pinecone/core-2020-05-10-deduplication huggingface dataset:
Construction and Characterization of a Replication-Defective Herpes Simplex Virus 2 ICP8 Mutant Strain and Its Use in Immunization Studies in a Guinea Pig Model of Genital Disease

AbstractA replication-defective mutant of herpes simplex virus 2 (HSV-2) was engineered by replacing the ICP8 gene of HSV-2 strain 186 with an ICP8–lacZ fusion gene from the herpes simplex virus 1 (HSV-1) HD-2 mutant strain. The resulting virus, HSV-2 5BlacZ, is defective for growth in Vero cells but is capable of growth in a cell line that expresses HSV-1 ICP8. In Vero cells, the mutant virus is defective for DNA synthesis but is able to express many viral proteins at levels similar to those of wild-type virus, including several of the late kinetic class. SDS–PAGE and Western blot analysis demonstrated the expression of glycoproteins B and D by 5BlacZ in Vero cells. Initial studies have shown that immunization with 5BlacZ protects guinea pigs from intravaginal HSV-2 challenge. Immunized animals had less severe genital skin disease and reduced replication of the challenge virus in the genital tract during primary infection and reduced episodes of recurrent disease. Thus, HSV-2 ICP8 shows gene regulatory properties similar to those of HSV-1 ICP8, and this HSV-2 ICP8 mutant virus shows a phenotype similar to those of HSV-1 ICP8 mutant strains. Replication-defective mutants of HSV-2 offer a potential vaccine approach for immune intervention against HSV-2 genital disease and latent infection
Mutations That Alter a Conserved Element Upstream of the Potato Virus X Triple Block and Coat Protein Genes Affect Subgenomic RNA Accumulation

AbstractThe putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation ofNicotiana tabacumNT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process
The Long Terminal Repeats of Human Immunodeficiency Virus Type-1 and Human T-cell Leukemia Virus Type-I Are Activated by 12-O-Tetradecanoylphorbol- 13-acetate through Different Pathways

AbstractThe LTRs of HIV-1 and HTLV-I have been shown by several laboratories to be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). This agent is a potent activator of protein kinase C (PKC). However, long exposure to TPA downregulates PKC in many cell types. We demonstrated that TPA treatment of Jurkat cells for more than 24 hr resulted in a sever depletion of this enzyme. Therefore, to explore the role of PKC in the effect of TPA on these LTRs, we transfected Jurkat cells with HIV-1 LTR-CAT or HTLV-I LTR-CAT construct after 72 hr of TPA pretreatment. While this TPA pretreatment considerably reduced the HIV-1 LTR basal expression, it strongly stimulated the expression of HTLV-I LTR. Furthermore, when TPA was added after transfection, a strong stimulation of HIV-1 LTR was observed, which could be abrogated by PKC inhibitors like H7 and chelerythryn. However, under these conditions TPA stimulated HTLV-I LTR to a lesser extent than did the long-term TPA pretreatment. Moreover, this stimulation was enhanced by the PKC inhibitors. Thus our data indicate that while the effect of TPA on HIV-1 LTR is strictly dependent on PKC activity, its effect on HTLV-I LTR is exerted via a different pathway that not only does not require PKC activation but rather seems to be antagonized by the activated PKC. Using a deletion mutant of HTLV-I LTR we mapped the PKC-independent effect of TPA to the c-ets responsive region 1 (ERR-1) located in U3 of this LTR
Replacement of Posttranscriptional Regulation in SIVmac239 Generated a Rev-Independent Infectious Virus Able to Propagate in Rhesus Peripheral Blood Mononuclear Cells

