pinecone/core-2020-05-10-deduplication huggingface dataset:
Cell Type-Dependent Regulation of the Activity of the Negative Regulatory Element of the Hepatitis B Virus Core Promoter

AbstractThe Hepatitis B virus core promoter regulates the expression of the core protein, the precore protein, and the viral DNA polymerase. This promoter is transactivated by HNF4, a liver-enriched transcription factor, through an HNF4 binding site located upstream of the core promoter. The transactivation activity of HNF4 on the core promoter is antagonized by a negative regulatory element (NRE) located upstream of the HNF4 binding site. While the NRE can effectively antagonize HNF4 to suppress the core promoter in HeLa cervical carcinoma cells, it has only a marginal suppressing activity on the core promoter in Huh7 hepatoma cells. By performing deletion-mapping experiments, we have found that the NRE contains at least three independent subregions named NREα, NREβ, and NREγ. Each of these three subregions possesses a weak suppressing activity, but together they generate a strong synergistic suppressing effect on the core promoter. The NREγ subregion is active in both HeLa and Huh7 cells and is bound by a protein factor slightly less than 130 kDa in molecular mass. The NREα and NREβ subregions are active in HeLa cells but not in Huh7 cells. Thus, the marginal suppressing effect of the NRE observed in Huh7 cells was mostly due to the activity of the NREγ subregion. No clear protein factor binding sites could be identified in the NREα and NREβ subregions when the HeLa nuclear extract was used for the DNaseI-footprinting analysis, indicating weak or no protein association with these two subregions in this cell type. However, extensive protein factor binding sites could be identified throughout the sequences of these two subregions when the Huh7 nuclear extract was used for the analysis. These results indicate that a different set of protein factors binds to the NREα and NREβ subregions in Huh7 cells and may account for the inactivity of these two subregions in this cell type. Thus, our results indicate that the cell type-dependent activity of the NRE is due to differential regulation of the activities of the NREα and NREβ subregions by the cell types. This regulation is most likely mediated by cell type-dependent protein factors
Hierarchal Constitutive Phosphorylation of the Vesicular Stomatitis Virus P Protein and Lack of Effect on P1 to P2 Conversion

AbstractIn vitroreconstitution of a transcriptionally active VSV polymerase complex (P:L) reportedly requires phosphorylation of the N-terminal domain of P by CKII. Two constitutively phosphorylated sites have been implicated in this activation for both VSV Indiana and New Jersey serotype P proteins. We show here that, in contrast to New Jersey, the Indiana P protein is constitutively phosphorylated on three sitesin vivo.The evidence rests on assessing the phosphorylation status of transfected P gene constructs containing all possible combinations of Ala substitutions at Ser60, Thr62, and Ser64. All mutants containing the T62A substitution showed a reduced level of phosphorylation and yielded no P-Thr. Surprisingly the S60A/S64A mutant behaved like the triple substitution and displayed no significant phosphorylation, while the S64A mutant yielded no P-Thr. Phosphorylation of Thr62 therefore depended on prior modification of Ser64. We also tested the ability of our mutant P proteins to convert to the more highly phosphorylated P2 species, a modification essential for transcription in the New Jersey serotype and thought to be carried out by an L-protein-associated kinase. All of our transfected mutant P proteins readily converted to P2 in the presence or absence of L cotransfection, and the latter had no significant effect on P phosphorylation. We conclude that VSV Indiana P protein differs in significant ways from New Jersey P. It is hierarchically and constitutively phosphorylated on a cluster of three sites, not two, suggesting that an additional kinase may be involved. Moreover, Indiana P1 to P2 conversion is independent of prior constitutive phosphorylation and does not require the presence of L protein
The Vaccinia Virus A38L Gene Product Is a 33-kDa Integral Membrane Glycoprotein

AbstractThe vaccinia virus gene A38L encodes a highly hydrophobic protein with amino acid similarity to mammalian integrin-associated protein (IAP). In this report we have identified the A38L protein of strain Western Reserve (WR), defined its membrane topology, and analyzed its role in virus production and virulence. An antiserum raised against an A38L peptide identified the A38L gene product as a 33-kDa protein which is expressed at low levels during virus infection. A serum from a rabbit previously infected with WR virus recognized the A38L protein, thus confirming that the A38L gene is expressedin vivo.Using a coupledin vitro-translation/membrane-translocation system the 33-kDa protein was shown to be a membrane-associated and glycosylated form of a 29-kDa polypeptide precursor. The membrane topology of the A38L protein was defined by its glycosylation and protease sensitivity when associated with microsomal membranes. The N-terminal immunoglobulin-like variable domain was protected from exogenous protease and was therefore in the lumen of the vesicle, whereas the C-terminus was sensitive and therefore cytoplasmic. A38L deletion and revertant viruses were constructed and were used to study the involvement of A38L in virus assembly, release, and virulence. Deletion of the A38L gene caused a slight reduction in virus plaque size but did not affect the production of intracellular mature virus or extracellular enveloped virus particles in tissue culture cells nor the virulence of the virus in the murine intranasal model. The A38L protein therefore possesses similar sequence and membrane topology to the mammalian IAP protein but is not required for virus particle production or virulence
Early Phase in the Infection of Cultured Cells with Papillomavirus Virions

AbstractThe fate of full bovine papillomavirus (BPV) virions and virus-like particles after binding to C127 or CV-1 cells was studied by electron microscopy and indirect immunofluorescence. After incubation at 4° for 1 hr, BPV virions were found to be bound to the plasma membrane, and most viruses were absorbed by the cells after 30 min incubation at 37°. Ninety minutes after the virions had been bound to the plasma membrane, the uptake of the virions was completed and most of the antigen was found to be localized in the nucleus. The viruses were transported in phagosomes and the uptake and transportation could be inhibited by cytochalasin B and taxol, suggesting the possible involvement of microfilaments and microtubules in the virus particle uptake and transportation. The capsid proteins could be detected for about 14 hr, until degradation and deposit of the viral antigen in the Golgi complexes. Although binding to the plasma membrane and uptake of virions into large cytoplasmic vesicles could be monitored by electron microscopy, no complete virions were observed in the nucleus of infected cells despite a very strong nuclear fluorescent staining for both L1 and L2 proteins. This may indicate that disintegration of the virions occurs in the cytoplasm and the L1/L2 proteins migrate to the nucleus via their nuclear localization signals
A Potyvirus Polymerase Interacts with the Viral Coat Protein and VPg in Yeast Cells

AbstractThe two-hybrid system was used to test for pairwise interactions between the tobacco vein mottling virus (TVMV)-encoded RNA-dependent RNA polymerase (or NIb protein) and two other TVMV-encoded proteins: the NIa protein, which consists of genome-linked protein (VPg) and proteinase domains, and the viral coat protein (CP). Using this approach, we find that the NIb protein interacts with both the NIa protein and the CP in yeast cells. Moreover, we find that a mutation in the conserved GDD domain of the NIb protein diminishes the NIb–CP interaction but not the NIb–NIa interaction. Likewise, mutations in the vicinity of the NIa protein to which the genomic RNA is covalently attached eliminate the NIb–NIa interaction. We conclude that the NIb protein interacts with the VPg domain of the NIa protein and that this interaction requires a functional RNA attachment site. This interaction may be important for the initiation of viral RNA synthesis in infected cells. We also conclude that the CP interacts with the NIb in a manner that is sensitive in changes in the highly conserved GDD motif. The role of this interaction in the functioning of the NIb protein or the CP is unclear, but may involve regulation of viral RNA synthesis in infected cells
Synthesis of a Full-Length Infectious cDNA Clone of Cucurbit Aphid-Borne Yellows Virus and Its Use in Gene Exchange Experiments with Structural Proteins from Other Luteoviruses

