pinecone/core-2020-05-10-deduplication huggingface dataset:
Identification of an RNA-Stimulated NTPase in the Predicted Helicase Sequence of the Rubella Virus Nonstructural Polyprotein

AbstractThe replicative proteins of Rubella virus are generated from a polyprotein that is translated from the 5′-terminal segment of the viral genome. The determination of the genome sequence and the description of amino acid sequence motifs which are proposed to be characteristic for helicase proteins have indicated that the polyprotein region located between amino acid residues 1300 and 1600 represents the Rubella virus helicase. We have expressed a segment comprising the sequences between the amino acid residues A (1225) and R (1664) as part of a glutathioneS-transferase fusion protein inEscherichia coli.We show that this protein contains a nucleoside triphosphatase activity which hydrolyses all eight ribonucleoside- and deoxyribonucleoside triphosphates. The activity of the protein, determined by ATP hydrolysis, was influenced by the presence of single-stranded RNA; it was stimulated about 1.7-fold in the presence of poly(U), poly(C), or poly(dT) and inhibited to half its activity in the presence of poly(G). These functions represent characteristic helicase partial functions and provide experimental support for the predicted localization of the helicase in the nonstructural polyprotein
The Mode of Death of Pig Kidney Cells Infected with Cowpox Virus Is Governed by the Expression of thecrmAGene

AbstractPig kidney cells (LLC-PK1) were infected with one of three viruses: wild-type cowpox virus (Brighton red strain) expressing thecrmAgene; recombinant cowpox virus A602, lacking thecrmAgene; or cowpox virus A604, a revertant of virus A602, expressing thecrmAgene. The wild-type virus and virus A604 produced identical cytopathic effects consistent with death by necrosis. In these cells, the structural features of the plasma membrane, the nuclear membrane, and the chromatin were maintained until lysis of the cells. In contrast, cowpox virus A602 produced cytopathic effects consistent with death by apoptosis. These effects included loss of microvilli on the cell surface, margination and condensation of the chromatin, progressive convolution of the nuclear membrane, release of dense chromatin masses on disintegration of the nucleus, fragmentation of the DNA, and the generation of apoptotic bodies. These results suggest that thecrmAgene is necessary to inhibit processes of apoptosis induced in LLC-PK1cells by infection with cowpox virus. Thus in cells of certain types, thecrmAgene can act with other viral genes to control the mode of death of the virus-infected cell. This capability may be advantageous to virus replicationin vivo,potentially facilitating both virus trafficking and interference with antiviral immune defenses
NEF-CD4 Physical Interaction Sensed with the Yeast Two-Hybrid System

AbstractHIV-1 Nef protein has been known to induce downmodulation of CD4 receptor. In order to test whether the two proteins physically interact, the yeast two-hybrid system was exploited. ASaccharomyces cerevisiaestrain carrying a GAL4-responsivelacZfusion gene was cotransformed with plasmids in which the Nef and the CD4 cytoplasmic domain (CD4cd) coding sequences were fused to either the DNA binding (DB) or the activation (A) moiety of the GAL4 transcriptional activator. Both the DB-Nef + A-CD4cd and the DB-CD4cd + A-Nef combinations activated the reporter gene, weakly but specifically, as inferred by comparison with a number of controls. Reporter activation was similarly observed when DB-Nef was cotransfected with the fusion A-CD4cd(aa 1–23). On the contrary, the combination DB-Nef + A-CD4cd(aa 24–40) was inactive. Also, mutating the CD4cd Leu20–Leu21motif (known to be essential for both physiological and Nef-induced CD4 endocytosis) to Ala20–Ala21abolished the GAL4 activity of DB-Nef + A-CD4cd. None of six DB-Nef derivatives in which Nef was partially deleted activated specifically the reporter when coexpressed with A-CD4cd. These findings suggest that CD4cd and Nef directly interact and that a largely complete Nef is required for the interaction. CD4cd aa 1–23 are sufficient for binding; in particular, the Leu20–Leu21motif is essential. One can infer from these data that: (i) Nef-induced CD4 downmodulation involves a direct CD4–Nef contact and (ii) CD4cd Leu20–Leu21is required in Nef-induced downmodulation, not simply as an endocytosis signal, but also as an essential component of the Nef-binding moiety
Study on Simian Virus 40 DNA Synthesis in Herpes Simplex Virus-Infected Cells

