pinecone/core-2020-05-10-deduplication huggingface dataset:
Analysis of the Contributions of the Equine Herpesvirus 1 Glycoprotein gB Homolog to Virus Entry and Direct Cell-to-Cell Spread

AbstractExperiments to analyze the functions of the equine herpesvirus 1 (EHV-1) glycoprotein gB were performed. Cell lines which stably expressed either the full-length EHV-1 gB or only the extracellular portion of gB (amino acids 1 to 844) were constructed and were termed TCgBf and TCgBΔ, respectively. Using the cell line TCgBf, a gB-negative viral mutant, L11ΔgB, was generated by replacing a 2.1-kbBglII–NruI fragment in the EHV-1 strain RacL11 gB with theEscherichia coli LacZgene. EHV-1 strain RacL11, the modified live vaccine strain RacH, and L11ΔgB were used for functional studies. It was shown that: (i) EHV-1 gB is essential for virus growthin vitrosince gB-negative L11ΔgB exhibited titers of <10 PFU/ml when grown and titrated on noncomplementing cells. (ii) The cell line expressing truncated gB (TCgBΔ) did not complement for the growth of L11ΔgB, but the RacH virus grew to titers comparable to those of RacL11 in all cell lines tested. Since RacH had amino acids 944–980 of gB replaced by 7 missense amino acids as determined by nucleotide sequence analysis, the extreme carboxyterminus but not a domain between amino acid residues 845 and 943, probably the transmembrane domain, of EHV-1 gB is dispensable for virus growth in cultured cells. (iii) Single infected cells but no plaque formation were observed after infection of noncomplementing cells with L11ΔgB, demonstrating the requirement of EHV-1 gB for direct cell-to-cell spread of infection. (iv) The attachment of gB-negative L11ΔgB virions to target cells was similar to both phenotypically complemented L11ΔgB and parent RacL11 virus. (v) L11ΔgB viral titers could be enhanced by using the fusogen polyethylene glycol (PEG). The increase of L11ΔgB titers by PEG treatment, however, was considerably lower compared to gB-negative pseudorabies virus, suggesting that EHV-1 gB might not be as stringently required for virus penetration as are its homologs in otherAlphaherpesvirinae
Identification of HIV-1 Determinants for Replicationin Vivo

AbstractPathogenic organisms are frequently attenuated after long-term culturein vitro.The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicityin vivo.To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenicin vivo.In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as anin vivomodel to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replicationin vivo.The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected forin vivoreplication or, alternatively, an attenuated genotype dominantin vitromay have reverted to become more infectiousin vivo.To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replicationin vivoin a NEF-independent mechanism. The specific determinants have been mapped to the V1–V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replicationin vivo
Recombinant Measles Viruses Defective for RNA Editing and V Protein Synthesis Are Viable in Cultured Cells

AbstractThe measles virus (MV) phosphoprotein (P) gene encodes three proteins, P, C, and V. The V protein is synthesized by pseudo-templated transcription, also designated as RNA editing: during P gene transcription one G residue is inserted at a defined position in about 50% of the mRNAs. To study the importance of sequence elements for the nontemplated G insertion, we generated recombinant MVs in which six different mutations were introduced within the region where editing occurs (3′ UUUUUCCC, template strand). These viruses were then analyzed for their ability to edit their P mRNA and to produce V protein. Single U to C changes within the U stretch abolished editing. Extending the template by three C residues at the site of G insertion resulted in a less precise editing phenotype and overproduction of V. None of these mutants were impaired in their multiplication behavior when analyzed in cultured cells. However, the syncytia of a recombinant MV overproducing V protein were in general smaller and lysed 1 to 2 days later than usual
Entry of Semliki Forest Virus into Cells: Effects of Concanamycin A and Nigericin on Viral Membrane Fusion and Infection

