pinecone/core-2020-05-10-deduplication huggingface dataset:
Dimerization of Rubella Virus Capsid Protein Is Not Required for Virus Particle Formation

AbstractRubella virus (RV) virions contain, in addition to the RNA genome, a capsid protein (C) and two envelope glycoproteins (E1 and E2). The C protein in isolated virions has been reported to be a disulfide-linked dimer (C2). There are two cysteine residues (Cys-152 and Cys-196) within the C protein. To define the role of disulfide-linked C2 dimer in virion formation, site-directed mutagenesis was used to alter the Cys-152 residue to serine. The association of mutant C protein with envelope glycoproteins was examined by transient expression of the cDNAs in COS cells. Mutation at the Cys-152 residue completely abolished the formation of C2 dimer. However, C2 dimerization does not appear to be important for the assembly of RV structural proteins into virus-like particles
Sendai Virus P Protein Is Constitutively Phosphorylated at Serine249: High Phosphorylation Potential of the P Protein

AbstractPreviously we showed that the Sendai virus P protein (568 aa) in virus-infected cells and in virions was primarily and constitutively phosphorylated on serine(s) in a single tryptic phosphopeptide TP1. By two-dimensional thin-layer electrophoresis and chromatography analysis of tryptic phosphopeptides of several deletion and point mutants of the P protein, we now show that the sole phosphorylation site in TP1 is serine249. Interestingly, when serine249 was deleted or mutagenized alternate potential serine sites were more heavily phosphorylated. A similar effect was observed when the deletion was very close to serine249 (Δ208–236). Mutagenesis of proline250 to alanine abrogated phosphorylation at serine249 suggesting that proline250 is essential for the primary phosphorylation of the P protein. Conceivably, serine249 phosphorylation is mediated by a proline-directed protein kinase. This finding is unusual because a majority of the P proteins from other negative-strand RNA viruses have been shown to be phosphorylated primarily by casein kinase II. Our results demonstrate that the P protein has a strong potency to remain phosphorylated. Based on our previous and present results, we suggest that the phosphorylation sites on P are dependent on the accessibility of phosphatases rather than kinases as all potential sites are about equally competent for phosphorylation. We propose that phosphorylation is important for maintaining the structural integrity of the Sendai virus P protein
Genomic Rearrangement in Genome Segment 12 of Rice Dwarf Phytoreovirus

AbstractThe genome segment 12 (S12) of rice dwarf phytoreovirus (RDV) isolated from the Philippines (RDV-P) and of a variant (RDV-S-6) of RDV severe strain (RDV-S) migrated abnormally slower during polyacrylamide gel electrophoresis than that of the isolate maintained at Hokkaido University (RDV-H). Nucleotide sequence analysis revealed that rearrangement had occurred in these segments, affecting the open reading frame. A polypeptide encoded by S12 (Pns12) of RDV-P had a duplication of 28 amino acids while 1/3 of the carboxyl terminus of Pns12 was deleted in RDV-S-6 by premature termination due to a frameshift. RDV-S is always present in plants infected with the RDV-S-6 variant, suggesting that Pns12 of RDV-S-6 is defective. On the other hand, Pns12 of RDV-P was expressed and appeared to be functional in infected cells in spite of the duplication, as demonstrated by immunoblot analyses using antibody raised against Pns12 expressed inEscherichia coli
Intracellular Immunization against SIVmacUtilizing a Hairpin Ribozyme

AbstractA hairpin ribozyme targeting the 3′ LTR region (9456) of SIVmac239was cloned into a murine retroviral vector. This target sequence is conserved among various SIV, as well as most HIV-2, strains. The ribozyme cassette is driven from a polymerase III promoter, that of the human tRNAval gene. Hybrid human B-/T-cell lines (CEM/174) were transduced with the retroviral constructs and selected for G418 resistance. Cells stably expressing the 9456 ribozyme exhibited long-term resistance to infection by a pathogenic molecular clone of SIV and two strains of HIV-2. The ribozyme was also able to effectively reduce the proviral DNA burden. Its efficient protection against SIV/HIV-2 infection constitutes an important step toward evaluating ribozyme gene therapy in a primate model
Down-Modulation of HIV-1 LTR Activity by an Extra-LTRnefGene Fragment

