pinecone/core-2020-05-10-deduplication huggingface dataset:
The Satellite RNA of Barley Yellow Dwarf Virus-RPV Is Supported by Beet Western Yellows Virus in Dicotyledonous Protoplasts and Plants

AbstractThe subgroup II luteovirus barley yellow dwarf virus-RPV (BYDV-RPV) acts as a helper virus for a satellite RNA (satRPV RNA). The subgroup II luteovirus beet western yellows virus (BWYV) and the ST9-associated RNA (ST9a RNA), a BWYV-associated RNA that encodes a polymerase similar to those of subgroup I luteoviruses, were assayed for their ability to support replication of satRPV RNA. SatRPV RNA was replicated in tobacco protoplasts in the presence of BWYV RNA or a mixture of BWYV plus the ST9a RNA, but not in the presence of ST9a RNA alone. ST9a RNA stimulated BWYV RNA accumulation which, in turn, increased the accumulation of satRPV RNA. SatRPV RNA was encapsidated in BWYV capsids primarily as circular monomers, which differs from the linear monomers found in BYDV (RPV + PAV) particles. SatRPV RNA was transmitted toCapsella bursa-pastorisplants by aphids only in the presence of BWYV and ST9a RNA. SatRPV RNA reduced accumulation of both BWYV helper and ST9a nonhelper RNAs in plants but did not affect symptoms. The replication of satRPV RNA only in the presence of subgroup II luteoviral RNAs but not in the presence of RNAs with subgroup I-like polymerase genes, in both monocotyledonous and dicotyledonous hosts, suggests that the specificity determinants of satRPV RNA replication are contained within the polymerase genes of supporting viruses rather than in structural genes or host plants
Cucumber Mosaic Virus Is Restricted from Entering Minor Veins in Transgenic Tobacco Exhibiting Replicase-Mediated Resistance

AbstractTransgenic tobacco plants expressing an altered form of the 2a replicase gene from cucumber mosaic virus (CMV) strain Fny exhibited a suppression of viral replication and restricted viral movement when inoculated mechanically or by insect vectors. Resistant plants could be infected, however, through a graft-union with an infected nontransformed plant. The infectious entity moved quickly through intergrafts of resistant tissue, indicating that it could move without replicating in the vascular system. Viral replication continued to be suppressed in systemically infected transgenic portions of grafted plants, as demonstrated by the synthesis of lower levels of viral RNA than in systemically infected nontransformed portions of the same grafted plants. Cell-to-cell spread within this tissue also occurred much more slowly than in nontransformed tobacco. Young inoculated leaves of transgenic-resistant plants exhibited limited cell-to-cell virus movement, revealed as chlorotic lesions, but no long-distance virus movement occurred. The results ofin situhybridization studies on these lesions indicated that CMV RNA does not traffic from bundle-sheath cells to vascular parenchyma or companion cells in chlorotic lesions on the inoculated leaves of transgenic-resistant tobacco plants. The inhibition of long-distance movement was a consequence of restricted entry of the infectious entity into the vascular system
A Cell-Free Stock of Simian–Human Immunodeficiency Virus That Causes AIDS in Pig-Tailed Macaques Has a Limited Number of Amino Acid Substitutions in Both SIVmacand HIV-1 Regions of the Genome and Has Altered Cytotropism