AbstractA full-length cDNA of cucurbit aphid-borne yellows virus (CABYV) has been constructed and expressed either as anin vitrotranscript, under control of a bacteriophage T7 RNA polymerase promoter, orin vivo,under control of the cauliflower mosaic virus 35S promoter in an agroinfection vector. The biological activity of the cloned cDNA was demonstrated by the ability of itsin vitrotranscript to replicate in protoplasts and of the agroinfection vector to infect agroinoculated plants. Virus in the agroinfected plants could be transmitted by the aphid vectorsMyzus persicaeandAphis gossypii.The specificity of luteovirus RNA packaging was investigated by replacing (1) the CABYV coat protein gene (and the overlapping ORF5) by the corresponding region of potato leafroll luteovirus or (2) the CABYV readthrough domain by the readthrough domain of beet western yellows luteovirus. The resulting chimeric transcripts replicated in protoplasts and produced virions
Paramyxovirus Phosphoproteins Form Homotrimers as Determined by an Epitope Dilution Assay, via Predicted Coiled Coils

AbstractWhen HA epitope-tagged and untagged Sendai virus (SeV) P proteins are coexpressed and the products reacted with anti-HA, the untagged P protein is also selected because this protein is found as an oligomer. The oligomer was determined to be a homotrimer by coselection studies in which increasing amounts of untagged versus tagged protein were coexpressed, and these findings were extended to mumps virus, a member of therubulavirusgenus. The region of the SeV protein responsible for the oligomerization was localized to residues 344–411. Computer analysis of the 13ParamyxovirusP proteins in the database revealed that all but one are predicted to form coiled coils in this region, the first of only two regions that can be aligned throughout the entire virus subfamily. The predicted coiled-coil region of the measles virus P protein, when grafted onto the C-terminus of the normally monomeric La protein, led to the efficient oligomerization of this reporter protein. The predicted coiled-coil region of these P proteins thus appears to be sufficient for oligomerization
A Conserved Motif at the 3′ End of Mouse Hepatitis Virus Genomic RNA Required for Host Protein Binding and Viral RNA Replication

AbstractA conserved 11-nucleotide sequence, UGAAUGAAGUU, at the 3′ end of the genomic RNA of coronavirus mouse hepatitis virus was required for host protein binding and viral RNA synthesis. An RNA probe containing this 11-nucleotide sequence bound four cellular proteins with a highly labeled protein of 120 kDa and three minor species with sizes of 103, 81, and 55 kDa. Mutation of the 11-nucleotide motif abolished cellular protein binding. The RNA–protein complexes observed with cytoplasmic extracts from MHV-JHM-infected cells in both RNase protection/gel mobility shift and UV cross-linking assays were indistinguishable from those observed with extracts from uninfected cells. Both negative-strand synthesis and positive-strand replication of viral defective interfering RNAs in the presence of helper virus were affected by mutations that disrupt RNA–protein complex formation, even though the 11 mutated nucleotides were converted to the wild-type sequence, presumably by recombination with helper virus. Kinetic analysis indicated that recombination between DI RNA and helper virus occurred relatively early in the MHV replicative cycle at 5.5 to 7.5 hr postinfection, a time when viral RNA synthesis and replication of positive-strand DI RNA were at barely detectable levels. A DI RNA with a mutation upstream of the protein binding element replicated as efficiently as wild type without undergoing recombination. Thus, the 11-nucleotide conserved host protein binding motif appears to play an important role in viral RNA replication
Characterization of Mengo Virus Neutralization Epitopes II. Infection of Mice with an Attenuated Virus

AbstractA panel of five neutralizing monoclonal antibodies was generated from mice immunized with an attenuated strain of Mengo virus. Four of the antibodies were used to select mutants of Mengo virus which were able to escape neutralization by the selecting antibody, but it was not possible to select mutants which could escape neutralization by the fifth antibody. The capsid coding region of the RNA genome of each mutant was directly sequenced to identify the mutation(s) responsible for the neutralization escape phenotype. These results are compared to those of a previous study in which immunogenic determinants recognized by neutralizing antibodies generated against pentameric capsid subunits were located on the external surface of the Mengo virion. We have confirmed the existence of the previously identified immunogenic determinant in VP3 (site 2) as well as an immunodominant determinant in VP2 (site 1). Two previously uncharacterized determinants, located in surface loops of VP1 (sites 3 and 4), were also identified. None of the mutations conferring the neutralization escape phenotype was found near the surface depressions on the virion which are believed to be the receptor binding sites
Antigenic Sites on the Receptor-Binding Domain of Human Adenovirus Type 2 Fiber