AbstractSemliki forest virus (SFV) was biosynthetically labeled with pyrene phospholipids and used to investigate two alternative routes of entry of SFV into BHK-21 cells: (1) receptor-mediated endocytosis followed by fusion of the viral envelope with the endosomal membrane and (2) direct fusion of SFV with the plasma membrane induced by low pH treatment. The selective inhibitor of the vacuolar proton-ATPase, concanamycin A, abolished fusion and subsequent infection only when the virus utilized the endocytic route to enter cells. The inhibitory effect of this macrolide antibiotic was bypassed by low pH treatment of cells. However, the ionophore nigericin was inhibitory irrespective of the route used by the virus to infect cells, suggesting the necessity of a transmembrane pH gradient for the entry process. According to our results, concanamycin A emerges as a suitable tool for selectively investigating the involvement of endosomal function in animal virus entry
Differential Infectivity of TwoAutographa californicaNucleopolyhedrovirus Mutants on Three Permissive Cell Lines Is the Result oflef-7Deletion

AbstractWe isolated twoAutographa californicanucleopolyhedrovirus (AcMNPV) mutants that have infectivity similar to that of wild-type (wt) AcMNPV in TN368 cells, but reduced budded virus and polyhedral inclusion body production in IPLB-SF-21 and SE1c cells. Restriction endonuclease analysis and sequence analysis indicated that 3.2-kb (77.0–79.4 m.u.) and 4.4-kb (76.7–80.1 m.u.) regions, the location of four major open reading frames (ORFs),pk2,ORF-247,lef-7,and chitinase, were deleted in mutant T295 and T297, respectively. Phenotypes of recombinant viruses vdel-AG, in which all four ORFs were deleted, and vlef7-AG, in which onlylef-7was deleted, were identical to the mutants. The phenotypes of recombinant viruses with deletions of the other ORFs were indistinguishable from wt AcMNPV. This demonstrated that the deletion oflef-7was responsible for the mutant phenotypes. Viral DNA synthesis in both mutant- and vlef7-AG-infected SF-21 and SE1c cells was reduced to less than 10% of that of wt AcMNPV-infected cells. In TN368 cells, DNA synthesis in mutant- and vlef7-AG-infected cells was delayed relative to wt-infected cells. Althoughlef-7is not essential for AcMNPV infection in TN368 cells, it is expressed in TN368, SF-21, and SE1c cells in a similar manner
Complementation of and Interference with Sindbis Virus Replication by Full-Length and Deleted Forms of the Nonstructural Protein, nsP1, Expressed in Stable Transfectants of Hela Cells

AbstractStable transfectants of Hela cells were isolated which expressed either the full-length 540-amino-acid Sindbis virus (SV) nonstructural protein, nsP1 (the form encoded by the mutant, SVLM21), or one of four forms with a carboxyl-terminal deletion. SVLM21, in contrast to standard SV (SVSTD), can replicate in Hela cells maintained in low-methionine (LM) medium. Expression of full-length nsP1(1–540), nsP1(1–492), or nsP1(1–448) resulted in complementation of SVSTDwhen infected Hela cells were kept in LM medium after infection. In contrast, when cells were infected with SVLM21and maintained in LM medium, stable expression of any of the deleted forms of nsP1 interfered with the replication of virus. The ability of “cellular” nsP1 to complement SVSTDin LM medium correlated with its methyltransferase activity
Nucleotide Sequence of the Barmah Forest Virus Genome

AbstractBarmah Forest virus (BFV) is an atypical alphavirus [Dalgarno, L., Short, N. J., Hardy, C. M., Bell, J. R., Strauss, J. H., and Marshall, I. D. (1984).Virology133, 416–426] and has been classified as the sole known member of a seventh alphavirus serocomplex. The complete nucleotide sequence of BFV genomic RNA is 11,488 nucleotides in length excluding the poly(A) tail. Two long open reading frames in the RNA encode a nonstructural polyprotein of 2411 amino acids and a structural polyprotein of 1239 amino acids, respectively. The BFV envelope protein E2 is unique among sequenced alphaviruses in having no N-linked glycosylation sites; E1 carries two glycosylation sites. From amino acid sequence comparisons with sequenced alphaviruses BFV is most closely related to Ross River and Semliki Forest viruses. Sequence homology between BFV and other alphaviruses is relatively uniform along the length of the nonstructural and structural polyproteins, providing no evidence that BFV has arisen from recombination between ancestral alphaviruses in the coding region of the genome. The BFV 3′ noncoding region of 445 nucleotides has unusual features. There are two unrelated sequence blocks of 48 nucleotides (sequence I) and 47 nucleotides (sequence II) both of which are repeated once. Sequence I is closely related to a repeat in the 3′ noncoding region of Ross River and Getah viruses; sequence II is unrelated to repeat blocks in other sequenced alphaviruses. Thus, recombination between ancestral viruses may have played a role in the evolution of the BFV 3′ noncoding region
Two Amino Acid Changes at the N-Terminus of Transmissible Gastroenteritis Coronavirus Spike Protein Result in the Loss of Enteric Tropism

AbstractTo study the molecular basis of TGEV tropism, a collection of recombinants between the PUR46-MAD strain of transmissible gastroenteritis coronavirus (TGEV) infecting the enteric and respiratory tracts and the PTV strain, which only infects the respiratory tract, was generated. The recombinant isolation frequency was about 10−9recombinants per nucleotide and was 3.7-fold higher at the 5′-end of the S gene than in other areas of the genome. Thirty recombinants were plaque purified and characterized phenotypically and genetically. All recombinant viruses had a single crossover and had inherited the 5′- and 3′-halves of their genome from the enteric and respiratory parents, respectively. Recombinant viruses were classified into three groups, named 1 to 3, according to the location of the crossover. Group 1 recombinants had the crossover in the S gene, while in Groups 2 and 3 the crossovers were located in ORF1b and ORF1a, respectively. The tropism of the recombinants was studied. Recombinants of Group 1 had enteric and respiratory tropism, while Group 2 recombinants infected the respiratory, but not the enteric, tract. Viruses of both groups differed by two nucleotide changes at positions 214 and 655. Both changes may be in principle responsible for the loss of enteric tropism but only the change in nucleotide 655 was specifically found in the respiratory isolates and most likely this single nucleotide change, which leads to a substitution in amino acid 219 of the S protein, was responsible for the loss of enteric tropism in the closely related PUR-46 isolates. The available data indicate that in order to infect enteric tract cells with TGEV, two different domains of the S protein, mapping between amino acids 522 and 744 and around amino acid 219, respectively, are involved. The first domain binds to porcine aminopeptidase N, the cellular receptor for TGEV. In the other domain maps a second factor of undefined nature but which may be the binding site for a coreceptor essential for the enteric tropism of TGEV
Activation of Collagenase IV Gene Expression and Enzymatic Activity by the Moloney Murine Leukemia Virus Long Terminal Repeat

AbstractMoloney murine leukemia virus (Mo-MuLV) is a thymotropic and leukemogenic retrovirus which causes T lymphomas and leukemias, yet does not contain a transforming gene product. Mo-MuLV has been shown totrans-activate cellular genes via a polymerase III-generated transcript, designatedlet,from the long terminal repeat (LTR). Here we demonstrate that introduction of the Mo-MuLV LTR stably, or transiently, into murine or human cultured cells resulted in an 8- to 15-fold increase incollagenase IV(92-kDa gelatinase, gelatinase B, matrix metalloproteinase-9) gene expression. Collagenase IV protein expression was induced 9-fold by stable integration of MuLV LTR, as measured by immunoblot analysis using an anti-collagenase IV polyclonal antibody. The MuLV LTR coordinately stimulated the proteolytic activity of collagenase IV by 14-fold. The AP-1-binding site in thecollagenase IVpromoter was required for transactivation by the LTR. Collagenase type IV degrades type IV collagen, a major component of basement membrane, which constitutes the first step of the metastatic cascade. The activation of proteolytic enzymes by the MuLV LTR may thus play a contributory role in the development or spread of virus-induced lymphomas or leukemias
Inhibition of HIV-1 Transcription and Virus Replication Using Soluble Tat Peptide Analogs

AbstractThe human immunodeficiency virus type 1 (HIV-1) transactivator Tat protein is essential for efficient viral gene expression and virus replication. The Tat core domain, a stretch of 12 amino acids between the cysteine-rich and the basic domain, is conserved in all HIV isolates and required for interaction with a number of cellular transcriptional regulatory proteins. Here we demonstrate that soluble peptide analogs of the Tat core domain (amino acid 36–50) are able to effectively block LTR transactivation. In transfection experiments, Tat core peptide analogs containing amino acid substitutions at position 41 and 44 inhibited Tat transactivation of an HIV-1 LTR-CAT reporter construct up to 80-fold. In contrast, inhibition of other promoters such as HTLV-I and CMV was approximately 2-fold. Tat peptide analog 36–50 (41/44) inhibited HIV virus replication by 85% in latently infected U1 cells induced with Tat. Furthermore, U1 cells treated with the Tat peptide 36–50 (41/44) analog showed markedly delayed virus transmission when cocultivated with parental U937 cells. Interestingly, while both short and long peptide analogs (amino acids 36–50 vs 36–72) inhibited Tat transactivation in transient assays, the short peptides were more effective inhibitors of virus replication in U1 cells. The Tat peptide analog did not decrease expression of cellular genes including β-actin, GAPDH, and histone H2B
Initiation of SV40 DNA Replicationin Vitro:Analysis of the Role Played by Sequences Flanking the Core Origin on Initial Synthesis Events

AbstractReplication initiation events are suppressed over the SV40 core originin vitro;they are also greatly reduced over sequences flanking the origin which contain binding sites for several transcription factors. To address the biochemical basis for the gap in initiation events over the flanking sequences, initial synthesis events have been characterized on templates lacking these sequences. Herein, it is demonstrated that previously functional initiation sites are nearly inactive when moved to positions that are proximal to the core origin. Thus, the gap in initiation events depends, in part, on the proximity of the initiation sites to the SV40 core origin. Additional experiments demonstrate that removal of the flanking sequences had little or no effect on DNA unwinding or on the efficiency of initiation of DNA synthesisin vitro.These results indicate that, under ourin vitroconditions, initiation of SV40 DNA synthesis is not enhanced by binding of transcription factors to the flanking sequences
Sequence Close to the N-terminus of L2 Protein Is Displayed on the Surface of Bovine Papillomavirus Type 1 Virions

AbstractThe bovine papillomavirus type 1 (BPV1) L2 protein purified fromEscherichia coliwas used as an antigen to produce monoclonal antibodies (MAbs). A total of 26 individual clones which recognized the BPV1 L2 protein were obtained. Using infectious BPV1 virus particles, 3 of the MAbs were found to interact with BPV1 virus particles. Binding of the MAbs to BPV1 was confirmed by immunoelectron microscopy. A set of 92 13-mer peptides overlapping by 8 amino acids spanning the entire BPV1 L2 protein was synthesized on a membrane and used to map the epitopes recognized by these antibodies. Seventeen linear epitopes were identified. Our results revealed that a sequence toward the N-terminus of the L2 protein (aa 61–123) is displayed on the virus surface, while the remaining L2 sequences are hidden inside the virus capsid. Although the polyclonal antisera raised against BPV1 L2 neutralized the BPV1 virus, we failed to detect any neutralizing activity for the 3 L2-specific monoclonal antibodies which bound to the BPV1 particles. This suggests that extra binding sites may be needed for neutralization. This study prompted us to propose a model about how L1 and L2 proteins may interact during infectious papillomavirus assembly
Adenovirus E1B 19K Protein Is Required for Efficient DNA Replication in U937 Cells