AbstractWe have examined both the sequence changes in the LTR,gag, vif, vpr, vpx, tat, rev, vpu, env,andnefgenes and the cell tropism of a cell-free stock of chimeric simian–human immunodeficiency virus (SHIV) isolated from the cerebrospinal fluid of a pig-tailed macaque (PNb) that developed AIDS. This virus (SHIVKU-1) is highly pathogenic when inoculated into other macaques. DNA sequence analysis of PCR-amplified products revealed a total of 5 nucleotide changes in the LTR whilevifhad 2 consensus amino acid changes. Thegag, vif,andvpxhad no consensus amino acid substitutions, whereasvprhad 1 consensus substitution. Thetatandrevgenes of the HXB2 region of SHIVKU-1had 2 and 1 consensus amino acid changes, respectively. Thevpugene of the HXB2 region of SHIV, which originally had an ACG codon at the beginning of the gene, reverted to an initiation ATG codon and in addition contained a consensus amino acid substitution at position 69 of this protein. As expected, the majority of the nucleotide substitutions were found in theenvandnefgenes. Thirteen and 5 amino acid changes were predicted for the corresponding Env and Nef proteins, respectively. In addition, one-third of theenvgene clones isolated from the SHIVKU-1stock had a 5-amino-acid deletion in the V4 region. Using three independent assays, we determined that the changes in the SHIVKU-1were associated with an increase in the efficiency of replication in macrophages. The strikingly few consensus changes in the virus suggest that conversion of this virus to one capable of causing AIDS in pig-tailed macaques was associated with relatively few changes in the viral envelope and/or accessory genes. These results will provide the basis for the development of a pathogenic, molecular clone of SHIV capable of causing AIDS in pig-tailed macaques
Protection against Influenza Virus Encephalitis by Adoptive Lymphocyte Transfer

AbstractCD8+ cytotoxic T lymphocytes (CTL) have an established role in anti-viral immunity, but whether CTL function efficiently in the brain remains unclear. In particular, virus-infected neurons, which express only low levels of MHC class I antigens and are resistant to the induction of apoptosis, could constitute a relatively intractable CTL target. We have used immune lymphocytes adoptively transferred into the CSF to protect naive mice against an intracerebral infection with influenza A/WSN, a virus that infects neurons in the brain parenchyma and causes a lethal encephalitis. Afterin vitrorestimulation, heterotypically immune spleen cells protected against A/WSN encephalitis in an H-2-restricted, CD8-dependent, CD4-independent manner. Adoptively transferred CTL clones were also protective. Homotypically immune spleen cells additionally mediated CD8-independent, H-2-unrestricted protection, probably due to the generation of A/WSN-specific plasma cells from memory B cells duringin vitrorestimulation. Thus afterin vitrorestimulation, either CTL or B cells adoptively transferred into the CSF protected against an acutely lethal intracerebral virus infection
A Hybrid Baculovirus-T7 RNA Polymerase System for Recovery of an Infectious Virus from cDNA

AbstractWe established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells. Two recombinant baculoviruses carrying cDNA of bacteriophage T7 RNA polymerase, with or without a nuclear localization signal, under the control of a mammalian promoter were constructed. High level expression of T7 RNA polymerase was observed in various mammalian cell lines after infection with the recombinant baculoviruses. After transfection of plasmids containing the luciferase gene under the control of the T7 promoter, high luciferase activity was detected in cells infected with the recombinant baculoviruses. We also constructed a plasmid containing an entire cDNA clone of type 1 poliovirus under the T7 promoter. Two days after transfection of the plasmid into the cells infected with the recombinant baculoviruses, a high titer of poliovirus was recovered. The use of the recombinant baculoviruses did not cause any cytopathic effects even at a high multiplicity of infection. The lack of replication ability and low toxicity are the advantageous features of the hybrid baculovirus–T7 polymerase system in comparison with the widely used vaccinia–T7 polymerase system for gene expression and recovery of infectious viruses from its cDNA
Amplification of Recombinant Adenoviral Transgene Products Occurs by Inhibition of Histone Deacetylase

Abstractn-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5–5 mM,and that the amplification required an exposure of 12–24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level
Late Promoter Selection in the Baculovirusgp64 Envelope Fusion ProteinGene

AbstractThe upstream promoter region of theAutographa californicamulticapsid nuclear polyhedrosis virus (AcMNPV)gp64gene contains five copies of TAAG, the conserved sequence found at the transcriptional initiation sites of almost all baculovirus late genes. In AcMNPV-infected Sf9 cells, late transcription initiation is detected from only two upstream TAAG sites and not from three downstream TAAG sites. To examine several models for preferential TAAG site utilization, we constructed a series of recombinant AcMNPV baculoviruses that contain promoter region sequences from thegp64gene fused to achloramphenicol acetyl transferasereporter gene. Promoter–reporter constructs were inserted in the polyhedrin locus. To test a scanning model in which TAAG sites are sequentially selected according to their location in the region, we generated recombinant viruses in which the highly transcribed sites were inactivated by point mutations. Transcription from the mutant promoter constructs was compared qualitatively and quantitatively to transcription from the wild-typegp64promoter. Inactivation of the upstream TAAG sites did not result in increased transcription from the downstream TAAG sites, suggesting that immediate context, rather than position, determines promoter utilization. To test this hypothesis, we made a series of minimal promoter constructs containing decreasing quantities of the sequences immediately flanking one of the activegp64TAAG sites. Reporter constructs containing agp64TAAG site and ≥12 bp of flanking sequence on both sides were transcribed at near wild-type levels. Constructs with less flanking sequence (9 or 6 bp of flanking sequence) were accurately transcribed, but at substantially lower levels, and transcription was not detected from constructs containing only 3 bp of flanking sequence. These results suggest that nucleotides immediately flanking the TAAG site (4–6 bp) are necessary for basal promoter activity while additional flanking sequences (≥12 bp) are required for late promoter activation and regulation. To further examine late promoter selection, we constructed recombinant AcMNPV baculoviruses that contain heterologous late promoters from thegp64gene of the related virusOrgyia pseudotsugataMNPV (OpMNPV). TAAG sites that serve as functional late promoters in OpMNPV were found to mediate transcription initiation at only basal levels in the context of the AcMNPV genome, suggesting that late promoter activation may be virus specific within the familyBaculoviridae
Phosphorylation of Both Phosphoacceptor Sites in the HIV-1 Vpu Cytoplasmic Domain Is Essential for Vpu-Mediated ER Degradation of CD4

AbstractHuman immunodeficiency virus type 1 (HIV-1) Vpu is phosphorylated at two serine residues (Ser52 and Ser56) present within the acidic dodecapeptide region of the 54-aa cytoplasmic domain. Previous experiments have shown that Vpu phosphorylation is critical for the degradation of CD4 in the endoplasmic reticulum. In this study, we carried out experiments to elucidate the role of individual phosphoacceptor sites in CD4 proteolysis. We show here that acidic amino acids could not functionally substitute for phosphoserines in Vpu that is capable of inducing the degradation of CD4. Our studies have further revealed that phosphorylation of either of the two phosphoacceptor sites is not sufficient to generate a functional Vpu protein. When tested for functional complementation, inactive phosphorylation-proficient Vpu mutants failed to generate Vpu proteins that had the ability to induce the degradation of Vpu-sensitive glycoproteins. The failure to complement was not due to assembly defects in the Vpu protein as unphosphorylated Vpu formed oligomeric complexes in the cell. We also showed that Vpu expression inhibits protein transport in a phosphorylation-dependent manner. Our studies have thus revealed that both phosphoserines in Vpu are critical participants in a pathway that leads to the proteolysis of CD4 in the ER and that these phosphoserines should be present on the same subunit of the Vpu protein
Mutations Affecting Lysine-35 of gpNu1, the Small Subunit of Bacteriophage λ Terminase, Alter the Strength and Specificity of Holoterminase Interactions with DNA