AbstractThe objective of this study was to assess the inhibitory effect of potential negative regulatory elements on human immunodeficiency virus (HIV)-1 long terminal repeat (LTR) activity. This was carried out by pairwise comparisons of reporter gene activities of HIV–LTR–CAT constructs differing in the presence and absence ofnefsequences in transient transfection assays. Parallel transfections were performed in two persistently HIV-infected cell lines and the uninfected parental cell lines. The negative regulatory element (NRE) of the LTR did not suppress HIV LTR activity in any of the cell lines examined. However, a non-LTR-derived fragment of thenefgene had a distinct suppressive effect on activity of the full-length LTR in chronically infected astrocytoma cells. A weaker negative effect of thisnefpartial sequence (nps) was detected in the other cell lines with constructs lacking the NRE. The nps was capable of suppressing LTR activityin transin chronically infected astrocytoma cells in a concentration-dependent manner. These results stress a negative role of a non-LTRnefpartial sequence in a cell-specific manner. In addition, our data indicate that nps functionsin transwith promoters unrelated to HIV LTR such as SV40 early and CMV immediate–early promoters
A Temperature-Sensitive Mutation of the Vaccinia Virus E11 Gene Encoding a 15-kDa Virion Component

AbstractThets49 mutation of vaccinia virus WR was mapped by marker rescue to the E11 gene encoding a 15-kDa polypeptide. During synchronous infection of BSC40 cells with wild-type virus, immunoreactive E11 protein accumulated in parallel with the onset of late protein synthesis. Immunoblotting of extracts of wild-type virions showed that the E11 protein was encapsidated within the virus core. A normal temporal pattern of viral protein synthesis was observed in cells infected withts49 at the nonpermissive temperature (40°) and normal-appearingts49 progeny virions were observed by electron microscopy. Sequencing of the E11 gene ofts49 revealed a single amino acid substitution, Gly(66)Arg, in the mutant E11 polypeptide. The steady-state level of E11 protein duringts49 infection was much lower than that observed during infection with wild-type virus. This was the case at both the permissive and nonpermissive temperatures. We discuss two possible explanations for the thermosensitive growth ofts49: (i) that virus infectivity requires a threshold level of active E11 protein or (ii) that E11 function is conditionally essential
DNA Polymerase δ-Dependent Formation of a Hairpin Structure at the 5′ Terminal Palindrome of the Minute Virus of Mice Genome

AbstractThe parvovirus the minute virus of mice (MVM) has a linear single-stranded DNA genome with unique palindromic sequences at both termini which enable it to fold back on itself and form hairpin or cruciform-type structures. The purpose of this study was to examine the primary events occurring during MVM replication mediated solely by the host cell replication machinery. In anin vitroDNA replication system using HeLa cell extracts, we found that there was a distinct activity that utilized the 5′ terminal palindrome sequence of MVM to produce a secondary structure from a duplex extended form, in a time-dependent fashion. The secondary structure was due to the formation of a hairpin rather than a stem-plus-arms type structure and was associated with initiation of DNA synthesis, performed specifically by DNA polymerase δ. Inhibition of DNA polymerase α had no effect upon this activity. Removal of all but 13 base pairs of the hairpin arm abolished the synthesis of DNA, indicating that there is a minimal length requirement for the duplex region of DNA or that this region contains regulatory genetic elements. These data are consistent both with the role of DNA polymerase δ in extending the synthesis of DNA from a DNA primer and with unidirectional continuous DNA synthesis, initiating from a hairpin, as a mode of replication for MVM
Sequence Diversity within the Reovirus S3 Gene: Reoviruses Evolve Independently of Host Species, Geographic Locale, and Date of Isolation

AbstractTo better understand genetic diversity of mammalian reoviruses, we studied sequence variability in the S3 gene segment of 17 field-isolate reovirus strains and prototype strains of the three reovirus serotypes. Strains studied were isolated over a 37-year period from different mammalian hosts and geographic locations. A high degree of variability was observed in the nucleotide sequences of the S3 gene, whereas the deduced amino acid sequences of the S3 gene product, σNS, were highly conserved. When variability among the S3 nucleotide sequences was analyzed using pairwise comparisons, we found that 5′ and 3′ noncoding regions were significantly more conserved than the remainder of the gene. This high degree of sequence conservation was also observed within the first 15 nucleotides of the 5′ coding region. Phylogenetic analyses showed that multiple alleles of the S3 gene cocirculate and that genetic diversity in the S3 gene does not correlate with host species, geographic locale, or date of isolation. Phylogenetic trees constructed from variation in the S3 sequences are distinct from those previously generated from sequences that encode attachment protein σ1, core protein σ2, and outer capsid protein σ3, which supports the hypothesis that reovirus gene segments reassort in nature. These findings suggest that reovirus gene segments are well-adapted to mammalian hosts and that reovirus evolution has reached an equilibrium
Domains of the Measles Virus N Protein Required for Binding to P Protein and Self-Assembly

AbstractThe nucleocapsid protein (N, 525 amino acids) of measles virus plays a central role in the replication of the viral genomic RNA. Its functions require interactions with itself and with other viral components. The N protein encapsidates genomic RNA, a function reflected in its ability to self-assemble into nucleocapsid-like particles in the absence of other viral proteins. The substrate for the packaging of nascent RNA during RNA replication is a complex between the N and phosphoprotein (P). The domains on the N protein that promote binding to P protein and self-assembly have been identified utilizing a series of N protein deletions. Two noncontiguous regions, amino acids 4–188 and 304–373 of N protein, are required for the formation of the soluble N–P complex, while deletion of amino acids 189–239 did not affect N–P binding. Amino acids 240–303 appear to be necessary for the stability of the protein. The N-terminal 398 amino acids are all required for the formation of organized nucleocapsid-like particles, since deletion of the central region from amino acids 189–373 completely abolished N–N interaction, and deletion of amino acids 4–188 and 374–492 caused the formation of unstructured aggregates
Characterization of an Essential HSV-1 Protein Encoded by the UL25 Gene Reported to Be Involved in Virus Penetration and Capsid Assembly

AbstractThe 2.3-kbBamHI-U DNA fragment (map units 0.319–0.335) of herpes simplex virus type 1 (HSV-1) genome contains the complete UL25 open reading frame (ORF). It specifies an essential viral protein reported previously to be involved in virus penetration and capsid assembly (C. Addison, F. J. Rixon, J. W. Palfreyman, M. O'Hara, and V. G. Preston,Virology138, 246–259, 1984). To identify the protein encoded by the UL25 gene, the UL25 ORF was cloned in a eukaryotic expression vector (p91023) downstream of the adenovirus major late promoter to generate the expression plasmid p9-UL25. Synthesis of a 60-kDa protein was observed in COS-7 cells transfected with p9-UL25 plasmid DNA, but not in cells transfected with p91023 control plasmid DNA. To identify and characterize the UL25 protein from HSV-1-infected cells, we prepared a rabbit antiserum by using UL25-GST fusion protein expressed inEscherichia colias immunogen. This rabbit antiserum readily immunoprecipitated the 60-kDa UL25 protein from HSV-1-infected cells. In HSV-1-infected cells, UL25 protein was expressed as a late (γ) or a leaky late (γ1) viral protein. The rabbit antiserum raised against HSV-1 UL25 protein immunoprecipitated a UL25-homologue of identical size from HSV-2-infected cells. However, the reactivity of the antiserum with HSV-2 UL25-homologue was weaker than compared to the corresponding HSV-1 protein. Consistent with its classification as a virion component, the UL25 protein was found to be associated with purified HSV-1 virions
Differential Effects of IκB Molecules on Tat-Mediated Transactivation of HIV-1 LTR

