pinecone/core-2020-05-10-deduplication huggingface dataset:
Regulation of the Initiation of Coronavirus JHM Infection in Primary Oligodendrocytes and L-2 Fibroblasts

AbstractUpon maturation, primary rat oligodendrocytes become resistant to coronavirus JHM (JHMV) infection at an early stage. Involvement of cAMP-dependent protein kinase (PK) in the regulation of oligodendrocyte differentiation has been established (S. Beushausenet al.(1987).J. Virol.61, 3795–3803). An inducer which accelerates maturation, dibutyryl cyclic AMP (dbcAMP) also upregulates the expression of the regulatory subunit, R1 of PK1. Since (i) early block preventing infection of mature oligodendrocytes can be bypassed when transfection with genomic RNA is used and (ii) inhibitors of PKs counteract the dbcAMP effect, so as to alleviate the inhibition of JHMV, enhanced expression of R1 appeared to be connected with virus restriction. This idea was confirmed following upregulation of the R1 gene in fully permissive L-2 cells. There was a connection between an effect due to R1 and dephosphorylation of the nucleocapsid protein N by an endosomal phosphoprotein phosphatase (PPPase) having the properties of types 1 or 2A enzyme which occurs during penetration of inoculum virions. An inhibitionin vitro(cell free) of N dephosphorylation by R1 together with evidence thatin vivo(cell culture) overexpression of R1 inhibited the endosomal PPPase as well as replication of JHMV supports the hypothesis that uncoating of the JHMV inoculum occurs after dephosphorylation, a step obligatory for dissociation of the N protein from the genome. Thus inhibition by R prevents uncoating and thereby interferes with the commencement of replication. These observations intimate the existence of a novel mechanism controlling a virus infection of specific cell target(s) undergoing a process of differentiation and maturation in the central nervous system
Identification and Characterization of a Sixth Structural Protein of Lelystad Virus: The Glycoprotein GP2Encoded by ORF2 Is Incorporated in Virus Particles

AbstractPreviously we have shown that Lelystad virus (LV) contains five structural proteins, a nucleocapsid protein N, an integral membrane protein M, and three glycoproteins GP3, GP4, and GP5. In this study we identified a sixth structural protein of Lelystad virus. The protein has an apparent molecular weight of 29 to 30 kDa, was recognized by an ORF2-specific antipeptide serum in Western immunoblotting using sucrose gradient purified LV virions, and was shown to beN-glycosylated. It was therefore designated GP2. The GP2protein was also immunoprecipitated by ORF2-specific antipeptide serum from lysates and extracellular virus of CL2621 cells infected with LV. A fraction of the GP2protein present in these lysates contained an intrachain disulfide bond. Endoglycosidase H treatment of immunoprecipitates indicated that the endoglycosidase H-sensitiveN-glycans of the GP2protein become endoglycosidase H-resistant during passage through the Golgi compartment in cells infected with LV. In contrast, theN-glycans of the GP2protein expressed individually in a recombinant Semliki Forest virus remained endoglycosidase H-sensitive, indicating that the GP2protein was retained in the endoplasmic reticulum. LV is the first arterivirus which has been demonstrated to contain six virion-associated proteins, one nucleocapsid protein, one integral membrane protein, and four glycoproteins
Human T Cell Leukemia Virus Type 1 Tax Protein Increases NF-κB Dimer Formation and Antagonizes the Inhibitory Activity of the IκBα Regulatory Protein

AbstractHuman T cell leukemia virus type 1 (HTLV-1) encodes a strong transcriptional transactivator, the Tax protein, that stimulates viral transcription through the long terminal repeat and also stimulates many cellular genes via the activation of host transcription factors. Previous studies have demonstrated that Tax activates NF-κB through binding to the Rel homology domain of NF-κB proteins. Tax was also shown to increase degradation of IκBα resulting in the induction of NF-κB DNA binding activity. We addressed the specificity and function of Tax interaction with members of the NF-κB/IκBα family by using EMSA, protein affinity chromatography, protein–protein crosslinking and co-immunoprecipitation assays. The results of the present study demonstrate that: (1) Tax enhances NF-κB binding to DNA 40- to 100-fold by increasing NF-κB dimer formation which can be detected in the absence of DNA; (2) Tax binds to all NF-κB DNA binding subunitsin vitroand to IκBα; (3) Tax physically associates with IκBαin vivo;and (4) Tax and IκBα have antagonistic effects on NF-κB binding and gene activity. These results suggest that Tax interaction with IκBα interferes with the formation of NF-κB–IκBα complexes and may play a role in targeting IκBα for degradation
Structural Analysis of a Mutation in Canine Parvovirus Which Controls Antigenicity and Host Range

AbstractA single mutation in canine parvovirus (CPV) of VP2 residue 300 from alanine to aspartic acid causes a loss of canine host range and alters the antigenic properties of the virus. The three-dimensional structure of this mutant has been solved to 3.25 Å resolution. Crystals of full particles were triclinic, with cell dimensions ofa= 267.6,b= 268.5,c= 274.3 Å, α = 61.9, β = 62.6, and γ = 60.2°. The native structure of CPV was used as an initial model. Phases were improved by real-space electron density averaging. In spite of the relative low percentage of observed reflections (32.5% of the data between 15.0 and 3.25 Å resolution), the presence of 60-fold noncrystallographic redundancy allowed the averaging procedure to converge smoothly. The mutant aspartic acid at residue 300 forms a salt bridge with Arg81 in an icosahedrally threefold-related subunit, inducing local changes within the antigenic site B on the CPV surface. In addition, the loop between residues 359 and 374 adopts a conformation similar to that displayed by feline panleukopenia virus. The ability of the Ala300 → Asp mutant to evade antibody binding can be associated with the change of charge distribution and structure in the antigenic binding site. The variation in host range behavior may be due to the increased stability as a result of formation of the salt bridge between adjacent subunits
Correlation between Herpes Simplex Virus Type 1 Rate of Reactivation from Latent Infection and the Number of Infected Neurons in Trigeminal Ganglia

AbstractThe presence of wild-type herpes simplex virus type 1 (HSV-1) and several latency associated transcript (LAT) region mutants within the trigeminal ganglia (TG) of latently infected mice was examined. A combination of methods including conventionalin situhybridization to detect viral LAT and anin situDNA polymerase chain reaction (PCR) to detect viral DNA was used. These data show that, for all virus strains in which a comparison was possible, the population of neurons expressing detectable levels of LAT was approximately one-third the total number of viral DNA-containing cells. In addition,in situPCR analysis revealed that mutants such as 17ΔSty, 17ΔBstE, and 17ΔS/N, which contain deletions within the LAT locus which do not affect the kinetics of viral reactivation from explanted murine TG, are present in as many neurons as wild-type virus. This was true regardless of the ability to induce accumulation of intact 2.0-kb LAT. On the other hand, mutant 17ΔN/H, which contains a deletion removing the LAT promoter and surrounding genomic region and reactivates slowly from explanted TG, was present in only one-sixth as many neurons as wild-type virus. These data show that detection of mutants unable to synthesize or accumulate 2.0-kb LAT (such as 17ΔN/H) is possible within situDNA PCR and that the slow reactivation phenotype of 17ΔN/H correlates with a reduced number of HSV DNA-containing neurons
Scaffolding Mutants Identifying Domains Required for P22 Procapsid Assembly and Maturation

AbstractAssembly of the icosahedral shells of the dsDNA bacteriophages, herpesviruses, and adenoviruses requires proteins not found in the mature virion, termed scaffolding proteins. The bacteriophage P22 precursor procapsid contains approximately 300 scaffolding molecules within a shell composed of 420 coat protein subunits. Though nonsense mutants are common, few mutants affecting the functions of the scaffolding protein have been recovered. We report here the isolation and characterization of new missense mutants unable to form infectious virions under restrictive conditions. These mutant scaffolding subunits were competent for protein folding and capsid assembly under restrictive conditions. Two mutants were defective in assembly into the procapsid of the portal complex, which serves as the channel through which DNA is packaged. These mutations may identify a region of the scaffolding protein required for interaction with the portal subunits. Two mutants in a different region of the sequence were impaired in scaffolding release from the procapsid bothin vivoandin vitro.These mutations may identify a new domain required for scaffolding release. Scaffolding release appeared to be required for capsid expansion; in turn, scaffolding release seemed to depend upon the presence of a portal. This may help to order the pathway of events in phage maturation
Production and Characterization of Murine IgA Monoclonal Antibodies to the Surface Antigens of Rhesus Rotavirus