AbstractThe small subunit of λ terminase, gpNu1, contains a low-affinity ATPase activity that is stimulated by nonspecific dsDNA. The location of the gpNu1 ATPase center is suggested by a sequence match between gpNu1 (29-VLRGGGKG-36) and the phosphate-binding loop, or P-loop (GXXXXGKT/S), of known ATPases. The proposed P-loop of gpNu1 is just downstream of a putative helix–turn–helix DNA-binding motif, located between residues 5 and 24. Published work has shown that changing lysine-35 of the proposed P-loop of gpNu1 alters the response of the ATPase activity to DNA, as follows. The changes gpNu1 K35A and gpNu1 K35D increase the level of DNA required for maximal stimulation of the gpNu1 ATPase by factors of 2- and 10-fold, respectively. The maximally stimulated ATPase activities of the mutant enzymes are indistinguishable from that of the wild-type enzyme. In the present work, the effects of changing lysine-35 on thecos-cleavage and DNA-packaging activities of terminase were examined.In vitro,the gpNu1 K35A enzyme cleavedcosas efficiently as the wild-type enzyme, but required a 2-fold increased level of substrate DNA for saturation, suggesting a slight reduction in DNA affinity. In a crude DNA-packaging system using cleaved λ DNA as substrate, the gpNu1 K35A enzyme had a 10-fold defect.In vivo,λNu1K35A showed a 2-fold reduction incoscleavage, but no packaged DNA was detected. The primary defect of the gpNu1 K35A enzyme was concluded to be in a post-cos-cleavage step of DNA packaging. Inin vitro cos-cleavage experiments, the gpNu1 K35D enzyme had a 10-fold increased requirement for saturation by substrate DNA. Furthermore, thecos-cleavage activity of gpNu1 K35D enzyme was strongly inhibited by the presence of nonspecific DNA, indicating that the gpNu1 K35D enzyme is unable to discriminate effectively betweencosand nonspecific DNA. Nocoscleavage was observedin vivofor λNu1K35D, a result consistent with the discrimination defect foundin vitrofor the gpNu1 K35D enzyme. In a crude packaging system the gpNu1 K35D enzyme had a 200-fold defect; in a purified packaging system, the gpNu1 K35D enzyme was found to be unable to discriminate between λ DNA and nonspecific phage T7 DNA, a result indicating that the gpNu1 K35D enzyme is also defective in discriminating between λ DNA and nonspecific DNA during DNA packaging
The Structure of Cucumber Mosaic Virus: Cryoelectron Microscopy, X-Ray Crystallography, and Sequence Analysis

AbstractThe three-dimensional structure of cucumber mosaic virus (CMV) was analyzed at 23 Å resolution by cryoelectron microscopy and image reconstruction, demonstrating structural similarity to cowpea chlorotic mottle virus (CCMV), another member of theBromoviridaefamily. The CMV structure was determined at 8 Å resolution by X-ray crystallography with phases determined by single isomorphous replacement and refined by fivefold noncrystallographic symmetry averaging. The X-ray structure agreed with the electron microscopy reconstruction; the electron density is consistent with β-barrel subunits arranged withT= 3 quasi-symmetry in an orientation similar to that observed in CCMV. Strong density surrounding the icosahedral threefold axes (quasi sixfold axes in theT= 3 particle) between 80 and 100 Å from the particle center formed a cylinder of radius 11 Å, similar to the density observed in the same region of CCMV. This density corresponds to the β-annulus of CCMV, which differentiates hexamers from pentamers and determines the formation of theT= 3 particles. The CMV and CCMV amino acid sequences were aligned, providing information (based on the CCMV atomic model) about the probable distribution of residues in the three-dimensional structure of CMV
Effects of Coat Protein Mutations and Reduced Movement Protein Expression on Infection Spread by Cowpea Chlorotic Mottle Virus and Its Hybrid Derivatives

AbstractPreviously we have reported that the essential 3a movement gene of icosahedral cowpea chlorotic mottle virus (CCMV) can be functionally replaced by the 30-kDa movement gene of rod-shaped sunn-hemp mosaic virus (SHMV). Because plant RNA viruses differ in requiring or not requiring coat protein for systemic infection, we have now investigated whether systemic spread by this CCMV/SHMV hybrid is dependent on its CCMV coat protein as well as its SHMV movement protein. We find that either deletion or frameshift mutations in the coat protein gene block systemic spread. Thus, like wild-type CCMV, systemic infection by the hybrid is dependent on both movement protein and coat protein. These results further support the conclusion that the required functions of the coat and movement proteins in CCMV spread do not depend on sequence-specific interaction between these proteins. Additional features of the hybrid also motivated testing the effects of modulating movement protein expression. Creating an extra, out-of-frame translational start codon (AUG) shortly upstream of the 3a movement protein gene in CCMV downregulated its expression 18-fold. Nevertheless, for CCMV derivatives bearing either the CCMV 3a gene or the SHMV 30-kDa gene, the extra AUG resulted in only a minor delay in the onset of viral spread and little or no effect on the subsequent rate of cell-to-cell spread. Thus, under normal circumstances, the rate of CCMV cell-to-cell spread in cowpea plants appears to be limited primarily by factors other than movement protein synthesis
The NS2 Polypeptide of Parvovirus MVM Is Required for Capsid Assembly in Murine Cells