AbstractThetatgene product of the human immunodeficiency virus type 1 (HIV-1) strongly induces the transcription directed by the viral long terminal repeat (LTR). Tat acts by interacting with a target RNA element located immediately downstream of the initiation site. In addition, the action of Tat appears to be assisted by the upstream DNA enhancer elements, including the binding sites for the NF-κB/Rel family of host transcription factors. In the present study, we demonstrate that Tat transactivation of the HIV-1 LTR is markedly inhibited by several cytoplasmic inhibitors of NF-κB/Rel, suggesting the critical involvement of these host transcription factors in the function of the viral Tat protein. Furthermore, the various NF-κB inhibitors appear to have differential effects on Tat. While IκBα, IκBβ, and p100 potently inhibit Tat-mediated transactivation, p105 fails to inhibit, but even moderately synergizes, the action of Tat. We further demonstrate that the action of these NF-κB/Rel inhibitors on Tat correlates with their inhibitory activities on the RelA subunit of NF-κB. Finally, we show that a degradation-resistant IκBα mutant is able to potently inhibit Tat-mediated activation of the HIV-1 LTR in both untreated and tumor necrosis factor α-stimulated T cells, thus suggesting that such an IκBα mutant may serve as a constitutive repressor of HIV-1 LTR
Inhibition of Na+K+ATPase Activity in Membranes of Sindbis Virus-Infected Chick Cells

AbstractAlterations in intracellular concentrations of Na+and K+in Sindbis virus-infected cells result largely from inhibition of ouabain-sensitive Na+K+ATPase (Na+pump) activity. Here we report that membrane preparations derived from Sindbis virus-infected chick cells exhibit reduced Na+K+ATPase activity, indicating that limitation of cellular factors is not responsible for inhibition of ion transport.In vitrophosphorylation of the Na+K+ATPase by [32P]orthophosphate or [γ-32P]ATP is unaltered in membranes of Sindbis virus-infected cells, indicating that a loss of specific enzymatic functions unrelated to formation of Na+pump phosphoenzyme intermediates occurs during the course of viral infection. However, phosphoenzyme intermediates of the Na+K+ATPase prepared from membranes of Sindbis virus-infected cells are inherently less stable than those prepared with membranes of uninfected cells. The instability of these intermediatesin vitrois correlated with an altered capacity of the Na+pump to transport monovalent cations into virus-infected cells.22Na+transport studies reveal enhanced ouabain-sensitive Na+uptake by Sindbis virus-infected cells, suggesting that the Na+pump may catalyze enhanced Na+-Na+exchange in the infected cells. These results indicate that the capacity of the Na+K+ATPase to discriminate between binding of extracellular Na+and K+is specifically altered during the course of infection by Sindbis virus
Normal Replication of Vesicular Stomatitis Virus without C Proteins

AbstractThe expression of two small basic proteins (C and C′) encoded by a second open reading frame of the New Jersey serotype of vesicular stomatitis virus (VSV) P gene was reported previously (Spiropoulou and Nichol,J. Virol.,67, 3103–3110, 1993). Here we found that the Indiana serotype virus also expressed C and C′ proteins from this reading frame. We eliminated C and C′ expression by making a single base change that introduced a stop codon in the C and C′ coding sequence, but left the P-protein sequence unchanged. This mutated P gene supported normal replication and packaging of VSV minigenomes encoding G and M proteins. The mutated P gene was also recombined into an infectious clone of VSV that was used to recover virus. The mutant virus no longer expressed the C and C′ proteins but showed growth kinetics identical to wild-type virus. The amounts of viral mRNAs and proteins synthesized were indistinguishable in mutant and wild-type virus infected cells as were the yields and composition of mutant and wild-type virus particles. The kinetics of host protein-synthesis shut-off were also identical for both viruses. Although the C and C′ proteins were dispensable for VSV growth in tissue culture, they are known to be conserved in all vesiculoviruses, and thus perhaps play a role in viral pathogenesis or transmission by insect vectors
Recombinant Dengue Type 1 Virus NS5 Protein Expressed inEscherichia coliExhibits RNA-Dependent RNA Polymerase Activity

AbstractThe complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed inEscherichia colias a glutathioneS-transferase (GST) fusion protein (126 kDa). The GST–NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA
Defective RNAs Inhibit the Assembly of Influenza Virus Genome Segments in a Segment-Specific Manner

AbstractFour avian influenza viruses have been generated, each containing a single extra defective RNA segment in addition to the eight standard segments. Three of the extra RNAs were derived from segment 1 and the fourth from segment 2. Chick embryo fibroblast cells were infected with each virus, and a wild-type virus. Virus RNA was quantified in extracts of virus-infected cells and in virus released by 10 hr postinfection using reverse transcription and by Northern blot analysis. In the case of two of the viruses the presence of the defective RNA did not markedly affect the accumulation of virus RNA within the infected cell, but significantly and selectively reduced the amount of the “parent” segment in released virus. This effect was reduced in a third virus. In a fourth virus, defective RNA was found to be present at a low-input multiplicity and results were varied. Mixed infections of one of the viruses with a closely related wild-type virus resulted in reduction of the corresponding vRNA segment of the nondefective virus. We conclude that assembly of influenza virus segments is not a purely random process
Analysis of the Determinants of Neurotropism and Neurotoxicity of HFV in Transgenic Mice

AbstractThe Bel-1 protein of human foamy virus (HFV) is a transactivator acting on the U3 region of the long terminal repeat and on an internal promoter (IP) immediately upstream of the bel genes. An HFV transgene called Δgpe, containing both promoters and all bel genes, is expressed in the central nervous system and induces neurodegeneration in mice. To dissect the role of individual promoters and bel genes on transgene expression and neurotoxicity we generated transgenic mice with a construct termed pL-bel1, which lacks the IP and the ancillary genes except bel-1. L-bel1 mice transcribed the HFV transgene in more tissues than Δgpe mice, suggesting that CNS specificity is dictated bycis-acting elements not present in the pLbel-1 construct. Unlike Δgpe mice, L-bel1 mice did not develop neurodegenerative changes and did not show induction of nitric oxide synthase expression, although both strains expressed Bel-1 in the brain. Therefore, Bel-1 expression is not sufficient for neurotoxicity. Our results suggest that Bet, a fusion protein between bel-1 and bel-2 which is highly expressed in Δgpe but not in L-bel1 mice, is a candidate for neurotoxicity
Two Novel Variants of the v-srcOncogene Isolated from Low and High Metastatic RSV-Transformed Hamster Cells

AbstractFour different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70–200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-srcprotein in cell lines was not correlated with their metastatic potentialin vivo.Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-srcisoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and v-srcLM: 62 kDa. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-srcsequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the oncoprotein. Both v-srcvariants and chimeric v-srcwith mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretina cells were associated with the mutations in the carboxy-terminal region of the v-srconcogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM
Identification of a Cellular Protein That Specifically Interacts with the Essential Cysteine Region of the HIV-1 Tat Transactivator

AbstractThe Tat protein of the human immunodeficiency virus (HIV) is a powerful activator of HIV gene expression. Genetic and biochemical evidence suggests that one or more cellular cofactors may be important for Tat activity. We have used two-hybrid interactive cloning in yeast to identify a partial cDNA clone (clone 10) from a human B-lymphoblastoid library that specifically interacts with the N-terminal 31 amino acids of HIV-1 Tat which contains the essential cysteine-rich portion of the Tat activation domain. The encoded protein also binds to purified Tatin vitro.Mutation of single essential cysteine residues in Tat abolishes interaction between Tat and clone 10, suggesting that interaction with the encoded protein is important for Tat activity. We have identified the full-length cDNA for the Tat binding protein and shown that overexpression of the encoded protein, Tip60 (Tat interactive protein, 60 kDa), results in a fourfold augmentation of Tat transactivation of the HIV-1 promoter in transient expression assays without increasing the basal activity of the HIV promoter or activating the heterologous RSV promoter. These data together with the genetic andin vitrobinding data support the notion that Tip60 might be a cofactor of Tat involved in the regulation of HIV gene expression