AbstractLentiviruses control virion production via posttranscriptional regulation mediated by the viral Rev protein. In this study, we demonstrate that the Rev regulation of SIVmac239 can be replaced by the presence of the cis-acting transport element (CTE) of the type D simian retroviruses 1 (SRV-1). To avoid the possibility of generating revertants, the Rev-independent SIV clones have bothrevand the Rev responsive element (RRE) destroyed by multiple point mutations that do not affect the overlappingtatandenvopen reading frames. Virus stocks generated from these Rev-independent SIV molecular clones can infect and can be propagated in rhesus peripheral blood mononuclear cells (PBMCs). Therefore, the Rev/RRE system of SIVmac239 can be replaced by the SRV-1 CTE as previously shown for HIV-1. In both rhesus and human primary cells, the replicative capacity of the Rev-independent SIV is 10- to 20-fold lower than that of the wild-type virus. Rhesus PBMC-derived virus stocks of the Rev-independent SIV have lower infectivity. Interestingly, in CEM × 174 cells, no difference in replicative capacity between wild-type and Rev-independent SIV has been observed. The Rev-independent SIV has a stable genotype after several passages in primary cells. The availability of such Rev-independent viruses will allow the study of the role of Rev in pathogenesis and the potential generation of attenuated SIV strains
The Assembly of the Measles Virus Nucleoprotein into Nucleocapsid-like Particles Is Modulated by the Phosphoprotein

AbstractMeasles virus nucleoprotein encoded from the vaccinia virus genome assembles into nucleocapsids similar in many respects to those observed during a natural measles virus infection. The influence of the measles virus phosphoprotein on nucleocapsid assembly has been studied using a vaccinia virus recombinant encoding both the nucleoprotein and the phosphoprotein. Infection of cells with the virus recombinant resulted in the formation of cytoplasmic inclusions in which the nucleoprotein and the phosphoprotein colocalized. Electron microscopic examination suggested that these inclusions contained characteristic nucleocapsid filaments. The buoyant density of nucleocapsids assembled in the presence of the phosphoprotein was found to be slightly higher than that of nucleocapsids assembled in its absence. Furthermore, the phosphoprotein partially inhibited the formation of nucleocapsids, a process which was extremely efficient when the nucleoprotein was expressed alone. Analysis of the nucleic acid content of nucleocapsids showed that they packaged heterologous RNA into a micrococcal nuclease-resistant form. These experiments demonstrate that the measles virus phosphoprotein regulates the efficiency with which the nucleoprotein assembles into nucleocapsids and the structural conformation they acquire
The N-Terminal Portion of the 216-kDa Polyprotein ofCommelinaYellow Mottle Badnavirus Is Required for Virus Movement but Not for Replication

AbstractCommelinayellow mottle virus (CoYMV) is the type member of the badnaviruses, a genus of plant pararetroviruses. The N-terminus of the polyprotein encoded by ORF III has limited similarity to known cell-to-cell movement proteins. To test the hypothesis that the N-terminus is required for viral movement, the phenotypes caused by mutations constructed in this region were determined. Similar to mutants affected in the reverse transcriptase, mutants affected in the putative movement protein were unable to cause a systemic infection. However, when the abilities of the mutated viral genomes to direct virion assembly and replication were tested using anin vitrostem-culture system, the mutants affected in the putative movement protein were found to assemble virions, whereas the reverse transcriptase mutants were unable to do so. Moreover, the putative movement protein mutants were shown to be replication competent by detection and mapping of one of the genomic discontinuities that are the hallmark of replication by reverse transcription. Thus the N-terminal region of ORF III is required for the systemic movement but not for the replication of CoYMV
Changes in the Dengue Virus Major Envelope Protein on Passaging and Their Localization on the Three-Dimensional Structure of the Protein

AbstractTo help define the molecular events involved in dengue virus adaptation during serial passagein vivoand in cultured cells, we have sequenced the structural protein genes of three dengue type 3 isolates after intracerebral passage in mice and after passage in cultured monkey kidney (Vero) andAedes albopictus(mosquito) cells. Passaging in each host selected for amino acid changes in the envelope protein E and occasionally in prM but not in the capsid protein. Most changes were first apparent within five passages. Nineteen of twenty mutations in the structural protein genes resulted in amino acid changes concentrated on 12 residues; 9 of the 12 amino acid changes were at residues which are conserved between the four dengue virus serotypes. Certain amino acid changes were repeatedly selected on passage in cell culture. In six independent Vero cell passage series, changes were observed in E at residues 191 (four times), 202 (twice), 266 and 268 (three times), and 291; change in prM was seen in two passage series at residue 26. Two independent passage series in mosquito cells each resulted in the loss of a conserved glycosylation site at Asn153 in E. Passage in mouse brain selected for mutations at E residues 18, 54, 277, 401, and 403. Residues which altered on passaging have been localized on the three-dimensional structure of the tick-borne encephalitis virus E protein soluble fragment (F. A. Rey,et al.,1995,Nature375, 291–298). Residues 54, 191, 202, 266, 268, and 277 map to a postulated “hinge” region between domains I and II which may be involved in fusion of flaviviruses with cell membranes. The oligosaccharide at Asn153 also appears to be involved in flavivirus fusion. Changes in the fusion characteristics of the passaged viruses were demonstrated
Replication Signals in the Genome of Vesicular Stomatitis Virus and Its Defective Interfering Particles: Identification of a Sequence Element That Enhances DI RNA Replication

AbstractWe have analyzed the role of terminal sequences of a defective interfering (DI) particle RNA of vesicular stomatitis virus (VSV) in replication. A series of internal deletion mutants of DI cDNA was generated to obtain DI genomic RNAs that differed from one another by the presence of different lengths of 3′-terminal and/or 5′-terminal sequences. Analyses of the mutant RNAs for their ability to replicate in cells transfected with the corresponding plasmids suggested that distinct regions at the termini of DI RNA are important for RNA replication. Region I, encompassing nucleotides 1–24, is absolutely required for replication since DI RNA genomes lacking any part of this region failed to replicate. Region II, spanning nucleotides 25–45, is not essential for replication but it functions as an enhancer of replication in that the presence of these specific sequences confers high efficiency of replication to the template. Deleting these specific sequences from both termini of DI RNA but maintaining the length of terminal complementarity as seen in wild-type DI RNA resulted in a template that replicated poorly (about 20-fold less efficiently). Furthermore, insertion or substitution of these sequences into the 3′-terminus of a VSV minigenome resulted in a template that replicated more efficiently (at least 4-fold to as high as 15-fold) than the parental minigenome. These results strongly support the conclusion that the presence of specific sequences rather than the extent of complementarity at the termini of DI RNA is a major determinant of the efficiency of replication. The presence of the specific sequences at the 3′-terminus of both genomic and antigenomic DI RNAs may explain in part the replicative dominance of DI RNA over the full-length VSV genome which contains these sequences only at the 3′-terminus of the antigenome
DNA Replication Promotes High-Frequency Homologous Recombination duringAutographa californicaMultiple Nuclear Polyhedrosis Virus Infection

AbstractThe relative ease with which foreign genes can be incorporated into the genome of the baculovirusAutographa californicanuclear polyhedrosis virus (AcMNPV) indicates that a highly efficient recombinational process exists within infected cells. However, it is unclear whether this is due to marker transfer mediated by host cell enzymes or recombination events promoted by AcMNPV itself. To address the latter possibility, a pair of inverted repeat IS50 elements derived from the bacterial transposon Tn5 was inserted into the polyhedrin gene locus of the AcMNPV genome. Inversion of Tn5 sequences arising from recombination between its IS50 repeats could be readily detected in this virus, indicating that AcMNPV DNA undergoes high-frequency recombination during infection. To further characterize this process, a transient recombination assay was developed and used to identify thecis- andtrans-acting requirements for Tn5 inversion in AcMNPV. A transfected Tn5-containing plasmid was found to undergo the same sequence inversion events seen in the viral genome, but only if it also contained a putative AcMNPV origin of replication (homologous region 2)in cisand was replicated by AcMNPV gene products suppliedin trans.Taken together, these results indicated that recombination events which occur in infected cells were strictly dependent upon AcMNPV-mediated DNA replication. Direct support for this hypothesis was provided by the observation that the minimal set of AcMNPV genes that was essential for plasmid DNA replication also promoted recombination events leading to Tn5 inversion in the absence of any other viral function. Finally, using a panel of deletion mutants of the IS50 elements in Tn5, sequence inversion was shown to be the result of homologous rather than site-specific recombination, since it occurred independently of a discrete sequence within the transposon. These results demonstrate that the AcMNPV DNA replication machinery exhibits a strong propensity to promote homologous recombination events during infection and is likely to play a role in the high frequency of marker transfer observed in this virus
Long Sequences in the 5′ Noncoding Region of Plum Pox Virus Are Not Necessary for Viral Infectivity but Contribute to Viral Competitiveness and Pathogenesis