AbstractRotavirus is the single most important cause of severe diarrhea in humans and is diffuse in most animal species worldwide, and an understanding of the antigenic properties of the virus is essential to the design of rational vaccine strategies. To better understand the localization of viral epitopes involved in antibody-mediated neutralization of virus infectivity, we have orally immunized mice with live rhesus rotavirus (RRV) and generated a panel of hybridoma cell clones secreting IgA class monoclonal antibodies. A total of 12 neutralizing IgA MAbs to VP4 and VP7 proteins were studied for their epitope specificity and topographical relationships by hemagglutination-inhibition assays, neutralization assays, and competitive-binding assays with previously mapped MAbs. In addition, neutralization-escape virus mutants were selected and gene segments for each variant were cloned and sequenced. Two IgA MAbs were found to be directed to the antigenic region A of the VP7 protein at amino acid 94, and 10 MAbs were directed at the VP8 trypsin cleavage fragment of VP4. Five of the VP4-specific MAbs identified the same neutralization epitope on the RRV VP8 protein, not previously associated with RRV neutralization. All neutralization-escape variants selected by this antibody group contained mutations at amino acids 132–135 of VP4. One IgA MAb selected for a mutation at amino acid 190 of VP4, and the corresponding viral mutant failed to agglutinate erythrocytes. This MAb mapped to an epitope recognized by 2 additional IgA MAbs. These results suggest that oral immunization of mice with RRV elicits an IgA immune response which is predominantly directed toward antigenic determinants on the VP8 portion of VP4. As a consequence, the route of immunization may alter immunodominant neutralization responses elicited to rotavirus
Identification of the Human Cytomegalovirus G Protein-Coupled Receptor Homologue Encoded by UL33 in Infected Cells and Enveloped Virus Particles

AbstractHuman cytomegalovirus (HCMV), strain AD169, contains four genes (US27, US28, UL33, and UL78) that encode putative homologues of cellular G protein-coupled receptors (GCRs). GCRs transduce extracellular signals to alter intracellular processes, and there is evidence that HCMV may elicit such changes at early times following infection. The US27, US28, and UL33 genes are transcribed during infection, and the US28 gene product has been found to be a functional receptor for the β-chemokine class of immune modulators. The US27, UL33, and UL78 gene products have not been described and we have concentrated on identifying the UL33 protein because it is the most highly conserved of the GCR homologues among the human β and γ herpesviruses. We report here cloning UL33 into a recombinant baculovirus (rBV) and expressing it in insect cells; constructing a mutant HCMV with a disrupted UL33 gene; and identifying the UL33 protein in HCMV-infected cells and virus particles. Our results demonstrate that the UL33 protein (i) is expressed as a ≈36-kDa, heat-aggregatable protein in rBV-infected cells, (ii) is modified heterogeneously by asparagine-linked glycosylation and expressed as a ≥58-kDa glycoprotein that is present in the region of the cytoplasmic inclusions in HCMV-infected fibroblasts, (iii) is present in virions and two other enveloped virus particles, and (iv) is not essential for growth of HCMV in human foreskin fibroblast cultures
Identification and Characterization of the Bovine Herpesvirus 5 US4 Gene and Gene Products

AbstractThe BHV-5 strain N569 (BHV-5/N569) homolog to the BHV-1 US4 gene was sequenced and characterized. RNA analyses showed that a 1.8-kb mRNA which contains the BHV-5/N569 US4 open reading frame initiates 55 nucleotides upstream from the predicted translational start codon and terminates 17 nucleotides downstream from the consensus sequence for polyadenylation. Comparison of the deduced amino acid sequences of the predicted US4 encoded proteins of BHV-5/N569 and BHV-1 strain Schönböken (BHV-1/Schö) revealed 75% identity. An antiserum, raised in rabbits after infection with a BHV-5/N569 US4 ORF expressing recombinant vaccinia virus, specifically precipitated a 65-kDa protein and a diffusely migrating protein species with an apparent molecular mass between 90 and >240 kDa from the supernatant of BHV-5/N569 infected cells. Treatment of immmunprecipitated proteins with chondroitinase AC demonstrated that the latter contains glycosaminoglycans. The mobility of the BHV-5/N569 US4 gene products was identical to the BHV-1 US4 ORF encoded glycoprotein G (gG) and glycoproteoglycan G (gpgG; G.M. Keil, T. Engelhardt, A. Karger, and M. Enz,J. Virol.70, 3032–3038, 1996) and were therefore named BHV-5 gG and BHV-5 gpgG. Immunoprecipitations with sera from BHV-1 infected cattle indicated a type-specific immune response to gG, since these sera failed to react with vaccinia virus-expressed gG-5 but recognized vaccinia virus-expressed gG-1
Immunization with Dissociated Neuraminidase, Matrix, and Nucleoproteins from Influenza A Virus Eliminates Cognate Help and Antigenic Competition

AbstractWhen hemagglutinin (HA) and neuraminidase (NA) are presented together on an intact influenza virus particle, the antigens are competitive, with HA dominant over NA in T- and B-cell priming. Immunization with mixtures of purified HA and NA eliminates antigenic competition between HA and NA, as well as between N1-N2 NA mixtures. Evidence that vaccine preparations contain influenza virus matrix (M1) and nucleoprotein (NP) prompted the investigation of possible competing effects of these proteins on the anti-HA and anti-NA immune response. However, in BALB/c mice immunized with mixtures of purified NA, M1, and NP no antigenic competition was demonstrated in either the primary or the secondary response. When mice were immunized with intact virus or by infection, a lesser antibody response to M1 and NP was observed. Furthermore, as measured by mean pulmonary virus titers after infection, no additional protective effects were conferred on mice immunized with M1 and NP either alone or in conjunction with other antigens. These studies of influenza virus antigen mixtures have implications for vaccination against influenza and other vaccines consisting of combinations of antigens
Intracellular Transport and Processing of the Marburg Virus Surface Protein in Vertebrate and Insect Cells

AbstractThe surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oligosaccharides leading to a mature protein of 170–200 kDa approximately 30 min after pulse labeling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slightly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein
The Mumps Virus SH Protein Is a Membrane Protein and Not Essential for Virus Growth

AbstractBy immunoprecipitation analysis using antisera against oligo peptides synthesized based on the deduced N-terminal and C-terminal amino acid sequences of the SH proteins of the mumps virus, the SH protein was detected in mumps virus-infected cells. The SH protein expressed from cDNA by the vaccinia-T7 expression system was recovered in the membrane fraction. Association of the SH protein with the membrane was resistant to high salt, EDTA, and alkaline treatment but sensitive to detergents. Indirect immunofluorescence experiments showed that the SH protein is involved in the exocytotic pathway. These data indicate that the SH protein is a membrane protein. Treatment of microsomes with TPCK-trypsin suggested that the SH protein is oriented in the membrane with its C-terminal facing the cytoplasm. Furthermore the SH protein was not detected in a particular strain (Enders strain) of mumps virus, indicating that the mumps virus SH protein is not essential for virus replication
The Efficiency of Sendai Virus Genome Replication: The Importance of the RNA Primary Sequence Independent of Terminal Complementarity

AbstractFrom the cDNAs of two defective RNAs naturally exhibiting a large difference in replication efficiency, a series of Sendai virus RNA chimeras were constructed by reciprocal exchanges of their 3′ end primary sequences. Using a reverse genetics system, the ability of these RNAs to replicate when expressed from cDNAs in the context of the viral proteins N, P, and L, also expressed from plasmids, was analyzed. First the extent of potential RNA 3′/5′ end complementarity was tested by disrupting and restoring the terminal 110-nucleotide complementarity of a copy-back RNA. Alternatively, this base pairing potential was gradually increased from 12 to 57 or to 98 nucleotides by continuous substitutions. In all cases, the restoration or the creation of more extended base pairing potential had no effect on RNA replication. Reciprocal exchanges were then made in order to identifycis-acting sequences that could induce high replication efficiency. It was found that nucleotides 1–31 of the antigenome 3′ end were sufficient to confer a high replication property (more than a 10-fold increase), regardless of the sequence adjacent to these terminal nucleotides. It is concluded that one of the most important features that modulate replication efficiency is contained in the promoter end primary sequence and that this feature is likely to operate independently of the ability to form a potential terminal base pairing
Potency of Wild-Type and Temperature-Sensitive Vesicular Stomatitis Virus Matrix Protein in the Inhibition of Host-Directed Gene Expression

AbstractThe matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and also appears to be involved in the inhibition of host gene expression that is a characteristic cytopathic effect of VSV infection. Previous studies have shown that expression of M protein inhibits host-directed transcription in the absence of other viral gene products and have suggested that only small amounts of M protein are required for the inhibition. In experiments described here, the potency of M protein in inhibition of host-directed gene expression was determined by cotransfecting different amounts ofin vitro-transcribed M protein mRNA together with a target gene encoding chloramphenicol acetyl transferase (CAT) into BHK cells or PC12 cells that had been cultured in the presence or the absence of nerve growth factor. The results of these experiments showed that the potency of M protein was similar in the two cell types and was not affected by the extent of differentiation of PC12 cells. Inhibition of CAT gene expression by M protein was also independent of the nature of the promoter activating sequences of several different RNA polymerase II-dependent promoters. The amount of M protein needed to give 50% inhibition of CAT expression was estimated to be 6700–11,000 copies per cell. Earlier data that temperature-sensitive (ts) M gene mutants of VSV inhibit host transcription had been interpreted to indicate that M protein was not involved in the inhibition. When the amount of M protein expressed was taken into account, ts M protein was as effective as wild-type M protein in the inhibition of host-directed transcription at the nonpermissive temperature. Thus, inhibition of host transcription by ts M mutants of VSV is due to the potent activity of M protein, which is evident even at the low levels produced at the nonpermissive temperature
Genetic Stability of a Porcine Rotavirus RNA Segment during Repeated Plaque Isolation

AbstractThe porcine group A rotavirus CC86 was characterized to explore its utility as a tool for mutation analysis. It has a semiduplication of the gene 11 RNA segment. Nucleotide sequence determination of cDNA confirmed that the NSP5 coding sequence and the conserved nontranslated termini of the RNA segment were retained. A comparison of the NSP5 genes of CC86 and CN86 that were isolated from the same fecal specimen showed eight base pair changes, suggesting that CN86 was not the immediate progenitor of CC86. Synthesis of NSP5 in monkey MA104 cells infected with CC86, CN86, or simian rotavirus SA11 was compared by one- and two-dimensional polyacrylamide gel electrophoresis. NSP5 from all three viruses had similar posttranslational modifications, and no difference in the expression levels was observed. To experimentally address the genetic stability of CC86 segment 11, the virus was passaged by serial plaque to plaque transfer. The repeated genetic bottlenecking led to a gradual loss of fitness. This effect is not observed when virus is passaged by the standard method of moderate dilution. Nucleotide sequence analysis of cDNA clones isolated from viral segment 11 RNA of virus from plaque-to-plaque passage numbers 0, 1, 4, and 8 showed occasional base substitutions, mostly in the NSP5 coding sequence. Two mutations, leading to His-to-Arg and Lys-to-Arg replacements, respectively, in NSP5 were established in the virus population. Forward and reverse base pair changes (A-U[formula]G-C) at the two sites appeared to be concerted and take place at a very high frequency, suggesting that a mechanism equivalent to RNA editing might operate. The overall mutation rate of segment 11 was much lower, having a calculated maximal value of 5 × 10−5per replicated base