AbstractThe trimeric fiber of adenovirus type 2 (Ad2) mediates the first stage of virus–cell attachment, and the distal head region of the fiber has been implicated as the receptor-binding domain. To locate regions on the primary polypeptide sequence of the fiber which may be involved in virus–cell interaction, peptide-based epitope mapping was performed using (1) polyclonal antibodies prepared against both native Ad2 fiber and Ad2 head protein expressed inEscherichia coliand (2) 18 monoclonal antibodies prepared against trimeric Ad2 head protein expressed in baculovirus. The approach using polyclonal antibodies revealed eight domains on the primary sequence of the head which contain one or more continuous epitopes. At least two of these regions were also recognized by monoclonal antibodies reacting against both monomeric and trimeric fiber head protein. The majority of monoclonal antibodies which did not recognize Ad2 head-specific peptides in ELISA were also nonreactive against the monomeric form of protein in Western blot, suggesting that their recognition of trimer is due to the existence of as yet undefined discontinuous epitopes or to alterations in monomer configuration. Our results correspond well with the recently published X-ray crystallographic model of Ad5 fiber head (D. Xia, L. J. Henry, R. D. Gerard, and J. Deisenhofer,Structure2, 1259–1270, 1994), since most antigenic determinants containing linear epitopes mapped to the outer loops or uppermost β-sheets in this structure. Four of five neutralizing monoclonal antibodies recognized trimer only and none recognized linear peptides. This might suggest that the trimeric form of fiber is necessary for making contact with the receptor(s) and that discontinuous epitopes on the head domain may be involved in fiber–cell interaction
Deletion Analysis of the Promoter for the Cucumber Necrosis Virus 0.9-kb Subgenomic RNA

AbstractSequences comprising the core promoter for the cucumber necrosis virus (CNV) 0.9-kb subgenomic RNA have been determined using deletion analysis and site-directed mutagenesis. The deletion studies indicate that the promoter lies within a region located 20 nucleotides upstream and 6 nucleotides downstream and including the subgenomic start site. Sequences further upstream or downstream of the core promoter do not appear to strongly affect promoter activity or viral RNA accumulation. Results of site-directed mutagenesis studies indicate that nucleotides immediately surrounding the subgenomic start site regulate promoter activity. Comparison of sequences within the CNV promoter region with the corresponding region of other tombusviruses shows that the tombusvirus promoter shares a region of near complete identity in 14 of the 26 core promoter nucleotides. Little similarity exists between the CNV 0.9-kb subgenomic RNA promoter and the region surrounding the transcription initiation site for the CNV 2.1-kb subgenomic RNA. Likewise, limited similarity occurs with the 5′ region of CNV genomic RNA. Sequences similar to the ICR2-like motifs found in the promoters of several alphavirus-like (supergroup III) plant and animal viruses are not apparent. This study represents the first analysis of a subgenomic promoter from a member of supergroup II of positive-strand RNA viruses
VP1 and VP2 Capsid Proteins of Theiler's Virus Are Targets of H-2D-Restricted Cytotoxic Lymphocytes in the Central Nervous System of B10 Mice

AbstractResistance to Theiler's virus-induced demyelination maps genetically to the MHC class I D region and is associated with up-regulation of class I products and the presence of MHC-restricted virus-specific cytotoxic CD8+T cells in the CNS. To determine the targets of the cytotoxic response, transfected C57SV (Kb, Db) cells expressing LP (including the leader peptide, VP4, VP2, and VP3 coding sequences), VP4 (including the leader peptide and VP4), VP2, VP3, VP1, or RP (including P2 and P3) were generated. CNS-infiltrating lymphocytes obtained from virus-infected B10, B10.K (Kk, Dk), B10.RBF (Kb, Df), B10.RFB3 (Kf, Db), and B10.RBQ (Kb, Dq) mice were used as effectors. Specific cytotoxicity to the capsid proteins encoded in the LP construct, VP2 and VP1, was demonstrated to be H-2Dbregion restricted and was mediated by CD8+T cells. No Kb-restricted virus-specific cytotoxicity response was observed. No specific cytotoxic response against RP-encoded proteins was observed in the CNS of B10 mice. Therefore, both VP1 and VP2 are targets for an H-2D-restricted cytotoxic immune response against Theiler's virus infection in the CNS of infected resistant B10 mice
Defective Point Mutants of the Encephalomyocarditis Virus Internal Ribosome Entry Site Can Be Complementedin Trans