AbstractThe 5′-terminal 31 nucleotides of the 146-nucleotides-long 5′ noncoding region of plum pox potyvirus (PPV) are highly conserved in all the members of thePotyvirusgenus. To map the sequences of the 5′ noncoding region that are necessaryin vivofor infectivity, we have constructed a nested set of substitution and deletion mutants. While we were not able to infectNicotiana clevelandiiplants with full-length PPV transcripts bearing mutations in the 5′-terminal 35 nucleotides of the viral genome, the deletion of long sequences located between nucleotides 39 and 145 did not alter either the rate of infection or viral accumulation. Nevertheless, these mutants were not able to compete with the wild-type strain in coinoculation experiments. Plants infected with a PPV mutant that lacked nucleotides 127 to 145 showed a very mild symptomathology; the wild-type symptom severity was recovered after spontaneous second-site mutations
Experimental Measles. II. Infection and Immunity in the Rhesus Macaque

AbstractMeasles infection and the host immune response to measles virus were compared using naive and immunized rhesus monkeys. The monkeys were experimentally challenged with a wild-type strain of measles virus inoculated intranasally. After pathogenic virus challenge, measles virus was detected in mononuclear cells of peripheral blood, lymph node, and spleen in naive monkeys and viremia peaked on Day 7. However, only one of five vaccinated monkeys had a low virus titer in peripheral blood mononuclear cells at one time point after challenge. No virus was detected in the lymphoid tissues from an immunized monkey that was euthanized on Day 7 of infection. Measles-specific IgM, IgG, neutralizing antibody, and cytotoxic T lymphocytes were detected in vaccinated monkeys before challenge, but antibody titers were significantly lower in immunized monkeys than in naive monkeys after challenge. Measles-specific IgG antibody and cytotoxic T cell responses were still detected more than 1 year after vaccination or infection. This animal model is useful for the further study of measles pathogenesis, immunosuppression, and immunologic memory
Experimental Measles. I. Pathogenesis in the Normal and the Immunized Host

AbstractAn animal model to study measles pathogenesis and the correlates of protective immunity was established using rhesus monkeys. A measles isolate, obtained during an epidemic of measles in the primate colony at the University of California, Davis, was passaged through rhesus monkeys and amplified in rhesus mononuclear cells to create a pathogenic virus stock. Sequence analysis of the nucleoprotein and hemagglutinin genes of this isolate revealed strong homology with the Chicago 89 strain of measles virus. Conjunctival/intranasal inoculation of juvenile rhesus monkeys with this virus resulted in skin rash, pneumonia, and systemic infection with dissemination to other mucosal sites and to the lymphoid tissues. Inflammation and necrosis occurred in the lungs and lymphoid tissues and many cell types were infected with measles virus on Day 7 postinoculation (p.i.). The most commonly infected cell type was the B lymphocyte in lymphoid follicles. Measles antigen was found in follicular dendritic cells on Day 14 p.i. In contrast to naive monkeys infected with measles virus, animals vaccinated with the attenuated Moraten strain did not develop clinical or pathologic signs of measles after challenge. However, moderate to marked hyperplasia occurred in the lymph nodes and spleen of a vaccinated animal on Day 7 after pathogenic virus challenge, suggesting that an effective measles vaccine limits but does not prevent infection with wild-type measles virus
Measles Virus Infection of Human T Cells Modulates Cytokine Generation and IL-2 Receptor Alpha Chain Expression