AbstractReplication of simian virus 40 (SV40) DNA occurs in SV40 nonpermissive hamster cells upon infection with herpes simplex virus (HSV), leading to concatemeric replication products characteristic for HSV DNA replication. This SV40 origin (ori)-dependent process is governed by SV40 large T antigen and HSV-encoded DNA replication factors; e.g., DNA polymerase, single-strand binding protein (SSB), and helicase–primase. In this study, we show that specific interaction of SV40 T antigen with SV40 ori is crucial for HSV-directed SV40 DNA synthesis and that the property of T antigen to bind and unwind the ori is not sufficient for this process. A T antigen with the mutation T217S, affecting a hypothetical novel DNA replication subfunction, is able to support DNA synthesisin vitrobut not in cultured primate cells. This subfunction is also necessary in HSV-infected hamster cells. Using temperature-sensitive mutants, we demonstrate that the T antigen acts at early stages of DNA synthesis while HSV helicase is required continuously as has been shown for HSV DNA polymerase. HSV SSB is also continuously involved in heterologous SV40 DNA synthesis. However, a HSV mutant, temperature-sensitive in SSB function, showed residual synthesis of SV40 DNA but not of HSV DNA at the nonpermissive temperature. The nature of this dichotomy between HSV SSB function on SV40 DNA and HSV DNA will be discussed
The Nonstructural C Protein Is Not Essential for Multiplication of Edmonston B Strain Measles Virus in Cultured Cells

AbstractMeasles virus (MV) is a highly contagious agent which causes a major health problem in developing countries. Efficacious and safe live attenuated vaccine strains are available, but for the elimination of measles a better knowledge about the molecular biology of MV appears crucial. Whereas the roles of the six structural proteins in the replication cycle are known, the functions of the two nonstructural proteins C and V are unclear, which is also true for related viruses.In vitrostudies implicating Sendai virus suggest that the C protein might be involved in downregulating viral mRNA synthesis (J. Curran, J. B. Marq, and D. Kolakofsky,Virology189, 647–656, 1992). However, not all members of the Paramyxovirinae subfamily encode this protein, raising the question about its importance for the viral replication cycle. Taking advantage of a recently developed reverse genetics system allowing MV recovery from cloned DNA (F. Radecke, P. Spielhofer, H. Schneider, K. Kaelin, M. Huber, C. Dötsch, G. Christiansen, and M. A. Billeter,EMBO J.14, 5773–5784, 1995), the question was addressed whether the C protein is essential for the life cycle of MV. A plasmid was constructed to produce a derivative of the Edmonston B vaccine strain, MV C−EdB, having its C reading frame silenced by two point mutations. The C−mutant MV could indeed be rescued, and it multiplies in cultured cells without obvious impairment
The Protein Kinase Activity of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Fused to the Extracellular Domain of the Epidermal Growth Factor Receptor Is Ligand-Inducible

AbstractThe gene coding for the large subunit of herpes simplex virus type 2 ribonucleotide reductase (RR) (ICP10) has a unique 5′ terminal domain the product of which has a serine/threonine (Ser/Thr) protein kinase (PK) catalytic domain preceded by a transmembrane (TM) segment. Because ICP10 localizes on the cell surface and is internalized by the endocytic pathway like an activated growth factor receptor (Hunteret al.,1995,Virology210, 345–360), we asked whether it is ligand-inducible in order to examine whether it has intrinsic transphosphorylating activity. We constructed a chimeric expression vector that contains the extracellular and TM domains of the epidermal growth factor receptor (EGFR) joined to the intracellular PK and RR domains of ICP10 (pCH5) and established constitutively expressing cell lines in NIH3T3 2.2 cells that do not express EGFR. The chimeric protein, designated p210CH5, localized to the surface of these cells as determined by immunofluorescent staining with MAb EGFR, and it bound125I-EGF. p210CH5coprecipitated with protein species p170, p120, p88, p60, p44, p34, and p25. EGF treatment activated the PK activity of p210CH5, resulting in its autophosphorylation and the phosphorylation of the p120, p88, and p34 species. Immunoprecipitation/immunoblotting with anti-ras-GAP antibody and phosphoamino acid analysis indicated that p120 isras-GAP and it is phosphorylated on Ser/Thr residues. The identities of the phosphorylated p88 and p34 are still unknown. The data indicate that when fused to a ligand-regulated extracellular domain (EGFR), the ICP10 PK auto- and transphosphorylating activities are ligand-inducible. These findings support the interpretation that the ICP10 PK activity is intrinsic and indicate thatras-GAP is one of its phosphorylation substrates
Movement of a Barley Stripe Mosaic Virus Chimera with a Tobacco Mosaic Virus Movement Protein

AbstractThe tobacco mosaic virus (TMV) 30K movement protein (MP) gene was inserted into a full-length cDNA clone of barley stripe mosaic virus (BSMV) RNAβ replacing the triple gene block (TGB). The resulting recombinant ND-MPT genome, consisting of infectious wt transcripts of BSMV RNAs α and γ, together with the hybrid RNAβ transcript, was inoculated onto test plants to study the functional compatibility between the BSMV TGB-adapted genetic system and the tobamovirus transport gene. ND-MPT infected the inoculated leaves ofNicotiana benthamianaandChenopodium amaranticolor,which are common hosts for the parental viruses; the size, growth rate, and morphology of local lesions onC. amaranticolorwere influenced by the foreign MP gene. However, the hybrid virus failed to infect barley,N. tabacum(var. Samsun), andN. clevelandii,the selective hosts. Thus, the TMV MP was able to functionally substitute for the BSMV TGB-coded MPs, i.e., the 30K MP functioned independently of any other BSMV sequences. However, the TMV MP gene promoted the cell-to-cell movement in a host-dependent manner
The Equine Herpesvirus 1 IR6 Protein Is Nonessential for Virus Growthin Vitroand Modified by Serial Virus Passage in Cell Culture

AbstractThe IR6 protein of different plaque isolates from three passages of the equine herpesvirus 1 strain Rac was investigated. Southern blot and DNA sequence analyses revealed that plaque isolates from the 12th passage (RacL11 and RacL22) retained both copies of the IR6 gene, whereas two different genotypes were observed by the 185th passage: RacM24 still harbored both copies of the IR6 gene, whereas RacM36 deleted one of the two copies. In the 256th passage (RacH), both copies of the IR6 gene were absent. As compared to the wild-type IR6 protein, both the RacM24 and RacM36 IR6 protein displayed amino acid exchanges at positions 34, 42, 110, and 134 of the 272 amino acid polypeptide. It is shown that (i) the IR6 protein is nonessential for virus growthin vitro.(ii) In RacL11-infected equine and rodent cells, the typical rod-like appearance of the IR6 protein could be detected from 6 hr p.i., whereas in RacM24- and RacM36-infected cells formation of these structures was not observed. (iii) The RacL11 IR6 gene product was present in both the nuclei and cytoplasmic fraction of infected cells. In contrast, the IR6 protein of both RacM24 and RacM36 colocalized with cytoplasmic membrane vesicles. (iv) The RacL11 and RacL22 IR6 protein is present in viral nucleocapsids, whereas that of RacM24 and RacM36 is not incorporated into virions. (v) The RacL11 IR6 gene product aggregated to disulfide-linked oligomers, whereas the RacM24 and RacM36 IR6 protein showed only marginal oligomerization. (vi) In COS7 cells transfected with constructs expressing either the full-length RacL11-IR6 protein or a truncated form lacking the 81 carboxyterminal amino acids, the formation of rod-like structures was observed, indicating that another viral protein is not necessary for aggregation of the IR6 protein. In contrast, the IR6 protein expressed from constructs derived from either RacM24 or RacM36 failed to form these structures. (vii) Analyses of chimeric RacL11-RacM24 IR6 proteins suggest a crucial role for amino acid Leu-134 in the ability of the IR6 protein to form the rod-like structures
A Surface Plasmon Resonance Assay for the Binding of Influenza Virus Hemagglutinin to Its Sialic Acid Receptor

AbstractWe have developed a sensitive microscale binding assay to study the interaction between influenza hemagglutinin and its cell surface receptor sialic acid using real-time surface plasmon resonance. The glycoprotein fetuin was bound to a carboxymethylated-Dextran sensor surface usingN-hydroxysuccinimide andN-ethyl-N′-(dimethylaminopropyl) carbodiimide. Low-pH-induced BHA rosettes bind specifically to the fetuin-derivitized sensor surface, but not to an asialofetuin-derivitized sensor surface. Binding can be inhibited by preincubation of BHA rosettes with millimolar concentrations of inhibitors of the influenza hemagglutinin–sialic acid interaction. The association rate, dissociation rate, and dissociation constant for the multivalent interaction between BHA rosettes and the fetuin-derivitized sensor surface were also measured, allowing us to quantitate the tight binding achieved through the multivalent interaction between BHA rosettes and the fetuin-derivitized sensor surface
Poliovirus RNA Polymerase Mutation 3D-M394T Results in a Temperature-Sensitive Defect in RNA Synthesis

