pinecone/core-2020-05-10-deduplication huggingface dataset:
Identification of the Active-Site Residues of the 3C Proteinase of Foot-and-Mouth Disease Virus

AbstractTo identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates. Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, involved in substrate binding, or present in nonconserved regions. Wild-type and mutant 3C proteins were expressed inEscherichia coliand tested for their ability to cleave synthetic substrates corresponding to different portions of the viral genome. Substitutions at His-46 (catalytic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) produced enzymes unable to process P1, P2, or P3 substratesin trans,whereas a change in the conserved Asp-98 had no effect on enzyme activity. Substitution of Ser for Cys-163 (catalytic triad) yielded an enzyme that retained activity on some substrates, while a substitution of Gly at this position resulted in a completely inactive enzyme. The kinetics oftransprocessing of translation products from a transcript encoding the P1 and P2 coding regions and the 2C/3A cleavage site with wild-type 3C or a transcript encoding P1 with 3C mutants revealed that the order of cleavage was VP3-VP1, VP0-VP3, VP1-2A, 2C-3A, and 2B-2C. Mutations in 3C that resulted in a partially active enzyme were individually introduced into full-length FMDV cDNA and RNA transcripts were translated in a cell-free system and used to transfect cells. In all cases the virus that was rescued had reverted to the wild-type 3C codon
Isolation and Characterization of Marek's Disease Virus (MDV) cDNAs Mapping to theBamHI-I2,BamHI-Q2, andBamHI-L Fragments of the MDV Genome from Lymphoblastoid Cells Transformed and Persistently Infected with MDV

AbstractWe have isolated and sequenced two cDNAs of sizes 2674 and 677 bp from a cDNA library derived from MKT-1, a lymphoblastoid cell line transformed and latently infected with Marek's disease virus (MDV) using probes corresponding to the right-hand end of theBamHI-I2fragment of the MDV genome. The larger cDNA clone represents an abundant transcript, which extends from the right-hand end ofBamHI-I2to the adjacentBamHI-Q2andBamHI-L fragments of the MDV genome and contains the Meq (MDVEco-Q) open reading frame. The smaller cDNA clone represents a spliced transcript containing the putative DNA binding domain of Meq as well as sequences in theBamHI-L region. We prepared a polyclonal antibody against part of the protein sequence of Meq and detected a 44-kDa protein in MKT-1 cells and in cells lytically infected with MDV. In addition, riboprobes corresponding to sequences specific to each cDNA as well as shared sequences between cDNAs detected a number of transcripts in cells either lytically or latently infected with MDV. Our results indicate that the Meq transcriptional unit extends to theBamHI-L fragment and that the transcripts mapping to the right-hand end of theBamHI-I2and adjacentBamHI-Q2andBamHI-L fragments are not preferentially expressed during latency
Lymphocyte-Tropic Simian Immunodeficiency Virus Causes Persistent Infection in the Brains of Rhesus Monkeys

AbstractMolecularly cloned SIVmac239 is the prototypical SIVmaclymphocyte-tropic virus that replicates productively in lymphocytes but poorly in macrophages. In macaques, the virus causes activation and productive infection of T lymphocytes which invade the central nervous system (CNS) early after infection in the animal. However, infected animals develop immunosuppression and AIDS but rarely overt neurological disease. In this study, we examined multiple regions of the brain and spinal cord for the presence of SIVenvsequences and histological lesions in five macaques that had been infected with SIVmac239 for 1.7 to 2.25 years. Histopathological examination of the brain revealed no lesions consistent with encephalitis; however, viral DNA was found in all five brains. In one animal the virus caused infection in a widely disseminated pattern from the frontal cortex to the distal end of the spinal cord, whereas in the other four animals infection in the CNS occurred in a nonspecific, focal pattern. Sequence analyses were performed on gp120 sequences isolated from selected regions of the CNS and compared to gp120 sequences isolated from corresponding lymph nodes, a tissue known to support productive replication of SIVmac239. Examination of the viral sequences from the CNS tissue from two animals (macaques 10F and 14F) revealed a low mutation rate when compared to the sequences isolated from the lymph node tissues. The percentage change in the amino acid sequence was approximately 1% for CNS clones versus ≥3% for clones isolated from the lymph node. The majority of the CNS viral sequences of macaques 10F and 14F had none of the genetic markers shown in a previous study to be associated with macrophage-tropic variants and indeed retained a nucleotide sequence of similar to the original lymphocyte-tropic virus used for inoculation despite almost 2 years of persistent infection in the animals. Construction of chimeric viruses with V1–V5 regions of selected macaque 10F and macaque 14F CNS-gp120 clones confirmed the predicted lymphocyte-tropic nature of theseenvgenes. In contrast, the gp120 sequences isolated from the CNS tissue of one of the other three animals (macaque 13F) had a mutation rate comparable to that observed for the lymph node clones. The CNS clones from this animal had amino acid substitutions that were previously shown to be associated with macrophage tropism. Compared to the chimeric viruses constructed with V1–V5 sequences from macaques 10F and 14F, viruses constructed with the V1–V5 sequences of several macaque 13F brain clones did not yield infectious virus. These data suggest that following entry into the CSF early during infection in the animals, SIVmac239 caused infection in the CNS. In some animals, the viralenvsequences recovered by the PCR suggested that minimal replication had occurred, whereas in another macaque virus replication had progressed with gradual selection of a more macrophage-tropic genotype
Intracellular Localisation of Herpes Simplex Virus Type 1 Ribonucleotide Reductase Subunits during Infection of Cultured Cells