AbstractMeasles virus (MV) suppresses specific functions in cells of the immune system and causes a generalized immunosuppression by mechanisms which remain undefined. It has been previously established that mitogen-induced proliferation of peripheral blood mononuclear cells (PBMC) is suppressed by infection with MV. Our current study demonstrates that MV infection inhibits antigen-specific proliferation of T lymphocytes. The inhibition of proliferation was not due to a decrease in IL-2 production. IL-2 production in cultures of infected and uninfected antigen-specific T cells was similar. In contrast, we found that expression of the IL-2Rα subunit was decreased in mitogen-stimulated, MV-infected PBMC and antigen-stimulated, MV-infected T lymphocytes compared to stimulated but noninfected T cells. However, the expression of the IL-2Rβ subunit was not altered in MV-infected T cells. We also examined the influence of MV infection on the production of the cytokines IL-4, IL-6, IL-10, and IFN-γ by T lymphocytes. By comparing infected versus uninfected antigen-specific T cell lines, we found that MV infection of antigen-specific activated T cells caused no substantial change in generation of IFN-γ, IL-6, or IL-10. There was a 50% reduction in IL-4 generation following MV infection. These data indicate that the immunosuppression by acute MV infection is not associated with a generalized inhibition of cytokine production. One mechanism for the suppression of proliferation following acute MV infection may be a block in the expression of the IL-2Rα subunit by activated T cells
The Influenza A Virus M2 Channel: A Molecular Modeling and Simulation Study

AbstractThe M2 protein of influenza virus forms ion channels activated by low pH which are proton permeable and play a key role in the life cycle of the virus. M2 is a 97-residue integral membrane protein containing a single transmembrane (TM) helix. M2 is present as disulfide-linked homotetramers. The TM domain of M2 has been modeled as a bundle of four parallel M2 helices. The helix bundle forms a left-handed supercoil surrounding a central pore. Residue H37 has been implicated in the mechanism of low-pH activation of the channel. Models generated with H37 in a fully deprotonated state exhibit a pore occluded by a ring of H37 side chains oriented toward the lumen of the pore. Models with H37 in a fully protonated state no longer exhibit such occlusion of the pore, as the H37 side chains adopt a more interfacial location. Extended molecular dynamics simulations with water molecules within and at the mouths of the pores support this distinction between the H37-deprotonated and H37-protonated models. These simulations suggest that only in the H37-protonated model is there a continuous column of water extending the entire length of the central pore. A mechanism for activation of M2 by low pH is presented in which the H37-deprotonated model corresponds to the “closed” form of the channel, while the H37-protonated model corresponds to the “open” form. A switch from the closed to the open form of the channel occurs if H37 is protonated midway through a simulation. The open channel is suggested to contain a wire of H-bonded water molecules which enables proton permeability
Molecular Studies on Bromovirus Capsid Protein

AbstractTo determine whether the role of coat protein (CP) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (CCMV) is distinct from that of monocot-adapted brome mosaic bromovirus (BMV), two reporter genes, β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP), were substituted for the CP in a biologically active clone of CCMV RNA3 (C3). Primary leaves ofNicotiana benthamiana, Chenopodium quinoa,and cowpea were co-inoculated with wild-type (wt) CCMV RNA 1 and -2 and either C3/ΔCP-GUS or C3/ΔCP-EGFP and analyzed for GUS activity or the presence of green fluorescence. The visual appearance of infections caused by GUS or EGFP variants indicated that, in CCMV, epidermal cell-to-cell movement can occur without a functional CP. By contrast, inoculation of MP defective variants of C3/ΔCP-GUS or C3/ΔCP-EGFP resulted in subliminal infections. Additional experiments examining the infectivity of wt BMV RNA 1 and -2 and a BMV RNA3 variant bearing the EGFP in the place of CP (B3/ΔCP-EGFP) confirmed previous observations that, unlike CCMV, epidermal cell-to-cell movement of BMV is dependent on the expression of a functional CP. Taken together, the results demonstrate that BMV and CCMV use different mechanisms for initial epidermal cell-to-cell spread, and the individual role played by the respective CP genes in this active process is discussed