AbstractMutant ts10 is an RNA-negative temperature-sensitive mutant of Mahoney type 1 poliovirus. Mutant ts10 3Dpolwas purified from infected cells and was shown to be rapidly heat-inactivated at 45° when compared to wild-type polymerase. Sequencing of mutant ts10 genomic RNA revealed a U to C transition at nt 7167 resulting in an amino acid change of methionine 394 of 3Dpolto threonine. The 3D-M394T mutation was engineered into a wild-type infectious clone of poliovirus type 1. The resultant mutant virus, 3D-105, had a temperature-sensitive phenotype in plaque assays. The translation and replication of wild-type, ts10, and 3D-105 virion RNAs were all characterized in HeLa S10 translation-RNA replication reactionsin vitro.The optimum temperatures for the replication of the wild-type and mutant viral RNAs in the HeLa S10 translation-replication reactions were 37 and 34°, respectively. To characterize the temperature-sensitive defect in the replication of the mutant RNA, we used preinitiation RNA replication complexes which were formed in HeLa S10in vitroreactions containing guanidine HCl. Negative-strand RNA synthesis in 3D-M394T mutant preinitiation replication complexes was normal at 34° but was rapidly and irreversibly inhibited at 39.5°. To differentiate between the initiation and elongation steps in RNA replication, we compared the elongation rates in mutant and wild-type replication complexes at 39.5°. The results showed that the elongation rates for nascent negative strands in both the mutant and wild-type replication complexes were identical. Therefore, the results indicate that the heat-sensitive step in negative-strand synthesis exhibited by the 3D-M394T replication complexes is in the initiation of RNA synthesis and not in the elongation of nascent chains
Nitric Oxide Production Is Increased during Murine Vaccinia Virus Infection, but May Not Be Essential for Virus Clearance

AbstractRecent reports have highlighted a potential antiviral activity for nitric oxide (NO). The purpose of this study was to investigate the production of NO in mice during vaccinia virus (VV) or herpes simplex virus type 1 infection, and to assess the role of NO in clearance of VV. Reactive nitrogen intermediates (RNI; NO and its stable oxidation products, nitrite and nitrate) were significantly elevated in the plasma of mice infected with these viruses. Furthermore, spleen cells from virus-infected mice produced elevated RNI levels following stimulationin vitrowith LPS. NO production during VV infection was critically dependent on the cytokines tumor necrosis factor and interferon-γ, and on the presence of both CD4+and CD8+T lymphocytes. Treatment of VV-infected mice with the nitric oxide synthase inhibitorNG-methyl-L-arginine did not alter the course of infection, suggesting that NO may not be essential for the clearance of this virus
Phosphorylation State of the Cap-Binding Protein eIF4E during Viral Infection

AbstractThe eukaryotic translation initiation factor eIF4E, the cap-binding protein, seems to play an essential role in the establishment of the host shut-off after viral infection. Infection with adenovirus and influenza virus caused dephosphorylation of eIF4E and an involvement of a viral protein was suggested. In this report, we studied several other viruses for their ability to change the phosphorylation state of eIF4E, and we looked for the mechanism of eIF4E dephosphorylation. First, it was shown that after encephalomyocarditis virus (EMCV) and poliovirus infection, dephosphorylation of eIF4E occurred. Dephosphorylation of eIF4E was not observed after Semliki Forest virus and reovirus infection. An artificial increase of the level of phosphorylated eIF4E by treating the cells with the phosphatase inhibitor okadaic acid changed neither the kinetics of EMCV and poliovirus infection, nor that of host shut-off. Infections with uv-treated EMCV showed that virus binding or entry into the cell initiates eIF4E dephosphorylation. Besides this entry-induced eIF4E dephosphorylation, dephosphorylation was also induced by blocking protein synthesis with the initiation inhibitor pactamycin, or with the elongation inhibitor cycloheximide. We conclude that eIF4E is dephosphorylated by entry of EMCV, and the effect is strengthened by the decrease in cap-dependent translation
Characterization of a Novel Influenza Hemagglutinin, H15: Criteria for Determination of Influenza A Subtypes

AbstractTwo viruses with a novel hemagglutinin (HA), A/duck/Australia/341/83 and A/shearwater/West Australia/2576/79, have been isolated from a duck and a shorebird in Australia. Hemagglutination inhibition and double immunodiffusion assays failed to reveal cross-reactivity with any of the known subtypes (H1 to H14). We therefore propose that these viruses constitute a new HA subtype, H15. Sequence analysis of the HA genes confirmed the serologic findings. When compared at the amino acid level, the HA1 region of the H15 subtype differs from those of the other subtypes by 30% and more. This degree of heterogeneity is also found among HA genes of other subtypes. Thus we propose that amino acid sequence data should be evaluated when determining the HA subtypes of influenza A viruses. Sequence comparison and phylogenetic analysis suggested that the HA subtype H15 is most closely related to the H7. Compared to the H7 HA, the H15 acquired a 30-nucleotide insertion within HA1 at position 253 which is located in the globular head of the molecule. This finding suggests that RNA recombination, although a rare event in nature, may play an important role in the evolution of influenza viruses
Definition of a Human Herpesvirus-6 Betaherpesvirus-Specific Domain in Glycoprotein gH That Governs Interaction with Glycoprotein gL: Substitution of Human Cytomegalovirus Glycoproteins Permits Group-Specific Complex Formation

AbstractFormation of the glycoprotein gH/gL heterooligomer has important implications for understanding the pathology of human herpesvirus-6 (HHV-6)-associated disease because this complex is essential for infectivity and fusogenic cell-to-cell spread. Definition of the HHV-6 gH domain involved in protein–protein interactions was addressed by targeting regions defined by conserved cysteines identified by alignment of gH amino acid sequences representative of all herpesvirus subfamilies. Studies using site-directed mutagenesis and transient cellular expression showed that the N-terminus of HHV-6 gH includes a 230-amino-acid domain required for interaction with HHV-6 gL encompassing residues conserved specifically amongst betaherpesviruses. Interestingly, the human cytomegalovirus (HCMV) homologues, UL75 (gH) or UL115 (gL), can substitute for HHV-6 glycoproteins and participate in heterologous complex formation. Furthermore, the region which governs this heterologous gL binding also maps to the N-terminal portion of HHV-6 gH. Although both proteins can functionally substitute for complex formation there are also specific differences. Surprisingly, further deletion of HHV-6 gH to 145-amino-acid-domain residues abolishes complex formation with HHV-6 gL but allows interaction with HCMV gL. This may be related to requirements in HHV-6 for homodimer formation before complex formation between gH and gL. Under nonreducing conditions HHV-6 gH and gL form multimeric complexes consistent with intra- and intermolecular dimer formation stabilised by disulphide bonds whereas for HCMV there is no evidence for dimer formation for gH and multimeric complexes have only been observed between gH and gL. In summary, both HHV-6 and HCMV glycoproteins can interact and the heterologous complex between HHV-6 gH and HCMV gL is possibly more stable. This may result in important biological consequencesin vivoduring cellular coinfections by facilitating spread of the viruses, with applications to altered cellular tropisms and effects on reactivation from the latently infected cell
Mutagenesis of the Sindbis Virus nsP1 Protein: Effects on Methyltransferase Activity and Viral Infectivity

AbstractIt has been suggested that four amino acids which are absolutely conserved in the nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamily may constitute a “methyltransferase motif.” In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His39, Arg91, Asp94, and Tyr249. Earlier, in assays of methyltransferase (MTase) activity generated in SV-infected cells, we had shown that amino acid changes at positions 87 and 88 of SV nsP1 resulted in a 10-fold lowerKmfor S-adenosyl methionine, the methyl donor in MTase reactions. Using site-directed mutagenesis we now report the expression of nsP1 inEscherichia coli,and in the infectious clone of Sindbis virus, Toto/1101, in which His39, Arg91, Asp94, and Tyr249were changed one at a time to Ala. We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, inE. coli.Changing His39, Arg91, Asp94, or Tyr249to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile369to Val, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity. With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activityin vitro.However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity