown that the poly(A) polymerase stimulatory activity and the (nucleoside-2′-O-)methyltransferase activity are two independent functions of the protein that can be genetically separated through site-directed mutagenesis. In this article, the relationship between the J3-mediated transcription elongation activity and the two other functions of the protein was investigated by constructing several site-directed mutant viruses that contain specific defects in either methyltransferase or poly(A) polymerase processivity functions. The results demonstrate that the J3 positive transcription elongation factor activity is a third independent function of the protein that is genetically separable from its two other functions in mRNA modification. The results also show that neither the poly(A) polymerase stimulatory nor the methyltransferase activities of the J3 protein is essential for virus growth in cell culture
Generation of Velogenic Newcastle Disease Viruses from a Nonpathogenic Waterfowl Isolate by Passaging in Chickens

AbstractA benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background
Sorting of the Respiratory Syncytial Virus Matrix Protein into Detergent-Resistant Structures Is Dependent on Cell-Surface Expression of the Glycoproteins

AbstractThe interaction of the respiratory syncytial virus (RSV) Matrix (M) protein with the plasma membrane was investigated using polyclonal and monoclonal antisera raised against recombinant M expressed in bacteria. M bound mainly to the plasma membrane, although a significant proportion bound to internal membranes. However, no localisation of M with the Golgi was observed, suggesting that transport of M to the plasma membrane was independent of the transport mechanism for the viral glycoproteins. Expression from a recombinant baculovirus demonstrated the ability of M to bind membranes in the absence of viral glycoprotein expression. When cell-surface expression of the viral glycoproteins was prevented using Brefeldin A, M was still found in association with the plasma membrane, but the characteristics of M's membrane-binding ability were different to that found in untreated infected cells. In the presence of normal glycoprotein expression, M was sorted into lipid rafts and, in addition, formed structures that could only be disrupted by treatment with high salt buffers, a feature suggesting an interaction with the cytoskeleton or the formation of strong intramolecular associations. Brefeldin A prevented M from being sorted into lipid rafts or from forming strong intramolecular associations. Brefeldin A also affected the stability of M bound to the plasma membrane, as M was more readily dissociated in the presence of the inhibitor. Coexpression of M and F resulted in the incorporation of M into lipid rafts but did not cause the formation of the strong intramolecular bonds, suggesting that additional factors are required for this phenomena
Transcriptional Termination Modulated by Nucleotides Outside the Characterized Gene End Sequence of Respiratory Syncytial Virus

AbstractThe genes of respiratory syncytial (RS) virus are transcribed sequentially by the viral RNA polymerase from a single 3′-proximal promoter. Polyadenylation and termination are directed by a sequence at the end of each gene, after which the polymerase crosses an intergenic region and reinitiates at the start sequence of the next gene. The 10 viral genes have different gene end sequences and different termination efficiencies, which allow for regulation of gene expression, since termination of each gene is required for initiation of the downstream gene. RNA sequences within the previously characterized 13 nucleotide gene end, including a conserved sequence 3′-UCAAU-5′ and a tract of U residues, are important for termination. In this study, two additional sequence elements outside of the 13 nucleotide gene end were found to modulate termination efficiency: the A residue upstream of the 3′-UCAAU-5′ sequence, and the first nucleotide of the intergenic region when it follows a U4 tract
Telomerase Activation in Cervical Keratinocytes Containing Stably Replicating Human Papillomavirus Type 16 Episomes

AbstractRetroviral transduction and expression of the human papillomavirus type 16 (HPV-16) E6 gene has been shown to activate telomerase in human cervical and foreskin keratinocytes. There still remains some controversy, however, as to whether expression of E6 in the context of the whole HPV-16 genome can activate telomerase. In this study, we have generated human cervical keratinocyte clones that contain stably replicating HPV-16 episomes. Interestingly, the majority of the clones exhibited low or no telomerase activity at early passage and this was associated with low transcript levels of the reverse transcriptase component of telomerase, hTERT. The HPV-16-containing clones became immortal without a crisis and, at later passage, exhibited elevated levels of telomerase and higher levels of hTERT without any apparent increase in HPV-16 copy number, E6 transcript levels, or ability to degrade p53. These results indicate that HPV-16 by itself does not necessarily cause telomerase activation in cervical keratinocytes, but rather, supports a model in which HPV-16 facilitates telomerase activation in conjunction with other viral or cellular changes over time
Construction and Functional Characterization of Feline Foamy Virus-Based Retroviral Vectors

AbstractReplication-competent feline foamy or spuma virus (FFV) vectors were constructed and functionally tested. The unmodified FFV vector genome expressed by the strong human cytomegalovirus immediate early promoter encodes FFV particles that were replication-competent in cell cultures. Virus derived from the cloned FFV DNA replicated and persisted in experimentally infected cats similar to the FFV isolate FUV. A FFV vector partially deleted in the noncoding area of the U3 region was used to transduce the gene for the green fluorescent protein (Gfp) into cell cultures. Gfp was expressed either by an internal ribosomal entry site (IRES) or as C-terminal fusion protein linked to Bet that was recently shown to be essential for FFV replication. Whereas the genetic stability of the IRES–Gfp construct was comparably low, the Bet–Gfp fusion protein was detectable upon serial cell-free vector passages. However, genetic rearrangements also occurred leading to the concomitant loss of marker gene expression
Immunization of Macaques with Live Simian Human Immunodeficiency Virus (SHIV) Vaccines Conferred Protection Against AIDS Induced by Homologous and Heterologous SHIVs and Simian Immunodeficiency Virus

AbstractTo evaluate the vaccine potential of SHIVs attenuated by deletion of viral accessory genes, seven rhesus macaques were sequentially immunized with ΔvpuΔnefSHIV-4 (vaccine-I) followed by ΔvpuSHIVPPC (vaccine-II). Despite the absence of virological evidence of productive infection with the vaccine strains, based on analysis of infectivity among peripheral blood mononuclear cells (PBMC) of the vaccinated animals, all seven animals developed binding as well as neutralizing antibodies against both vaccine-I and -II. The animals also developed vaccine virus-specific CTLs that recognized homologous as well as heterologous pathogenic SHIVs and SIV, and also soluble inhibitory factors that blocked the in vitro replication of the vaccine strains and different challenge viruses. Virus-specific cellular and humoral responses were sustained throughout a 58-week prechallenge period. To model aspects of natural transmission, the animals received a mucosal (rectal) challenge, with a mixture of three challenge viruses, SHIVKU, SHIV89.6P, and SIVmacR71/17E. Two mock-vaccinated control animals inoculated with the same mixture of challenge viruses developed large numbers of infectious PBMC, high plasma viremia, and precipitous loss of CD4+ T cells. The control animals did not develop any immune responses and succumbed to AIDS between 6 and 7 weeks postchallenge. All seven vaccinated animals became infected with challenge viruses as indicated by the presence of infectious cells in the PBMC and/or viral RNA in plasma. However, peak plasma viremia in vaccinates was two to nearly five logs lower than in the control animals and later plasma viral RNA became undetectable in all vaccinates. Vaccinated animals maintained normal CD4+ T cell levels throughout the study. Challenge with pathogenic viruses caused massive anamnestic responses as determined by quantitation of virus-specific CD4+ and CD8+ T cells by intracellular IFN-γ staining, and these cells persisted for at least 74 weeks. The study is still in progress and at this time DNA of SIV has become undetectable in lymph nodes of six of the seven vaccinates, SHIV89.6P in five of the seven, and SHIVKU in three of the seven animals
TT Virus Replicates in Stimulated but Not in Nonstimulated Peripheral Blood Mononuclear Cells

AbstractTT virus (TTV) is an unenveloped, single-stranded, circular-DNA virus which resembles members of the Circoviridae, that is commonly found in humans and which lacks pathological consequences for the infected host. TTV replication has been demonstrated in bone marrow cells but not in peripheral blood mononuclear cells (PBMC), suggesting that hematopoietic cells must be activated to support TTV replication. To test this hypothesis, PBMC from two naturally TTV-infected individuals and from two healthy TTV-DNA negative donors infected in vitro with a TTV-DNA-positive serum were cultured in the presence (stimulated) or absence (unstimulated) of phytohemagglutinin, lipopolysaccharide, and interleukin-2. TTV-DNA was detected in both stimulated and unstimulated PBMC. However, TTV-DNA replicative intermediates and mRNA were detected only in stimulated PBMC. Furthermore, TTV-DNA and mRNA were detected in PBMC from two TTV negative donors reinfected with supernatants from TTV-infected stimulated cells but not when using culture supernatants from unstimulated cells. These results demonstrate that TTV replicates in PBMC only when stimulated
Fusion of Mature Dendritic Cells and Human T-Lymphotropic Virus Type I Infected T Cells: Its Efficiency as an Antigen-Presenting Cell

AbstractMonocyte-derived dendritic cells (DCs) from adult T cell leukemia are impaired in taking up exogenous antigens. To overcome this impairment, we fused unpulsed DCs to human T-lymphotropic virus type I (HTLV-I)-infected CD4+ T cells (fusion DC-T cells). The efficiency of fusion was 50% and the fusion cells expressed higher HLA-ABC and CD86 Ags than HTLV-I-infected DCs. The fusion DC-T cells stimulated autologous CD4+ and CD8+ T cells, but DCs fused to itself or PHA-blasts did not stimulate any subsets. The functionally highest fusion DC-T cells was obtained when a DC and HTLV-I-infected T cells were fused at a ratio of 3:1. Expression of HTLV-Igag Ag on CD4+ T cells was up-regulated when infected in the presence of 8-azaguanine, and these fusion DC-T cells were quite efficient in induction of higher CD8+ T cell response. The results suggest that fusion DC-T cells produce functionally competent Ag-presenting cells and may be a likely candidate for immunotherapeutic use
Enhanced Immune Responses After DNA Vaccination with Combined Envelope Genes from Different HIV-1 Subtypes

AbstractIn a multisubtype approach to HIV-1 vaccination, mice were immunized with HIV-1 envelope gp160 genes from subtypes A, B, and C. Subsequently the mice were challenged with syngeneic primary splenocytes infected with a HIV-1/MuLV pseudovirus carrying a subtype B genome. HIV-specific immune responses and protection were strongest in the group of animals immunized with a combination of subtype A, B, and C specific gp160 genes as compared to subtype B only. Immunization with the combination of the cross-reactive subtypes A and C envelope genes induced HIV-specific immune responses but did not result in significant protection to challenge with subtype B infected cells. From this we conclude that immunization with the envelope genes from several HIV-1 subtypes may indeed enhance immune responses. This study shows that by using a mix of subtype envelope genes, an enhanced protective immunity can be obtained experimentally, potentially also in humans
Systemic Infection and Limited Replication of SHIV Vaccine Virus in Brains of Macaques Inoculated Intracerebrally with Infectious Viral DNA

AbstractSHIV deleted in two accessory genes, ΔvpuΔnef SHIVPPC, functioned well as a vaccine against later challenge with highly pathogenic SHIVKU, and it was able to reach the brain after oral inoculation of live virus. In this study, the proviral genome cloned into a plasmid was inoculated as DNA intracerebrally and spread systemically. Few regions of the brain had detectable proviral DNA by real-time PCR. Two measures of virus replication, detection of viral mRNA expression and circular proviral DNA, were negative for those brain regions, with the exception of the infection site in the right parietal lobe, whereas lymphoid tissues were positive by both measures. Histopathological analyses of all the sampled brain and spinal cord regions did not reveal any abnormalities. Despite intracerebral inoculation of the viral DNA, the brain was not targeted for high levels of virus replication
Extinction and Rapid Emergence of Strains of Dengue 3 Virus during an Interepidemic Period

AbstractStrains of dengue 3 (DEN-3) virus circulating in Thailand prior to 1992 appear to have disappeared from that location and to have been replaced by two new lineages which have evolved locally, rather than being introduced. Similar DEN-3 virus extinctions may have occurred previously in Thailand in 1962 and 1973. Although no causal relationship could be shown, this strain replacement event was accompanied by DEN-3 replacing DEN-2 as the serotype recovered most frequently from patients in Thailand. Although this implies a change in selection pressure, we found no evidence for positive natural selection at the level of either the E protein or the E protein gene. Further, the extinction of the pre-1992 strains and the appearance of the new lineages occurred during an interepidemic period, suggesting that a genetic bottleneck, rather than selection, might have been important in the emergence of these two new strains of virus. The pre-1992 DEN-3 virus lineage could still be found in 1998, to the west, in Myanmar. The ratio of nonsynonymous-to-synonymous nucleotide changes within a DEN-3 virus population from a single patient was less than the ratio among the consensus sequences of DEN-3 viruses from different patients, suggesting that many of the nonsynonymous nucleotide changes which occurred naturally in the E protein were deleterious and removed by purifying selection
Mutational Analysis of the Replication Signals in the 3′-Nontranslated Region of Citrus Tristeza Virus

AbstractCitrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb messenger-sense RNA genome consisting of 12 open reading frames with nontranslated regions (NTR) at the 5′ and 3′ termini. The 273 nucleotide (nt) 3′-NTR is highly conserved (∼95%) among the sequenced CTV isolates in contrast to the highly diverse 5′-NTR sequences. The 3′ replication signals were mapped to the 3′ 234 nts within the NTR. This region of CTV does not contain a poly-A tract nor does it appear to fold as a tRNA-mimic. Instead, a computer-predicted thermodynamically stable secondary structure comprised of 10 stem-and-loop (SL) structures, referred to as SL1 to SL10 (5′ to 3′), was common to all CTV isolates. This putative structure was used as a guide to examine the 3′ requirements for replication in vivo. The resulting data suggest that a complex 3′ structure is required for those functions that provide for efficient replication of CTV in vivo such as minus-strand initiation, regulation of strand asymmetry, effective translation of the myriad of viral mRNAs, or stability of RNAs. Deletions into the 3′-NTR, up to 66 nts from the 5′ direction and 11 nts from the 3′ direction, deleting or disrupting putative SL1, SL2 and SL3, or SL10, resulted in continued replication, suggesting that these sequences are not essential for basal-level replication, but are required for efficient replication. Predicted stem loops 3 through 10 were examined by mutations designed to alter the primary structures while preserving the secondary structures. Mutations designed to disrupt the predicted stems of SL3, SL5, SL7, SL9, or SL10 resulted in substantially reduced levels of replication, while compensatory mutations resulted in partial restorations of replication, suggesting that these predicted secondary structures are involved in replication. Also, the putative loop sequences of SL5, SL6, SL7, and SL9 tolerated mutagenesis with continued but reduced levels of replication. In contrast, all mutations introduced into putative SL4, SL8, and the stem of SL6 prevented replication, suggesting that the primary structure of these regions make up the core of the 3′ replication signal. The 3′ triplet, CCA, was shown to be necessary for efficient replication, but deletion of eleven nts to expose an internal CCA resulted in continued replication
Murine Coronavirus Spike Glycoprotein Mediates Degree of Viral Spread, Inflammation, and Virus-Induced Immunopathology in the Central Nervous System

AbstractThe mouse hepatitis virus (MHV) spike glycoprotein is a major determinant of neurovirulence. We investigated how alterations in spike affect neurovirulence using two isogenic recombinant viruses differing exclusively in spike. S4R, containing the MHV-4 spike gene, is dramatically more neurovirulent than SA59R, containing the MHV-A59 spike gene (J. J. Phillips, M. M. Chua, E. Lavi, and S. R. Weiss, 1999, J. Virol. 73, 7752–7760). We examined the contribution of differences in cellular tropism, viral spread, and the immune response to infection to the differential neurovirulence of S4R and SA59R. MHV-4 spike-mediated neurovirulence was associated with extensive viral spread in the brain in both neurons and astrocytes. Infection of primary hippocampal neuron cultures demonstrated that S4R spread more rapidly than SA59R and suggested that spread may occur between cells in close physical contact. In addition, S4R infection induced a massive influx of lymphocytes into the brain, a higher percentage of CD8+ T cells, and a higher frequency of MHV-specific CD8+ T cells relative SA59R infection. Despite this robust and viral-specific immune response to S4R infection, infection of RAG1−/− mice suggested that immune-mediated pathology also contributes to the high neurovirulence of S4R
Human Immunodeficiency Virus Type 1 Reverse Transcription Is Stimulated by Tat from Other Lentiviruses

AbstractThe tat gene is required by HIV-1 for efficient reverse transcription and this function of Tat can be distinguished from its role in transcription by RNA polymerase II using tat point mutations that abrogate each function independently. The mechanism of Tat's role in reverse transcription, however, is not known, nor is it known whether this role is conserved among trans-activating factors in other retroviruses. Here we examine the abilities of heterologous viral trans-activating proteins from jembrana disease virus (jTat), HIV-2 (Tat2), and equine infectious anemia virus (eTat) to substitute for HIV-1 Tat (Tat1) and restore reverse transcription in HIV-1 carrying an inactivated tat gene. Natural endogenous reverse transcription assays showed that trans-activators from some retroviruses (Tat2 and jTat, but not eTat) could substitute for Tat1 in complementation of HIV-1 reverse transcription. Finally, we show that Y47 is critical for Tat1 to function in reverse transcription, but not HIV-1 gene expression. We mutated the homologous position in jTat to H62Y and found it did not improve its ability to stimulate reverse transcription, but an H62A mutation did inhibit jTat complementation. These data highlight the finding that the role of Tat in reverse transcription is not related to trans-activation and demonstrate that other tat genes conserve this function
Physical and Functional Interaction between the Varicella Zoster Virus IE63 and IE62 Proteins

AbstractThe varicella zoster virus (VZV) IE63 protein is required for growth of the virus in cell culture and is expressed during both lytic and latent phases of VZV infection. We have investigated the physical and functional interaction of this protein with the major VZV transactivating protein IE62. The region of the IE63 protein required for interaction with the IE62 protein has been identified and encompasses the N-terminal 142 amino acids. We have found that the interaction is stable at physiological ionic strength. We have also shown that a portion of the IE63 and IE62 proteins colocalize in VZV-infected cells at both 15 and 48 h postinfection. IE63 was found to have no transcriptional activating or repressing activity within the context of a minimal VZV glycoprotein promoter. The presence of the IE63, however, upmodulated the IE62 transactivation of this promoter. Finally, we show that the IE63 protein can be coimmunoprecipitated with the cellular RNA polymerase II from infected cell extracts, indicating that it is present in a complex with that enzyme