AbstractWe have analysed the intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase during infection of cultured cells by indirect immunofluorescence using polyclonal and monoclonal antibodies specific for the R1 and R2 subunits. Three different viruses were used to infect cells, wild-type strain 17+and two temperature-sensitive mutants,ts1222, which produces R1 only, andts1207, which expresses a normal R2 and an altered R1 that fails to interact with R2 at the nonpermissive temperature because of an amino acid substitution in R1. R1 was detected 2 hr postinfection with all three viruses and remained evenly distributed throughout the cytoplasm. R2 was not observed until 4 hr postinfection and, in contrast to the even distribution of R1, was localised in discrete cytoplasmic foci close to the nucleus. In double-labelling experiments both R1 and R2 were found in these foci where they presumably associate to form the active enzyme. As expected R2 was not detectable in cells infected withts1222. Ints1207-infected cells it formed wild-type-like foci, indicating that interaction with R1 is not required for R2 focus formation. R1 was present in a twofold excess over R2 in wild-type-infected cells. We suggest that the uncomplexed R1 could perform a role associated with the protein kinase present in the N-terminal domain
Induction by Herpes Simplex Virus of Free and Heteromeric Forms of E2F Transcription Factor

AbstractWe have determined that HSV causes rapid and large increases in cell-cycle-regulated free E2F and S-phase p107/E2F DNA binding activities in asynchronous cultures of C33A cells. Induction occurred by 4 hr postinfection and coincided with the appearance of viral encoded immediate-early and delayed-early proteins, i.e., when viral DNA replication normally commences. No increase in E2F activities occurred when cells were infected with viruses expressing mutant regulatory proteins ICP4 or ICP27, or mutant replication proteins ICP8, pol or helicase, or when cells were infected with wild-type virus in the presence of inhibitors of DNA synthesis. In contrast, ICP8 mutant-infected cells contained elevated amounts of NFκB activity equivalent to WT virus, no induction of Sp1 relative to WT virus, and reduced ATF/CREB activity relative to WT virus. Results of transient expression assays with E2F-responsive reporters indicated that the net effect of induction of both active (free E2F) and repressive (p107/E2F) complexes was a decrease in AdE2 promoter activity and an increase in c-myc promoter activity. Taken together these results suggest that HSV can cause unscheduled changes in the amount and functional status of a cell-cycle-regulated transcription factor. These results are discussed in light of possible roles for viral-induced alterations in E2F, especially as related to imposing or overriding cell-cycle checkpoints
Interaction between RNA-Dependent RNA Polymerase of Alfalfa Mosaic Virus and Its Template: Oxidation of Vicinal Hydroxyl Groups Blocksin VitroRNA Synthesis

AbstractIn the life cycle of a (+)-strand RNA plant virus the processes of template RNA recognition and initiation of the synthesis of a complementary strand by the viral RNA-dependent RNA polymerase (RdRp) are crucial early steps. Using a template-dependentin vitroRNA synthesizing system of alfalfa mosaic virus (AlMV) we were able to study the effect of small chemical modifications of the 3′ end of the template RNAs on product formation. After oxidation of the 3′-terminal nucleoside of the template no products could be detected. Presumably, RNA synthesis was blocked at the stage of initiation, since the promoter of the RdRp is internal (A. C. Van der Kuylet al., Virology176, 346–354, 1990). Blocking was probably due to an irreversible binding of the enzyme to the 3′ end of the modified RNA. Using this system it was shown that in template competition experiments the RdRp of AlMV displays a high specificity for its cognate template, either before or after the oxidation of the 3′-terminal nucleoside. From this it was concluded that periodate modification of the 3′-terminal nucleoside has little or no effect on template recognition. Furthermore, we showed that the viral coat protein, which forms a part of the viral polymerase (R. Quadtet al., Virology182, 309–315, 1991), was not the main target involved in the inhibition of RNA synthesis
Replication of Molluscum Contagiosum Virus

AbstractMolluscum contagiosum virus (MCV) infects preadolescent children and sexually active adults, frequently causing a disfiguring cutaneous disease in immunosuppressed HIV-infected individuals. The development of an efficacious treatment regime has been hampered by the failure to replicate the virus in the laboratory. Here we report the first demonstration of MCV replication in an experimental system. In human foreskin grafts to athymic mice, MCV induced morphological changes which were indistinguishable from patient biopsies and included the development and migration of molluscum bodies containing mature virions to the epidermal surface
Evidence That N-Linked Glycosylation Is Necessary for Hepatitis B Virus Secretion

AbstractHuman hepatitis B virus (HBV) envelopes contain three distinct glycoproteins called L, M, and S HBsAg. Each is posttranslationally modified to contain N-linked oligosaccharides. N-linked oligosaccharides, after attachment to a polypeptide backbone, are processed by enzymes within the endoplasmic reticulum (ER). There is uncertainty about what role, if any, these N glycans and their modification in the ER play in the function of the HBV envelope proteins. By treating hepatoblastoma cultures which secrete HBV (HepG 2.2.15 cells) with inhibitors of different steps of the glycosylation and glycan modifying pathway, we provide evidence that glycosylation and the first step in the processing pathway are necessary for virion, but not subviral particle, secretion. That is, using a highly sensitive immunoprecipitation/polymerase chain reaction system, enveloped HBV could not be detected in the medium of HepG2.2.15 cells incubated with tunicamycin. However, HBVsubviralparticle secretion was not prevented by tunicamycin. Moreover, inhibitors of α-glucosidase I (the first step in the glycan processing pathway) also prevented virion secretion. Inhibitors of mannose trimming (a later step) and glycolipid synthesis, did not prevent virion secretion, defining the limits of the glycosylation requirements in secretion. These results demonstrate a requirement for N-glycosylation and glucosidase processing in the secretion of virions and further distinguish between the requirements for virion and subviral particle secretion
Phenotypic Determinants of Adenovirus E1A Gene Autoregulation: Variable Region between Conserved Coding Domains 2 and 3

AbstractDifferent serotypes and evolutionary variants of human adenoviruses exhibit distinctive patterns of positive and negative autoregulation of the viral E1A gene. An autoregulatory E1A promoter mutation of the adenovirus type 3 (Ad3) E1A gene renders Ad3hr15 incapable of growth in normally permissive cells. The promoter mutation is complementedin transby E1A products of the heterologous helper adenovirus type 5 (Ad5). Second-site revertants of Ad3hr15 restore viability with high levels of E1A gene expression. The revertant E1A genotypes retain the mutant E1A promoter and have small in-frame deletions in the nonconserved region between the repression- and activation-associated conserved domains CR2 and CR3. Plasmid expression vectors were constructed as 12S and 13S cDNA forms of revertant E1A genes. These were used in cotransfection experiments with a reporter gene plasmid under transcriptional control of the mutant Ad3hr15 E1A promoter. The repression of the Ad3hr15 E1A promoter by helper Ad5 or revertant 12S E1A cDNA was consistently greater than that effected by wild-type Ad3 12S E1A cDNA expression. Significantly greater levels of positive transactivation were observed in cotransfections with 13S cDNAs of Ad5 or with the 13S E1A cDNA of Ad3hr15 revertants, compared to the transactivation observed with the mutant-encoded wild-type Ad3 13S E1A cDNA. The Ad5 helper anddl-revertant phenotype of Ad3hr15 appear to be related to transactivation activities of coexpressed E1A genes. The nonconserved region which separates the conserved coding regions CR2 and CR3 of the type 3 E1A gene acts to attenuate E1A-mediated repression and transactivation of transcription
The Proposed Gene for VP1 of HAV Encodes for a Larger Protein Than That Observed in HAV-Infected Cells and Virions

AbstractThe termini of hepatitis A virus (HAV) mature proteins have been assigned mainly by their homology to other picornaviruses and their apparent electrophoretic mobility; the proposed coding sequence for VP1 is supposed to encompass 900 nucleotides from position 2208 to 3107 of the HAV genome. In order to further characterize this protein, we analyzed thein vitro- andin vivo-synthesized translation products of the putative VP1 gene. cDNA coding for full-length VP1 was cloned under the control of a T7 promoter in pTF7-5; the resulting plasmid (pTF7-5/VP1) was used for both synthesis of RNA to program rabbit reticulocyte lysates and construction of a recombinant vaccinia virus (rvv/T7-VP1). Immunoblot analysis and immunoprecipitation using antisera raised against a synthetic peptide corresponding to amino acids 13 to 33 of VP1 (13-33/VP1) led to identification of a 37-kDa protein in lysates ofin vitrotranslated VP1 and rvv/T7-VP1-infected HFS cells, whereas a 33-kDa protein was detected with purified virions and in lysates of HAV-infected HFS cells. Because the antiserum used was directed against an amino-terminal part of VP1 and the amino terminus of VP1 is identified by sequence analysis, these results show that VP1 present in the HAV virions and infected cells is shorter than previously proposed and suggest that the real carboxy terminus of VP1 is approximately 40 amino acids upstream. In order to limit the possible carboxy-terminal sites in the predicted region, we investigatedin vitrosynthesized translation products of a set of constructs with C-termini ending at potential cleavage sites for viral proteases 3C. The construct containing the nucleotides from position 2208 to 3026 codes for a protein (1-273/VP1) which exhibits the same electrophoretic mobility as VP1 synthesized by HAVin vivo
Virus and Host-Specific Adaptations in theBL1andBR1Genes of Bipartite Geminiviruses

AbstractThe host range of individual geminiviruses may be quite narrow, and closely related viruses can exhibit distinct host adaptations. Two such bipartite geminiviruses are bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV). In both, theBL1andBR1genes are required for the spread of virus infection in plants. We have investigated the contributions ofBL1andBR1to host-specific phenotypes of BGMV and TGMV by constructing hybrid viruses in which these coding regions were exchanged. Hybrids were assayed on bean, a good host for BGMV, andNicotiana benthamiana,a good host for TGMV. A BGMV hybrid having TGMVBL1andBR1efficiently infected beans, but elicited attenuated symptoms. InN. benthamiana,this hybrid had slightly increased virulence and DNA accumulation relative to wild-type BGMV. A TGMV hybrid having BGMVBL1andBR1was virulent inN. benthamiana,but elicited attenuated symptoms. However, this hybrid exhibited no gain of function in beans relative to wild-type TGMV. Hybrid viruses with TGMVBL1and BGMVBR1had severely defective phenotypes in either viral or host background. Although exchangingBL1andBR1between BGMV and TGMV did not change host range, some host adaptation of these genes is suggested. However, virus-specific compatibility betweenBL1andBR1is of more importance for viability. Thus, these gene products may act in concert to potentiate virus movement
Tropism of Bunyaviruses: Evidence for a G1 Glycoprotein-Mediated Entry Pathway Common to the California Serogroup

AbstractThe California serogroup is composed of antigenically and biologically related viruses within theBunyavirusgenus of the Bunyaviridae. We used a large panel of murine cells to study their tissue tropisms and found virtually identical patterns of viral replication among all of the members of this serogroup, in contrast to other members of the family (Bunyamwera, Cache Valley, and Punta Toro viruses). By analyzing the nonpermissive infections with both an RNA dot-blot and a virus binding assay, we determined that tropism for cultured cells was determined at the level of entry. A truncated soluble form of the La Crosse G1 glycoprotein (sG1) was expressed in a baculovirus system and, despite slight differences in glycosylation, was shown to resemble native G1 by immunoprecipitation with six monoclonal antibodies. sG1 bound to permissive but not to nonpermissive cell lines, as demonstrated by flow cytometry. The sG1 effectively blocked infection of permissive cell lines with all of the California serogroup viruses, but did not block infection of two other bunyaviruses. These results indicate that the California serogroup bunyaviruses share a common receptor on vertebrate cells which may differ from the receptor used by other Bunyaviridae and demonstrate that the G1 glycoprotein is the virus attachment protein. sG1 will be a useful reagent in the search for a putative receptor molecule
Human Papillomavirus Type 16 Capsid Proteins Produced from Recombinant Semliki Forest Virus Assemble into Virus-like Particles

AbstractSeveral neutralizing sites of the human papillomavirus (HPV) capsid are known to be critically dependent on the conformation of the capsid. However, efficient production of HPV16 capsids in mammalian cells has been difficult, possibly because the HPV genome contains negative regulatory elements. To circumvent these problems, we cloned the HPV16 L1 and L2 genes from a healthy HPV16-infected woman into a Semliki Forest virus based expression vector (P. Liljeström and H. Garoff,Biotechnology9, 1356–1361, 1991). Recombinant HPV16 L1- or L2-producing Semliki Forest virus was generated and used for infection of mammalian cells. The HPV16 L1 and L2 proteins were efficiently expressed and the majority of the L1 protein self assembled into virus-like particles (VLPs). Coexpression of L1 and L2 resulted in incorporation of L2 into the VLPs. The particles had a density of ∼1.3 g/ml as determined by density gradient centrifugation. Transmission electron microscopy revealed that the particles had a morphology similar to native virions. The HPV16 VLPs produced by the Semliki Forest virus expression system may be useful as a conformationally correctly assembled target for studies of HPV attachment, assembly, serology, or vaccination
The Adeno-Associated Virus Rep78 Protein Is Covalently Linked to Viral DNA in a Preformed Virion

AbstractResolution of the covalently closed terminus of adeno-associated Virus (AAV) DNA is mediated by viral replication protein Rep78 or Rep68.In vitrostudies with purified Rep proteins indicate that concurrent with this resolution is a covalent attachment of one of the proteins to the 5′ end of the viral genome. Thein vivoexistence and fate of the covalently associated Rep protein during the virus life cycle has not yet been elucidated. In this report, we use immunoprecipitation analyses to demonstrate that the Rep78 protein is covalently attached to viral DNA in a preformed virion. The attached Rep78 is susceptible to antibody binding and protease digestion, and the DNA linkage is susceptible to nuclease digestion, therefore Rep78 is probably located on the outside of the particle. Rep proteins are also attached to double-stranded replicative-form monomer (RFM) DNA in extracts from AAV and adenovirus coinfected cells. Rep protein attachment to RFMand encapsidated AAV DNA suggest that the covalent complex is an intermediate in virus assembly. These observations are similar to those noted by others for the autonomous parvoviruses and provide additional insights into parvovirus assembly
HNF3 Binds and Activates the Second Enhancer, ENII, of Hepatitis B Virus

AbstractThe basic functional unit of hepatitus B virus (HBV) enhancer II (ENII) is located within nt 1687–1774, which is defined as the B fragment in our previous papers. A majortrans-acting factor binding site has been identified within the B fragment. The sequence corresponding to this binding site was named B2. In this paper, several point mutations were introduced into the B2 subunit by PCR-mediated site-directed mutagenesis. CAT analysis indicated that the TGTTTGTTT motif within the B2 subunit was critical for the activity of ENII. Mutations of individual nucleotides within this motif could decrease the activity of ENII. Electrophoresis mobility shift assay revealed that the liver-enriched transcription factors hepatocyte nuclear factor (HNF) 3α and HNF3β bound to the B2 subunit specifically and the TGTTTGTTT motif was essential for DNA–protein interaction. Anti-HNF3α and anti-HNF3β antisera could block such binding ability. Moreover, HNF3β could switch on the activity of ENII in HeLa cells and the activity of ENII could be suppressed by antisense HNF3α and antisense HNF3β mRNA in HepG2 cells. These results prompted the conclusion that HNF3 was crucial for the liver-specific activity of ENII, which in turn contributed significantly to the liver specificity of HBV