pinecone/core-2020-05-10-deduplication huggingface dataset:
Epithelial Cells Display Separate Receptors for Papillomavirus VLPs and for Soluble L1 Capsid Protein

AbstractWe examined the distribution of putative receptors for papillomavirus (PV) capsid proteins on various cell types, using either Hexahis HPV6b L1 fusion protein or synthetic HPV6b virus-like particles (VLPs). Specific, saturable binding of VLPs to CV-1 cells was demonstrated using35S-labeled VLPs, with an average receptor number of 1 × 104/cell and a binding affinity constant (Ka) of 4 × 107M.VLP binding was quantitated by flow cytometry using a monoclonal antibody to the L1 capsid protein. Intense staining of epithelial and mesenchymal cells was observed. Some immature bone marrow-derived cells bound VLPs weakly, while the majority of B lymphoma cells demonstrated no binding. Binding to 12 of 16 VLP receptor positive cell lines was abolished by trypsin pretreatment of cells. Removal of cellular sialic acid or O-linked oligosaccharides separately did not affect VLP binding, which was enhanced about 25% when cells were pretreated with both neuraminidase andO-glycosidase. Culture of cells with sufficient tunicamycin to inhibit Concanavalin A binding did not diminish the binding of VLPs. Denatured L1 protein, either from VLPs or expressed fromEscherichia colias a Hexahis fusion protein, bound to a trypsin-resistant structure on a range of cell types and did not block the binding of VLPs to cells. Dual-fluorescence assay with a Burkitt lymphoma line BL72 demonstrated that Hexahis L1 protein and VLPs bind to separate cell surface molecules on BL72 cells. We conclude that the first binding of PV virus to cells is via a widely distributed membrane protein receptor(s) and that subsequent processing of particles may involve other non-trypsin-sensitive structure(s) also displayed on the cell membrane
Pepscan Mapping and Fusion-Related Properties of the Major Phosphatidylserine-Binding Domain of the Glycoprotein of Viral Hemorrhagic Septicemia Virus, a Salmonid Rhabdovirus

AbstractThe binding of labeled phosphatidylserine (PS) to a collection of synthetic 15-mer peptides covering full-length glycoprotein G (G) of viral hemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus, showed three dominant overlapping reactive peptides. This major PS-binding region was contained in a 28-mer peptide (p2; aa 82–109) with consecutive hydrophobic amino acid a–d heptad repeats (putative amphipathic α-helix) and 2 carboxy-terminal arginines. This 28-mer peptide showed a 10-fold higher apparent specific activity for PS binding than the 15-mer peptides. Binding to PS was also detected with virion-purified protein G but was not detected with other viral proteins. The highest apparent specific activity for PS binding was found with purified VHSV particles by both solid-phase and liquid assays. In contrast to the pH-independent PS binding to peptide p2, binding to virions was optimal at pH 5.6. PS binding to purified VHSV was greatly reduced by protease or detergent treatments that removed protein G, by treatment at pH 7.6, or by anti-p2 mouse antibodies at pH 5.6. The PS-binding region seems to be related to viral–host cell fusion since anti-p2 mouse antibodies inhibited VHSV-infected cell to cell fusion (fusion from within) and the pH profile of the VHSV-infected cell to cell fusion was similar to the pH profile of PS binding to VHSV. Comparative analysis showed that sequences similar to the major PS-binding domain of VHSV were also present in other fish rhabdoviruses and in rabies and vesicular stomatitis viruses
Comparison of the Nucleic Acid- and NTP-Binding Properties of the Movement Protein of Cucumber Mosaic Cucumovirus and Tobacco Mosaic Tobamovirus

AbstractThe cucumber mosaic virus (CMV) 3a movement protein (MP) was compared directly to the well-characterized tobacco mosaic virus (TMV) 30K MP by cloning the genes encoding these proteins intoEscherichia coli,isolating theE. coli-expressed MPs, and characterizing them with regard to RNA- and NTP-binding activities. The two MPs were shown to bind single-stranded RNA and DNA cooperatively, but with no sequence specificity. However, discrete lengths of CMV RNA 3 could be protected against RNase digestion by the CMV 3a protein, indicating that the RNA was not uniformly covered by the MP after cooperative binding. The TMV 30K:RNA complex was more stable in NaCl than the CMV 3a:RNA complex; about 50% of the corresponding complexes were stable in 0.6 and 0.4MNaCl, respectively. Both MPs could bind GTP strongly and UTP weakly, but not ATP or CTP. The CMV 3a protein expressed either inE. coliorin plantafrom RNA 3 of CMV was tagged at its C-terminus with six histidine residues, which facilitated its purification by affinity chromatography on a matrix containing Ni2+-nitrilotriacetate. The soluble, His-tagged 3a proteins, affinity-purified fromE. coliand zucchini squash, both were able bind CMV RNA 3in vitro
Hepatitis B Virus X Gene Expression Is Activated by X Protein but Repressed by p53 Tumor Suppressor Gene Product in the Transient Expression System

AbstractHepatitis B virus (HBV) X gene is known to exhibit a transcriptional activation function and is considered to play a major role in hepatocarcinogenesis. We determined a 20-bp promoter element for the HBV X gene transcription and found a binding protein to this promoter element, designated as an X-PBP. We then examined the effects of HBV X protein and p53 tumor suppressor gene product on X gene transcription from the 20-bp promoter element using the transient expression technique. Activity of the X gene promoter was stimulated by X protein expression, but, in contrast, was repressed by transfected normal p53 gene. On the other hand, mutant p53 gene product exhibited no repression. Moreover, the p53 repression of X gene transcription was canceled by X protein coexpression. Thus, the effects of X protein and normal p53 product appear to be mutually antagonistic in the regulation of X gene expression. However, mutated promoter elements which failed to bind to X-PBP still responded to X protein or p53, indicating that the process of X transactivation or p53 respression may be independent of X-PBP binding to the promoter element. Our data suggest that X protein could disrupt function of normal p53 protein in X gene-transfected cells
Analysis of 94 kb of the Chlorella Virus PBCV-1 330-kb Genome: Map Positions 88 to 182

AbstractAnalysis of 94 kb of DNA, located between map positions 88 and 182 kb in the 330-kb chlorella virus PBCV-1 genome, revealed 195 open reading frames (ORFs) 65 codons or longer. One hundred and five of the 195 ORFs were considered major ORFs. Twenty-six of the 105 major ORFs resembled genes in the databases including three chitinases, a chitosanase, three serine/threonine protein kinases, two additional protein kinases, a tyrosine protein phosphatase, two ankyrins, an ornithine decarboxylase, a copper/zinc-superoxide dismutase, a proliferating cell nuclear antigen, a DNA polymerase, a fibronectin-binding protein, the yeast Ski2 protein, an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease, and an amidase. The genes for the 105 major ORFs were evenly distributed along the genome and, except for one noncoding 1788-nucleotide stretch, the genes were close together. Unexpectedly, a 900-bp region in the 1788-bp noncoding sequence resembled a CpG island
Separation and Characterization of Two Related Giardiaviruses in the Parasitic ProtozoanGiardia lamblia

AbstractGiardiavirus encapsidates a 6.2-kb double-stranded (ds) RNA within a capsid that consists of a major 100-kDa capsid protein (p100) and a minor 190-kDa protein (p190). In this study, two nonhomologous 6.2-kb ds RNAs cohabiting inGiardia lambliatrophozoites were found to be separately encapsidated into two distinct virions, one (designated GLV[p100]) whose capsid consists of p100 and p190, and the other (designated GLV[p95]) whose capsid consists of a 95-kDa protein (p95) and a minor p190-equivalent protein. Both types of virions were enriched in the membranous fraction of a lysate from virus-infectedG. lambliacells. Separation of these virions was achieved by CsCl gradient centrifugation following osmotic rupture of the viral particles. By these treatments, the 6.2-kb ds RNA was removed from GLV[p100] whereas that in GLV[p95] remained unchanged, and the two 6.2-kb ds RNAs that had been purified by this protocol displayed differential hybridization properties to viral cDNA probes. Western blotting and peptide mapping experiments show that p100 and p95 were closely related proteins, but each had distinct amino acid sequences. Virus purification and pulse–chase experiments show that GLV[p100] was selectively secreted into the medium whereas GLV[p95] remained within the trophozoites ofG. lambliatoward the late phase of cell growth. Secretion of GLV[p100] was not inhibited by Brefeldin A. These findings demonstrate the cohabitation of multiple Giardiavirus species inG. lamblia
Antibodies Specific for the Antigenic Domain 1 of Glycoprotein B (gpUL55) of Human Cytomegalovirus Bind to Different Substructures

AbstractGlycoprotein B (gB, gpUL55) is a major antigen for the induction of neutralizing antibodies against human cytomegalovirus, making it an attractive antigen for active and passive immunoprophylaxis. The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure which requires a minimal linear amino acid sequence of more than 75 amino acids (aa 552–635) for antibody binding. We have analyzed the fine specificity of neutralizing and nonneutralizing AD-1-binding monoclonal antibodies. Point mutations were introduced into AD-1 and mutants were expressed as bacterial fusion proteins. The antigens were analyzed in immunoblots using a panel of 13 human and murine monoclonal antibodies. Complete loss of binding of all antibodies was observed with mutations at cysteine residues 573 and 610 as well as with a combinatorial exchange of prolines at position 577 and 613. The remaining mutations had different effects on antibody binding. Six individual recognition patterns were observed, indicating various antigenic substructures on AD-1. Changing the Fc portions of 3 murine monoclonal antibodies to human IgG1 showed that neutralization of AD-1-binding immunoglobulins is exerted by different mechanisms. Dependent on the recognized substructure within AD-1, avidity-dependent as well as Fc portion-mediated effects were observed
Sequence, Function, and Phylogenetic Analysis of an Ascovirus DNA Polymerase Gene

AbstractWe have sequenced a 5.5-kb region of the DNA genome of theSpodopteraAscovirus (SAV) containing a DNA polymerase gene. The gene codes for a 1104-amino-acid polypeptide with seven motifs characteristic of DNA polymerases and three additional motifs associated with polymerases possessing 3′ to 5′ exonuclease activity. The SAV DNA polymerase gene was able to functionally substitute for a baculovirus DNA polymerase gene in a transient assay that relies on origin-specific reporter plasmid DNA replication. Analysis of the predicted DNA polymerase sequence using neighbor-joining and protein parsimony algorithms indicated that this gene was only distantly related to other known viral and cellular DNA polymerases. The SAV DNA polymerase gene is the first ascovirus gene to be identified and sequenced. The molecular phylogenetic analyses of this gene supports the placement of insect ascoviruses in a separate virus family
Functional Characterization of the P2 A Initiator Protein and Its DNA Cleavage Site

AbstractThe A protein of bacteriophage P2 initiates DNA replication by a single-stranded cut at the origin, and the DNA replication proceeds unidirectionally by a modified rolling circle type of replication. The P2 A protein belongs to a family of proteins involved in the initiation of rolling circle DNA replication, and the prototype for this family is the well-characterized A protein of phage φX174. One of the common motifs of this family contains two conserved tyrosine residues, which have been shown to be able to alternate in catalyzing the cleavage as well as joining reactions in the φX174 A protein. We investigated the role of the conserved tyrosine residues in P2 A protein byin vitromutagenesis. Only one of the two conserved tyrosine residues was found to be involved in the cleavage reaction. The tyrosine residue dispensable for cleavage and ligation is, however, required at some other stage of the P2 growth cycle, since viable recombinants containing this mutation could not be obtained. The sequence requirements for cleavage of the target site were analyzed with a set of oligonucleotides having single base alterations in the nick region, and the results indicate that only five core nucleotides need to be conserved for efficient cleavage
Genetic Variants of Human T-Lymphotropic Virus Type II in American Indian Groups

AbstractThe human T-lymphotropic virus type II (HTLV-II) is found in many New World Indian groups in North and South America and may have entered the New World from Asia with the earliest migration of ancestral Amerindians over 15,000 years ago. To characterize the phylogenetic relationships of HTLV-II strains infecting geographically diverse Indian populations, we used polymerase chain reaction to amplify HTLV-II sequences from lymphocytes of seropositive Amerindians from Brazil (Kraho, Kayapo, and Kaxuyana), Panama (Guaymi), and the United States (the Navajo and Pueblo tribes of the southwestern states and the Seminoles of Florida). Sequence analysis of a 780-base pair fragment (located between theenvgene and the second exons oftax/rex) revealed that Amerindian viruses clustered in the same two genetic subtypes (IIa and IIb) previously identified for viruses from intravenous drug users. Most infected North and Central American Indians had subtype IIb, while HTLV-II infected members of three remote Amazonian tribes clustered as a distinct group within subtype IIa. These findings suggest that the ancestral Amerindians migrating to the New World brought at least two genetic subtypes, IIa and IIb. Because HTLV-II strains from Amazonian Indians form a distinct group within subtype HTLV-IIa, these Brazilian tribes are unlikely to be the source of IIa viruses in North American drug users. Finally, the near identity of viral sequences from geographically diverse populations indicate that HTLV-II is a very ancient virus of man
Replication of Synthetic Defective Interfering RNAs Derived from Coronavirus Mouse Hepatitis Virus-A59

AbstractWe have analyzed the replication of deletion mutants of defective interfering (DI) RNAs derived from the coronavirus mouse hepatitis virus (MHV)-A59 in the presence of MHV-A59. Using two parental DI RNAs, MIDI and MIDIΔH, a twin set of deletion mutants was generated with progressively shorter stretches of 5′ sequence colinear with the genomic RNA. All deletion mutants contained in-frame ORFs. We show that in transfected cells and after one passage the DI RNAs were detectable and that their accumulation was positively correlated with the length of 5′ sequence they contained. However, accumulation of two twin mutants, Δ2, in which sequences from nucleotide position 467 were fused to those from position 801, was undetectable. In passage 4 cells, but not in transfected or in passage 1 cells, recombination with genomic RNA led to the appearance of the parental DI RNAs. The accumulation of these parental RNAs was inversely correlated with the length of 5′ sequence on the deletion mutants and was highest in the Δ2 samples. In sharp contrast to the data reported for MHV-JHM-derived DI RNAs, we show that MHV-A59-derived mutant RNAs do not require an internal sequence domain for replication. The data suggest that coronavirus replication involves an RNA superstructure at the 5′ end of the genome or one comprising both ends of the genomic RNA. We also conclude from the recombination data that in-frame mutants with impaired replication signals are more fit than out-frame mutants with intact replication signals
The Novel Protein Kinase of the RR1 Subunit of Herpes Simplex Virus Has Autophosphorylation and Transphosphorylation Activity That Differs in Its ATP Requirements for HSV-1 and HSV-2

AbstractThe large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR1) designated ICP6 and ICP10 for HSV-1 and HSV-2, respectively, has a novel protein kinase (PK) enzymatic activity. ICP10 is localized on the cell surface, a localization that depends on an intact transmembrane (TM) segment. We used immunocomplex PK assays to examine the PK activity of ICP10 in stably transfected eukaryotic cells. Activity was distinct from that of casein kinase II (CKII) in that it did not require monovalent ions and was not inhibited by zinc sulfate. PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the TM segment and it was significantly impaired by mutation of the invariant Lys (Lys176). Loss of PK activity by Lys176mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp291a1) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. PK activity was also absent in cells infected with a recombinant virus (ICP10ΔPK) deleted in the ICP10 PK catalytic motifs. In cells infected with HSV-1 or HSV-2, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and immunoglobulin G (IgG). Comparing the PK activity of ICP6 and ICP10 we found that ICP6 requires five-fold higher concentrations of [γ-32P]ATP than ICP10 and both enzymes are Mn2+dependent, which is also different from CKII that is primarily Mg2+-dependent. Similar results were obtained for various HSV strains and in different cell lines. The data are consistent with the conclusion that the RR1 PK activity is intrinsic
Cell-to-Cell Movement of Potato Virus X Is Associated with a Change in the Size-Exclusion Limit of Plasmodesmata in Trichome Cells ofNicotiana clevelandii

AbstractTo study the effect of cell-to-cell movement of potato virus X (PVX) on the size-exclusion limit of plasmodesmata we have microinjected virus particles and fluorescently labelled dextrans (F-dextrans) with average molecular mass of 4.4, 10, and 20 kDa intoNicotiana clevelandiitrichome cells. A vector construct of PVX expressing GUS (PVX.GUS) moved from the injected cell and modified the plasmodesmata to allow passage of the 4.4- and 10-kDa F-dextrans but not the 20-kDa F-dextran. A mutant PVX with a deletion in the gene for the 25-kDa protein accumulated in inoculated protoplasts to the same level as the wild-type PVX. This mutant virus did not move from the injected cell and did not modify the plasmodesmata to allow passage of the 10-kDa F-dextran. However, the 4.4-kDa F-dextran moved from the cells infected with the mutant virus. These data therefore illustrate that movement of PVX is associated with modification of plasmodesmata. The full plasmodesmatal modification requires a functional 25-kDa protein but partial modification occurs when this protein is present in a mutant form
Beet Soil-Borne Virus RNA 3—A Further Example of the Heterogeneity of the Gene Content of Furovirus Genomes and of Triple Gene Block-Carrying RNAs

AbstractThe complete nucleotide sequence of RNA 3 of the Ahlum isolate of beet soil-borne virus (BSBV) was determined from cDNAs obtained with immunocaptured virus particles and denatured preparations of dsRNA. BSBV RNA 3 is unique among the plant virus RNAs studied so far in containing apparently only the coding sequences of a triple gene block (TGB). The derived amino acid sequences of the three putative TGB-encoded proteins showed the highest level of sequence similarities with those of the corresponding proteins of potato mop top furovirus (PMTV) followed by those of peanut clump furovirus and barley stripe mosaic hordeivirus. Progressively fewer similarities were found with the TGB-encoded proteins of beet necrotic yellow vein virus (uncertain classification), potato X potexvirus, and potato M carlavirus. The 3′-terminal 78 nucleotides of BSBV RNA 3 can be folded into a tRNA-like structure and a high degree of sequence similarity exists between the 122 3′-terminal nucleotides of BSBV RNA 3 and PMTV RNA 2. In other regions, however, no pronounced sequence similarities were found between the two RNAs, and PMTV RNA 2 contains an additional putative gene for a cysteine-rich protein downstream of the TGB. The two viruses are unrelated serologically. BSBV RNA 3 adds a further variant to the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs
Cell-to-Cell Transport of Movement-Defective Cucumber Mosaic and Tobacco Mosaic Viruses in Transgenic Plants Expressing Heterologous Movement Protein Genes

AbstractTo determine if the movement proteins (MPs) of cucumber mosaic cucumovirus (CMV) and tobacco mosaic tobamovirus (TMV) are complementary in function, transgenic plants expressing genes encoding TMV or CMV MP were inoculated with movement-defective mutants of TMV and CMV. Transgenic plants expressing the MP gene of CMV strain S (subgroup II) complemented the cell-to-cell and systemic spread of a movement-defective mutant of CMV strain Fny (subgroup I) but not the local or systemic spread of a movement-defective mutant of TMV. Plants that contained the MP gene from CMV-S were not resistant to wild-type TMV infection. When inoculated with a movement-defective mutant of TMV that produced β-glucuronidase, transgenic plants with the CMV MP gene supported only subliminal infection. Conversely, immunodetection andin situlocalization techniques revealed that transgenic plants accumulating the TMV MP supported cell-to-cell spread, but not systemic transport, of a movement-defective CMV. These studies suggest that the transgenic TMV MP shares some of the functions with the CMV MP required to transport CMV, whereas the transgenic CMV MP is deficient in functions that are needed to mobilize the spread of TMV infection
The HBV-Producing Cell Line HepG2-4A5: A Newin VitroSystem for Studying the Regulation of HBV Replication and for Screening Anti-Hepatitis B Virus Drugs

AbstractAll tissue culture systems for propagating HBV employed so far make use of tandemly arranged HBV genomes usually under the control of strong foreign promoters. Thus these systems are helpful for virus production but are of limited value in the investigation of the regulation of HBV replication or of the extent to which the expression of viral genes might be influenced by cellular signal transduction pathways. To overcome this barrier we established an HBV-producing cell line (HepG2-4A5) by stably transfecting HepG2 cells with a replication-competent, terminally redundant HBV plasmid (pSPT1.2 ×HBV) that contains each of the four major HBV-ORFs only once and exclusively under the control of their own regulatory elements. HepG2-4A5 cells contain a single, nonrearranged, chromosomally integrated, replication-competent HBV genome. In the cytoplasm of HepG2-4A5 cells, all typical viral mRNAs were detectable, but no other viral transcripts were found. Furthermore, all viral gene products are synthesized in a balanced ratio, as close as possible to that found in anin vivoinfection. Dane-like particles released from HepG2-4A5 cells were indistinguishable from virions synthesizedin vivo,by all physical (electron microscopy, buoyant density) and biochemical (endogenous polymerase reaction, immunogenic behaviour) criteria. Because of the autologous genome organization in this system, the HepG2-4A5 cell line allows studies on the function of the HBV gene products with respect to their involvement in regulating HBV replication under conditions imitating as closely as possible the situationin vivo.Furthermore, this cell line might be a helpful tool in screening antiviral drugs and in studying their effect on regulating HBV replication