AbstractPoint mutations were introduced at random into cDNA corresponding to nucleotides 260–833 of the encephalomyocarditis virus (EMCV) 5′ noncoding region. This region contains the internal ribosome entry site (IRES). The mutations were identified by sequence analysis and the effect on the activity of the IRES was determined usingin vitrotranslation reactions in rabbit reticulocyte lysate. Significantly defective mutants each contained multiple point mutations. These mutants were constructed into a dicistronic mRNA expression plasmid and the activities of the mutant IRES elements were determined using the vaccinia virus/T7 RNA polymerase transient expression systemin vivo.The most severely defective of these mutants displayed about 5% of wild-type activity. The activities, relative to wild type, of these mutant IRES elements determined usingin vitroandin vivoassays were similar. Two deletion mutants, lacking sequences from the 5′ terminus to nt 411 and 484, were also constructed. Each of these deletions inactivated the IRESin vivo(to less than 1% of wild-type activity). Coexpression within cells of the wild-type EMCV IRES, either alone or linked to another coding sequence, enhanced the activity of each of the defective IRES elements except that deleted to nt 484. The results are consistent with a model in which different regions of the IRES participate in a discontinuous transfer of an initiation complex to the 3′ end of the IRES element for initiation of protein synthesis to occur
Functional and Molecular Characterization of African Swine Fever Virus Mutants Resistant to Phosphonoacetic Acid1

AbstractAfrican swine fever virus (ASFV) growth and plaque formation were inhibited by phosphonoacetic acid (PAA) concentrations of 200 μg/ml or more. One spontaneous mutant and two mutants isolated from mutagenized virus were resistant to PAA inhibition and showed practically normal viral DNA synthesis in the presence of PAA. DNA polymerase activity present in the cytoplasmic fraction from cells infected with the mutants required 10-fold higher concentrations of PAA for inhibition compared to equivalent inhibition of the wild-type enzyme. Like wild-type virus, the PAA-resistant mutants were resistant to inhibition by aphidicolin. Marker rescue analysis with mutant DNA fragments covering different regions of the ASFV DNA polymerase gene mapped the mutations within a fragment which was cloned and sequenced. A single nucleotide and amino acid change was assigned to each mutant. Two of the PAA-resistant mutations lie within the highly conserved region II common to alpha-like DNA polymerases, which has been implicated in pyrophosphate binding and probably also in dNTP binding. The other mutation was localized to within a region of moderate homology among viral DNA polymerases close to one of the motifs allegedly considered as constituting the 3′–5′ exonuclease active site
Biochemical and Genetic Analyses of the Interaction between the Helicase-like and Polymerase-like Proteins of the Brome Mosaic Virus

AbstractReplication of the three positive-strand genomic RNAs of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins. One hundred fifteen amino acids of the 2a N-terminus and the 1a helicase-like region of over 50 kDa are both necessary and sufficient for 1a–2a interaction. Requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. To explore the structural properties of 1a, we used limited proteolysis ofin vitro-translated 1a protein. Treatment of 1a and its deletion derivatives with papain or trypsin revealed that the C-terminal helicase-like segment of approximately 50–60 kDa is highly resistant under our assay conditions to proteolysis, while the N-terminus is rapidly degraded. All tested mutations in the helicase-like region that renders this region protease-sensitive have previously been found to be defective for RNA replicationin vivo.To complement thein vitrostudies, we examined the interaction of the 1a helicase-like domain and the 2a N-terminus in yeast using the two-hybrid system. Mutations previously known to disrupt 1a–2a interaction also prevented interaction in yeast. Furthermore, results from two-hybrid analysis suggest that the structural domain mappedin vitrois important for 1a–2a interaction. Finally, we found that the helicase-like proteins of three other tripartite RNA viruses also contain equivalently located protease-resistant domains