AbstractThe adenovirus E1B 19K gene plays an essential role in transformation of primary rodent cells in cooperation with E1A and in the inhibition of apoptosis during lytic infection. It has been shown that this E1B 19K protein is not necessary for viral DNA replication in human cell lines, such as HeLa and KB. We reported here that the E1B 19K mutant viruses were unable to replicate efficiently in a monocyte cell line, U937. Viral DNA synthesis and late gene expression were found to be defective in U937 cells infected with E1B 19K mutants compared with wild-type virus. Early viral RNA splicing patterns also differ between wild-type anddl337-infected cells. Furthermore, the defect in viral replication could be complemented bydl312 virus defective in E1A expression 4 days after infection with E1B mutants, suggesting persistence of the E1B mutant genome in the infected cells despite defective onset of the late phase of replication. These results imply that E1B 19K is required for efficient viral DNA replication in U937 cells. Inefficient DNA replication is also found in another monocyte cell line, THP-1
Genetic Analysis of the φX174 DNA Binding Protein

AbstractThe φX174 J protein is 37 amino acids in length and contains 12 basic residues. There are no acidic amino acids in the protein. The basic residues are concentrated in two clusters in the N-terminus which are separated by a proline-rich region. To investigate the morphogenetic functions of the J protein and possible mechanisms by which it may bind DNA, a genetic analysis was conducted. Lysine → leucine and arginine → leucine substitutions were generated within the basic amino acid clusters. At least three substitutions were required to eliminate viabilityin vivo.Lethal mutants with three or four substitutions exhibit dominant lethal phenotypes, indicating that the mutant proteins retain enough function to interfere with productive assembly. In cells infected with a dominant lethal mutant, noninfectious packaged particles were produced. Infectivity can be restored by second-site suppressors in the viral coat protein which disrupt polar interactions atop the threefold axis of symmetry in the capsid. The viability of strains containing compensating frameshift mutations within the proline-rich region suggests that only the proline residues in this segment are critical for efficient function
DNA Replication of Wheat Dwarf Geminivirus Vectors: Effects of Origin Structure and Size

AbstractWheat dwarf virus (WDV) is a representative member of subgroup I of the Geminiviridae, a unique plant DNA virus family. Since geminivirus DNA replication occurs in the host cell nucleus exclusively via double-stranded DNA intermediates, a considerable interest has arisen to use them as expression vectors. We have used particle bombardment to introduce WDV vectors into cultured wheat cells and to analyze the fate of input DNA and the accumulation of newly replicated DNA. Under our conditions, we have found that input DNA, which can be detected immediately after DNA delivery, is rapidly degraded. Newly replicated viral DNA appears ∼1 day after DNA delivery and reaches a maximum at Days 2–4. Afterward, the total amount of viral DNA is maintained for several days. We have observed a progressive decrease in the relative amount of supercoiled DNA and, concomitantly, an increase in plasmid forms migrating as open circular and nicked DNA. GUS expression from the virion-sense WDV promoter is also maximal 2–3 days after DNA delivery and then it declines to negligible levels 8 days afterward. These results support the conclusion that, under these conditions, reporter gene expression depends on the accumulation of newly replicated, supercoiled plasmid DNA and not on input plasmid DNA. We have also analyzed the effects of WDV origin structure and plasmid size on the accumulation of newly replicated plasmid DNA. Our results lead us to conclude that the replication efficiency of WDV-derived plasmids depends largely on plasmid size. Interestingly, sequences downstream of the initiation site, which in WDV confer an intrinsic curvature to the large intergenic region, seem to have a small effect on the efficiency of plasmid accumulation
Normal Cellular Replication of Sendai Virus without thetrans-Frame, Nonstructural V Protein

AbstractThe Sendai virus V protein is a nonstructuraltrans-frame protein in which a highly conserved cys-rich Zn2+-binding domain is fused to the N-terminal half of the P protein via mRNA editing. Using a recently developed system in which infectious virus is recovered from cDNA, we have engineered a virus in which a translation stop codon was placed at the beginning of the V ORF. Translation of the VstopmRNA yields a W-like protein, i.e., a protein composed of the N-terminal half of the P protein alone which is naturally expressed at low levels from the P gene. This V-minus but W-augmented virus was found to replicate normally in cell culture and embryonated chicken eggs. The Sendai virus V protein is thus an accessory protein, and the cys-rich Zn2+-binding domain is likely to function in a specialized role during virus propagation