pinecone/core-2020-05-10-deduplication huggingface dataset:
A Mutational Analysis of the Transforming Functions of the E8 Protein of Bovine Papillomavirus Type 4

AbstractThe E8 protein of BPV-4 contributes to transformation of primary bovine cells (PalFs) by inducing anchorage-independent growth and by down-regulating gap junction intercellular communication, likely due to its binding to 16K ductin. We show here that, in addition, E8 confers on PalF cells the ability to grow in low serum and to escape from contact inhibition (focus formation). E8 also transactivates an exogenous human cyclin A gene promoter, suggesting that overexpression of cyclin A is responsible for the transformed phenotype. Mutant forms of E8 were generated to establish whether the transforming functions of the protein could be segregated. Mutations were introduced both in the hydrophobic domain and in the hydrophilic C-terminal “tail”, and chimeras with BPV-1 E5 were constructed. Cells expressing either wild-type E8 or mutant forms were analyzed for their ability to grow in low serum and in suspension and to form foci. Wild-type E8 and its mutants were also analyzed for their ability to transactivate the cyclin A promoter. We show here that the transforming functions of E8 can be segregated and that both the hydrophilic C-terminal tail and the residue at position 17 in the hydrophobic domain are crucial for E8 functions and for the transactivation of the cyclin A promoter. These results support the hypothesis that the different aspects of cellular transformation brought about by E8 might be due to interaction with different cellular targets. They suggest that E8 might function differently from BPV-1 E5 and demonstrate that the separate domains of E5 and E8 are not functionally interchangeable
Noncytopathic Flavivirus Replicon RNA-Based System for Expression and Delivery of Heterologous Genes

AbstractNoncytopathic replicons of the flavivirus Kunjin (KUN) were employed for expression and delivery of heterologous genes. Replicon vector C20DX2Arep, containing a unique cloning site followed by the sequence of 2A autoprotease of foot-and-mouth disease virus, was constructed and used for expression of a number of heterologous genes including chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), β-galactosidase, glycoprotein G of vesicular stomatitis virus, and the Core and NS3 genes of hepatitis C virus. The expression and proper processing of these genes upon transfection of BHK21 cells with the recombinant replicon RNAs were demonstrated by immunofluorescence, radioimmunoprecipitation, and appropriate reporter gene assays. Most of these recombinant KUN replicon RNAs were also successfully packaged into secreted virus-like particles (VLPs) by subsequent transfection with Semliki Forest virus replicon RNA expressing KUN structural genes. Infection of BHK21 and Vero cells with these VLPs resulted in continuous replication of the recombinant replicon RNAs and prolonged expression of the cloned genes without any cytopathic effect. We also developed a replicon vector for generation of stable cell lines continuously expressing heterologous genes by inserting an encephalomyelocarditis virus internal ribosomal entry site-neomycin transferase gene cassette into the 3′-untranslated region of the C20DX2Arep vector. Using this vector (C20DX2ArepNeo), stable BHK cell lines persistently expressing GFP and CAT genes for up to 17 passages were established. Thus noncytopathic KUN replicon vectors with the ability to be packaged into VLPs should provide a useful tool for the development of noninfectious and noncytopathic vaccines as well as for gene therapy applications
Expression of Hepatitis C Virus cDNA in Human Hepatoma Cell Line Mediated by a Hybrid Baculovirus–HCV Vector

AbstractAlthough great progress has been made in the characterization of the biochemical and biological features of hepatitis C virus (HCV) gene expression, the elucidation of the HCV life cycle and the evaluation of novel antiviral strategies have been hindered by the lack of a suitable cell culture system. In this context, the development of an efficient HCV cDNA delivery method would contribute to the understanding of HCV replication. To assess the functionality of baculovirus mediated gene delivery for HCV expression, we have constructed recombinant baculoviruses encoding HCV cDNA under the control of the cytomegalovirus promoter. Transduction of the human hepatoma cell line Huh-7 with Bac–HCV vectors was efficient and HCV cDNA expression was enhanced by treatment of the infected cells with dexamethasone. HCV structural and nonstructural polypeptides were processed correctly and were found to localize in the cytoplasm in a pattern characteristic of the endoplasmic reticulum. The expression of the HCV proteins was detected for 49 days after infection. Thus, these results indicate that the recombinant Bac–HCV vectors are a useful tool for the delivery of HCV cDNA and can facilitate the analysis of structural and functional properties of the HCV proteins. In addition, the Bac–HCV vectors can provide important information on the evaluation of novel anti-HCV antiviral strategies
The Haplotype Distribution of Two Genes of Citrus Tristeza Virus Is Altered after Host Change or Aphid Transmission

AbstractGenetic variability of citrus tristeza virus (CTV) was studied using the haplotypes detected by single-strand conformation polymorphism (SSCP) analysis of genesp18andp20in six virus populations of two origins. The Spanish group included a CTV isolate and subisolates obtained by graft-transmission to different host species. The other included two subisolates aphid-transmitted from a single Japanese isolate. The homozygosity observed for genep20was always significantly higher than that expected under neutral evolution, whereas only three populations showed high homozygosity forp18,suggesting stronger host constraints forp20than forp18.Sequential transmissions of a Spanish isolate to new host species increased the difference between its population and that of the successive subisolates for genep18,as estimated by theFstatistic. Analysis of molecular variance indicated that variation between both groups of populations was not statistically significant, whereas variations between populations of the same group or within populations were significant for both genes studied. Our data indicate that selection affects the haplotype distribution and that adaptation to a new host can be as important or more as the geographical origin. Variation of the CTV populations after host change or aphid transmission may explain in part the wide biological variability observed among CTV isolates
The Cleavable Carboxyl-Terminus of the Small Coat Protein of Cowpea Mosaic Virus Is Involved in RNA Encapsidation

AbstractThe site of cleavage of the small coat protein of cowpea mosaic virus has been precisely mapped and the proteolysis has been shown to result in the loss of 24 amino acids from the carboxyl-terminus of the protein. A series of premature termination and deletion mutants was constructed to investigate the role or roles of these carboxyl-terminal amino acids in the viral replication cycle. Mutants containing premature termination codons at or downstream of the cleavage site were viable but reverted to wild-type after a single passage through cowpea plants, indicating that the carboxyl-terminal amino acids are important. Mutants with the equivalent deletions were genetically stable and shown to be debilitated with respect to virus accumulation. The specific infectivity of preparations of a deletion mutant (DM4) lacking all 24 amino acids was 6-fold less than that of a wild-type preparation. This was shown to be a result of DM4 preparations containing a much increased percentage (73%) of empty (RNA-free) particles, a finding that implicates the cleavable carboxyl-terminal residues in the packaging of the virion RNAs
Cis-Acting Functions of Alfalfa Mosaic Virus Proteins Involved in Replication and Encapsidation of Viral RNA

AbstractcDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to studycis- andtrans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to acis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required intransfor replication and encapsidation of RNAs 1 and 2 but is required incisfor replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis
Transfer RNA Modification Status Influences Retroviral Ribosomal Frameshifting

AbstractThe possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 3′ side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status. In addition, when AAU occurred immediately upstream of UUU as the penultimate frameshift site codon, addition of tRNAAsnwithout the Q base reduced the stimulatory effect of tRNAPhewithout the Y base, whereas addition of tRNAAsnwith the Q base did not alter the stimulatory effect. The addition of tRNAAsnwithout the Q base and tRNAPhewith the Y base inhibited frameshifting. The latter studies suggest an interplay between the tRNAs decoded at the penulimate frameshift and frameshift site codons that is also influenced by tRNA modification status. These data may be intrepreted as indicating that a hypomodified isoacceptor modulates frameshifting in an upward manner when utilized at the frameshift site codon, but modulates frameshifting in a downward manner when utilized at the penultimate frameshift site codon
Specific Binding of Recombinant Foamy Virus Envelope Protein to Host Cells Correlates with Susceptibility to Infection