AbstractMutants of minute virus of mice (MVM) which express truncated forms of the NS2 polypeptide are known to exhibit a host range defect, replicating productively in transformed human cells but not in cells from their normal murine host. To explore this deficiency we generated viruses with translation termination codons at various positions in the second exon of NS2. In human cells these mutants were viable, but showed a late defect in progeny virion release which put them at a selective disadvantage compared to the wildtype. In murine cells, however, duplex viral DNA amplification was reduced to 5% of wildtype levels and single-strand DNA synthesis was undetectable. These deficiencies could not be attributed to a failure to initiate infection or to a generalized defect in viral gene expression, since the viral replicator protein NS1 was expressed to normal or elevated levels early in infection. In contrast, truncated NS2 gene products failed to accumulate, so that each mutant exhibited a similar NS2-null phenotype. Expression of the capsid polypeptides VP1 and VP2 and their subsequent assembly into intact particles were examined in detail. Synchronized infected cell populations labeled under pulse–chase conditions were analyzed by differential immunoprecipitation of native or denatured extracts using antibodies which discriminated between intact particles and isolated polypeptide chains. These analyses showed that at early times in infection, capsid protein synthesis and stability were normal, but particle assembly was impaired. Unassembled VP proteins were retained in the cell for several hours, but as the unprocessed material accumulated, capsid protein synthesis progressively diminished, so that at later times relatively few VP molecules were synthesized. Thus in NS2-null infections of mouse cells there is a major primary defect in the folding or assembly processes required for effective capsid production
A Human IgG1 (b12) Specific for the CD4 Binding Site of HIV-1 Neutralizes by Inhibiting the Virus Fusion Entry Process, but b12 Fab Neutralizes by Inhibiting a Postfusion Event

AbstractThe human b12 IgG1, specific for the CD4 binding site of the gp120 of HIV-1, was prepared by recombinant DNA technology. It had a high neutralization rate constant (−3.5 × 105M−1sec−1), although this is about 10-fold less than the values for the best poliovirus or influenza A virus MAbs. The recombinant b12 Fab neutralized well, with about one-tenth of the activity of b12 IgG. The mechanisms by which b12 IgG1 and its Fab neutralize HIV-1 IIIB on C8166 cells have been investigated. Neither inhibited attachment of virus to the target cell as judged by FACS, immunofluorescence, and ELISA data. This was controlled using MAb F105, another human IgG1, that did neutralize by inhibiting attachment under our conditions. The interactions of b12 IgG- and Fab-neutralized virions with target cells were compared with those of nonneutralized virus using a number of different techniques (fluorescence dequenching of R18-labeled virions, immunofluorescence of virion gp41 and p24 antigens, and acquisition of resistance to removal of virions from the cell by protease). These and the inhibition of HIV-1-mediated cell–cell fusion all demonstrated that b12 IgG neutralized by inhibiting the primary fusion-uncoating mechanism. However, the interactions of b12 Fab-neutralized and nonneutralized virions with C8166 cells were indistinguishable. Thus b12 Fab did not inhibit fusion uncoating, and by inference inhibited a stage of infection that occursafterthe entry of the virion core into the cytoplasm. It is therefore possible that b12 IgG kills HIV-1 twice over, by fusion-inhibition and by inhibiting the postentry event proposed for the Fab. The mechanism of neutralization of b12 Fab and of other MAbs that neutralize in a similar way and why b12 Fab and IgG neutralize by different mechanisms are discussed