AbstractThe interaction of simian foamy viruses (FVs) with their putative cellular receptor(s) was studied with two types of recombinant envelope protein (Env). Transient expression of full-length Env in BHK-21 cells induced syncytia formation. However, selected stable transfectants fused with naive cells but not with each other. A soluble fusion protein of the Env surface domain with the Fc fragment of a human IgG1 heavy chain (EnvSU–Ig) was produced in the baculovirus expression system, purified to homogeneity, and used for binding and competition analyses. EnvSU–Ig but not unrelated Ig fusion proteins bound to cells specifically. Neutralizing serum blocked binding of EnvSU–Ig and, vice versa, serum-mediated neutralization was abrogated by the chimeric protein. Concomitant reduction of EnvSU–Ig binding and FV susceptibility was seen in Env-expressing target cells. Although EnvSU–Ig did not inhibit FV infection, very likely due to its displacement by multivalent virus–cell interactions, this divalent ligand should help to characterize functionally and to identify the ubiquitous FV receptor
Antisense Downregulation of a Mouse Mammary Tumor Virus Activated Protooncogene in Mouse Mammary Tumor Cells Reverses the Malignant Phenotype

AbstractActivation of the protooncogeneWnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice.Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state.Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuousWnt-1 expression for their ability to form tumorsin vivo.To address this question, we constructed antisense and senseWnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenousWnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and senseWnt-1 RNAs, changes in endogenousWnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cellsin vitroandin vivowere also examined. Our results show that (1) the levels of the expression of endogenousWnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisenseWnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors (≃0.2 cm in 16 weeks) compared to the tumors (≃2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest thatWnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity
Natural Selection of thePolGene of Bovine Immunodeficiency Virus

AbstractGenetic variability is a salient feature of lentiviruses, contributing to the pathogenesis of these viruses by enabling them to persist in the host and to resist anti-retroviral treatment. Bovine immunodeficiency virus (BIV), a lentivirus of unknown pathology, infects cattle in the United States and worldwide. Genetic diversity of BIV that is associated with naturally infected cattle is not well studied. We examined the genetic diversity and natural selection of a segment of the BIVpolgene amplified from the leukocyte DNA of naturally infected cattle. A portion of the reverse transcriptase domain (183 bp) of thepolregion was targeted for amplification by PCR. PCR products were sequenced directly and aligned. When compared to the sequences of BIV R29-127, a molecular clone of the original BIV R29 isolate, all isolates were greater than 91% identical in nucleotide sequences and 77% identical in amino acid sequences.Polgenotypes were polymorphic at 14% of the nucleotide sites. The ratio of nonsynonymous to synonymous nucleotide substitutions (relative to the number of respective sites, Ka/Ks) was 0.16, indicating that this region of the BIV genome, like that of HIV-1, is subject to purifying selection. Based on the McDonald–Kreitman analysis, this region also was under positive Darwinian selection as HIV-1 and BIV diverged from a common progenitor. Phylogenetic analysis revealed that genotypes were geographically distinct, possibly indicating a common source of infection for animals within a herd
Tagging the Human Immunodeficiency Virus Gag Protein with Green Fluorescent Protein Minimal Evidence for Colocalisation with Actin

AbstractThe assembly and budding of human immunodeficiency virus type 1, encoded solely in the Gag protein precursor Pr55Gag, occur at the plasma membrane of infected cells. However, little is known about the routing of the Gag molecule from its site of synthesis in the cytoplasm to the site of budding, with past studies suggesting that the cytoskeleton, particularly actin, may be involved in the translocation. We have constructed a T7 promoter-driven gag gene fusion with green fluorescent protein (GFP) that expresses Gag-GFP in both cells and supernatant. The distribution of Gag-GFP was the same as Gag only, suggesting that cellular routing was not affected by fusion to GFP, and using colabelling techniques, Gag-GFP was shown to have no particular colocalisation with actin. After detergent extraction of expressing cells, Gag and Gag-GFP remained cell associated, whereas GFP only was wholly released. These data suggest that Gag may associate with other cytoskeletal components or, perhaps more likely, that a partial assembly to a large-molecular-weight intermediate occurs before localisation at the plasma membrane
Poliomyelitis in Intraspinally Inoculated Poliovirus Receptor Transgenic Mice

AbstractMice transgenic with the human poliovirus receptor gene develop clinical signs and neuropathology similar to those of human poliomyelitis when neurovirulent polioviruses are inoculated into the central nervous system (CNS). Factors contributing to disease severity and the frequencies of paralysis and mortality include the poliovirus strain, dose, and gender of the mouse inoculated. The more neurovirulent the virus, as defined by monkey challenge results, the higher the rate of paralysis, mortality, and severity of disease. Also, the time to disease onset is shorter for more neurovirulent viruses. Male mice are more susceptible to polioviruses than females. TGM-PRG-3 mice have a 10-fold higher transgene copy number and produce 3-fold more receptor RNA and protein levels in the CNS than TGM-PRG-1 mice. CNS inoculations with type III polioviruses differing in relative neurovirulence show that these mouse lines are similar in disease frequency and severity, demonstrating that differences in receptor gene dosage and concomitant receptor abundance do not affect susceptibility to infection. However, there is a difference in the rate of accumulation of clinical signs. The time to onset of disease is shorter for TGM-PRG-3 than TGM-PRG-1 mice. Thus, receptor dosage affects the rate of appearance of poliomyelitis in these mice
Phenotypic and Functional Characterization of CD8+T Cell Clones Specific for a Mouse Cytomegalovirus Epitope

AbstractA series of CD8+T cell clones, specific for the IE1 epitope YPHFMPTNL, of the immediate-early protein 1 of the murine cytomegalovirus (MCMV) were generated in order to determine their protective activity against this infection and correlate their phenotypic markers with antiviral activity. We found that the adoptive transfer of three of these anti-MCMV CD8+T cell clones into irradiated naive mice resulted in protection against challenge, while another CD8+T cell clone, of the same specificity, failed to confer protection. The clones that conferred protection against lethal challenge reduced greatly viral replication in the lung and other organs of the mice. Using one of the protective anti-MCMV CD8+T cell clones we found that in order to be fully protective the cells had to be transferred to recipient mice no later than 1 day after MCMV challenge. The adoptive transfer of these CD8+T cell clones also protected CD4+T-cell-depleted mice. Phenotypic characterization of the anti-MCMV clones revealed that the nonprotective clone expressed very low levels of CD8 molecules and produced only small amounts of TNF-α upon antigenic stimulation. Most importantly, our current study demonstrates that this MHC class I-restricted IE1 epitope of MCMV is efficiently presented to CD8+T cell clonesin vivoand further strengthens the possibility of the potential use of CD8+T cell clones as immunotherapeutic tools against cytomegalovirus-induced disease
Immune Response to the Immunodominant Epitope of Mouse Hepatitis Virus Is Polyclonal, but Functionally Monospecific in C57Bl/6 Mice

AbstractMutations in an immunodominant CD8 CTL epitope (S-510-518) are selected in mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus. These mutations abrogate recognition by T cells harvested from the infected CNS in directex vivocytotoxicity assays. Previous reports have suggested that, in general, an oligoclonal, monospecific T cell response contributes to the selection of CTL escape mutants. Herein, we show that, in MHV-JHM-infected mice, the CD8 T cell response after intraperitoneal infection is polyclonal and diverse. This diverse response was shown to include both polyclonal and oligoclonal components. The polyclonal data were shown to fit a logarithmic distribution. With regard to specificity, we used a panel of peptide analogues of epitope S-510-518 and spleen-derived CD8 T cell lines to determine why only a subset of possible mutations was selected in persistently infected mice. At a given position in the epitope, the mutations identified inin vivoisolates were among those that resulted in the greatest loss of recognition. However, not all such mutations were selected, suggesting that additional factors must contribute to selectionin vivo.By extrapolation of these results to the persistently infected CNS, they suggest that the selection of CTL escape mutants requires the presence of a monospecific T cell response but also show that this response need not be oligoclonal
The Nucleoprotein of Marburg Virus Is Target for Multiple Cellular Kinases

AbstractThe nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404–432; II*, amino acids 446–472; III*, amino acids 484–511; IV*, amino acids 534–543; V*, amino acid 549; VI*, amino acids 599–604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*–V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis