pinecone/core-2020-05-10-deduplication huggingface dataset:
Increased Incorporation of Chimeric Human Immunodeficiency Virus Type 1 gp120 Proteins into Pr55gagVirus-like Particles by an Epstein–Barr Virus gp220/350-Derived Transmembrane Domain

AbstractNoninfectious Pr55gagvirus-like particles containing high quantities of oligomeric human immunodeficiency virus type 1 (HIV-1) envelope (Env) proteins represent potential candidate immunogens for a vaccine against HIV-1 infection. Thus, chimericenvgenes were constructed encoding the HIV-1 exterior glycoprotein gp120 which was covalently linked at different C-terminal positions to a transmembrane domain (TM) from the Epstein–Barr virus (EBV) major Env glycoprotein gp220/350. All chimeric Env-TM polypeptides as well as the wild-type HIV Env proteins were equally produced and incorporated at the outer surface of insect cells using the baculovirus expression system. In the presence of coexpressed HIV Pr55gagpolyproteins significantly decreased amounts of wild-type Env proteins were presented at the cell surface, whereas the membrane incorporation of the Env-TM chimeras was not affected. Biochemical and immunoelectron microscopical analysis of particles that were efficiently released from these cells displayed the incorporation of both wild-type Env and chimeric Env-TM proteins on the surface of VLPs. However, the quantities of particle-associated chimeric Env-TM proteins exceeded those of incorporated wild-type Env proteins by a factor of 5–10. Chemical cross-linking and subsequent polyacrylamide gel electrophoresis of VLP-entrapped Env proteins revealed that the chimeric Env-TM proteins form homodimers and a higher-order oligomer, similar to that observed for wild-type Env proteins. Thus, the results of this study clearly demonstrate that the replacement of the gp41 transmembrane protein of gp160 by a heterologous, EBV gp220/350-derived membrane anchor provides an effective strategy to incorporate high quantities of oligomeric HIV gp120 proteins on the surface of Pr55gagvirus-like particles
Type 1 HERV-K Genome Is Spliced into Subgenomic Transcripts in the Human Breast Tumor Cell Line T47D

AbstractTwo types of HERV-K genomes exist which differ in the absence (type 1) or the presence (type 2) of a sequence of 292 nucleotides between the putativepolandenvgenes. Previously published results from teratocarcinoma cell studies had firmly concluded that the type 1 HERV-K genome was defective in splicing and that only the nondeleted type 2 HERV-K genome containing the 292-nucleotide sequence was capable of being spliced. We now show that in the T47D human breast tumor cell line it is the type 1 HERV-K genome, and not the type 2, which is spliced to subgenomic transcripts
Oligomerization of HIV-1 Rev Mutants in the Cytoplasm and during Nuclear Import

AbstractOligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a newin vivoassay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev
Distinct Transcriptional Pathways of TAR-Dependent and TAR-Independent Human Immunodeficiency Virus Type-1 Transactivation by Tat

AbstractTat stimulates HIV-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR RNA and several putative cellular cofactors to increase the processivity of RNA polymerase II complexes during HIV-1 transcription elongation. Although HIV-1 transactivation by Tat in most cell types requires intact TAR sequences, previous reports demonstrate that Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that κB elements within the HIV-1 LTR mediate TAR-independent transactivation by Tat and demonstrate additionally that distinct amino acid residues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87-MG cell lines expressing a transdominant negative mutant of IκBα, IκBαΔN, fail to support TAR-independent transactivation by Tat, suggesting that binding of NF-κB to κB enhancer elements within the HIV-1 LTR is necessary for Tat-mediated transactivation in the absence of TAR. Ribonucleic acid protection analyses of promoter-proximal and -distal transcripts derived from TAR-deleted and TAR-containing HIV-1 LTR reporter constructs in U87-MG cells indicate that the predominant effect of Tat during TAR-independent transactivation occurs at the level of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transactivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interaction of Tat with NF-κB-binding sites in the HIV-1 LTR
HIV-1 Coreceptor Activity of CCR5 and Its Inhibition by Chemokines: Independence from G Protein Signaling and Importance of Coreceptor Downmodulation

AbstractHIV-1 infection requires the presence of specific chemokine receptors on CD4+ target cells to enable the fusion reactions involved in virus entry. CCR5 is a major fusion coreceptor for macrophage-tropic HIV-1 isolates. HIV-1 entry and fusion are mediated by the viral envelope glycoprotein (Env) and are inhibited by CCR5 ligands, but the mechanisms are unknown. Here, we test the role of G protein signaling and CCR5 surface downmodulation by two separate approaches: direct inactivation of CCR5 signaling by mutagenesis and inactivation of Gi-type G proteins with pertussis toxin. A CCR5 mutant lacking the last 45 amino acids of the cytoplasmic C-terminus (CCR5306) was created that was expressed on transfected cells at levels comparable to cells expressing CCR5 and displayed normal chemokine binding affinity. CCR5 ligands induced calcium flux and receptor downmodulation in cells expressing CCR5, but not in cells expressing CCR5306. Nevertheless, CCR5 or CCR5306, when coexpressed with CD4, supported comparable HIV-1 Env-mediated cell fusion. Consistent with this, treatment of CCR5-expressing cells with pertussis toxin completely blocked ligand-induced transient calcium flux, but did not affect Env-mediated cell fusion or HIV-1 infection. Also, pertussis toxin did not block chemokine inhibition of Env-mediated cell fusion or HIV-1 infection. However, chemokines inhibited Env-mediated cell fusion less efficiently for CCR5306than for CCR5. We conclude that the C-terminal domain of CCR5 is critical for G protein signaling and receptor downmodulation from the surface, but that neither function is required for CCR5 fusion coreceptor activity. The contrasting phenotypes of CCR5 and CCR5306suggest that coreceptor downmodulation and direct blockage of Env interaction sites both contribute to chemokine inhibition of HIV-1 infection
How Viruses Escape from Cytotoxic T Lymphocytes: Molecular Parameters and Players

AbstractViruses that persist in infected hosts must evolve successful strategies to avoid recognition by the immune system. The primary player in antiviral immune surveillance is the CD8+cytotoxic T lymphocyte (CTL), and the battle drawn between the CTLs and viruses is the focus of this review. In this struggle, viruses can follow multiple distinct pathways. For example, DNA viruses often adopt the strategy of encoding proteins that interfere with the immune response along routes of antigen presentation. Such interference prevents the viral peptide from binding to the major histocompatibility complex (MHC) class I glycoprotein; therefore, no virus–MHC complex forms for recognition by antiviral CTLs. RNA viruses, having fewer genes, generate swarms of quasispecies that can contain mutated viral proteins. When such mutants occur in viral peptides presented to the MHC protein or the residue recognized by the CTL receptor, CTL recognition and activation fail. If, instead, the mutation occurs in the viral peptide flanking sequence, the infected cell may not process the viral peptide from the cytosol to the endoplasmic reticulum. Viruses can also directly or indirectly attack dendritic cells and CD4+or CD8+T lymphocytes, other routes that interfere with immune functions. Dendritic cells are the primary professional antigen-presenting cells and are critical for the activation of CTL responses. CD4+T lymphocytes provide help for long-term CD8+CTL activity and are necessary for its maintenance. Consequently, interference with either dendritic or CD4+cell types constitutes yet another way that viruses can disable the immune response and persistently infect their host
Adsorptive Endocytosis of California Encephalitis Virus into Mosquito and Mammalian Cells: A Role for G1

AbstractThe G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) andAedes albopictus(C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAbs, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells
Naturally Occurring Hepatitis B Virus Genomes Bearing the Hallmarks of Retroviral G → A Hypermutation

AbstractTwo hypermutated genomes of hepatitis B virus (HBV) were cloned from sera of chronic virus carriers. Twelve percent and 26% of guanosine residues were replaced by adenosine, with the transitions being erratically distributed along the genome. G → A substitutions showed a strong dinucleotide preference, decreasing in the order GpA > GpG ⪢ GpC ≥ GpT. Such traits are typical of retroviral G → A hypermutation which results from cDNA synthesis coinciding with fluctuations in the intracellular [dTTP]/[dCTP] ratio. The observations offer an explanation for the high prevalence of HBV variants bearing a tryptophan 28 → stop codon in the pre-core region of carriers with chronic active or fulminant hepatitis. The HBV hypermutants indicate that a small proportion of hepatocytes have distorted dNTP pools, which might have implications for the fidelity of hepatocyte DNA replication or repair
Functional Characterization of the Major Late Promoter of Mouse Adenovirus Type 1

AbstractDuring the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mouse adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenced and compared to that of the human virus MLP. The general organization of the transcriptional elements of the putative MAV-1 MLP is similar to that of the human virus counterpart, with some interesting differences. We wished to investigate the function of the predicted MLP of MAV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both Northern blot and primer extension analyses were performed on intracellular RNA isolated from cells infected with MAV-1. The results show that late RNA can be detected 48 hr postinfection and increases up to 6 days p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue of the predicted TATA box. To analyze the functional significance of the predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequence, was established. The predicted MLP sequence was capable of directing luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 site, and the TATA box, either singly or in combination. Each single-element mutation causes a marked reduction in luciferase gene expression, with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1-binding site was examined by gel mobility shift assay and shown to interact with purified SP1 protein specifically, supporting the functional significance of this transcriptional element. These findings contribute to a better understanding of gene expression in MAV-1 and to its development as an appropriate model for the study of the molecular basis of pathogenesis in a natural host animal
Use of Synthetic Peptides to Identify Measles Nucleoprotein T-Cell Epitopes in Vaccinated and Naturally Infected Humans

AbstractRecombinant measles nucleoprotein (N) and synthetic peptides spanning the length of the N-protein-coding region were used with a proliferation assay to identify human T-cell epitopes in vaccinated and naturally infected adults. A number of epitopes were mapped to specific regions of the measles virus N. The proliferative response of at least two donors was mediated by CD4+T cells in association with HLA DR antigens. Over 70% of all donors tested responded to peptides representing amino acids 271–290, 367–386, 400–420, and 483–502, suggesting that these peptides may be broadly recognized within an HLA diverse population. The most frequently recognized T-cell epitopes in both naturally infected and vaccinated donors were located in the genetically heterogeneous carboxy-terminal half of the N. Analysis of patterns of peptide reactivity among vaccinated and naturally infected subjects identified several regions of potential difference between these two groups. Peptides 221–240 and 237–256 were recognized among 100% of naturally infected donors but among only 37.5% of vaccinated donors and therefore may be of further interest in studies to investigate induction of lifelong versus transient immunity to measles. Use of chimeric molecules containing multiple well-characterized T- and B-cell epitopes or genetic alteration of attenuated vaccine virus to enhance critical T-cell responses may eventually lead to the development of a vaccine candidate that can more closely model the patterns of immune response elicited by wild-type virus
In SituLocalization of the Putative Movement Protein (pr17) from Potato Leafroll Luteovirus (PLRV) in Infected and Transgenic Potato Plants

AbstractThe potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement
A Cottontail Rabbit Papillomavirus Strain (CRPVb) with Strikingly Divergent E6 and E7 Oncoproteins: An Insight in the Evolution of Papillomaviruses

AbstractWe previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.5% identical amino acids and differed in size by three and two amino acids, respectively. This divergence involved (i) a great number (4.4%) of nucleotide substitutions and a high rate (83.3%) of nonsynonymous mutations; (ii) mutations changing the E6 and E7 stop codons; and (iii) in-frame sequence insertions in the E6 ORF (18 nucleotides) and downstream of the mutated E7 stop codon (6 nucleotides), both likely to result from a duplication of adjacent sequences. These extensive differences could account for distinct biological and antigenic properties. Strikingly, only four (0.8%) amino acids of the L1 major capsid protein were variable. Thus, it seems likely that sequence duplications and mutations affecting stop codons exert a strong selection pressure on the fixation of nonsynonymous mutations and that phylogenetic calculations based only on point mutations may misevaluate the time scale of the evolution of papillomaviruses
The Three Subunits of the Polymerase and the Nucleoprotein of Influenza B Virus Are the Minimum Set of Viral Proteins Required for Expression of a Model RNA Template

AbstractThe genes encoding the nucleoprotein, PB1, PB2, and PA proteins of the influenza virus strain B/Panamá/45/90 have been cloned under control of the T7 RNA polymerase promoter of plasmid pGEM-3. Transfection of the recombinant plasmids obtained into mammalian cells, which had been infected with a vaccinia virus encoding the T7 RNA polymerase, resulted in expression of the expected influenza B virus polypeptides. Moreover, it is shown that coexpression of the four recombinant core proteins in COS-1 cells reconstituted a functional polymerase capable of expressing a synthetic influenza B virus-like CAT RNA. By using the influenza B virus recombinant plasmids and a set of pGEM-derived plasmids encoding the homologous core proteins of the influenza A virus A/Victoria/3/75 (I. Menaet al.(1994).J. Gen. Virol.75, 2109–2114), the capabilities of homo- and heterotypic mixtures of the four core proteins to express synthetic type A and B CAT RNAs were analyzed. Both the influenza A and B virus polymerases were active in expressing, albeit with reduced efficiencies, the heterotypic model CAT RNAs. However, none of all possible heterotypic mixtures of the core proteins reconstituted a functional polymerase. In order to fully characterize the recombinant plasmids obtained, the nucleotide sequences of the cloned genes were determined and compared to sequences of other type B virus isolates. The results obtained from these latter analyses are discussed in terms of the conservation and evolution of the influenza B virus core genes
In VitroReconstitution of an Intermediate Assembly Stage of Vaccinia Virus

AbstractA novel method is described which facilitates thein vitroassembly of one step in the life cycle of vaccinia virus, the formation of the spherical immature virus (IV). For this, advantage was taken of the ability of rifampicin to reversibly block the assembly of the IV. Rifampicin-treated, vaccinia virus-infected HeLa cells were permeabilized with streptolysin O (SLO) and the endogenous cytosol was allowed to exit the cells at 4°. Subsequently, exogenous cytosol from infected or uninfected HeLa cells as well as an ATP-regenerating system were added and the cells were incubated for different times at 37° in the absence of rifampicin. The preparations were then evaluated by thin section EM. Our data show that in the presence of infected or uninfected cell cytosol and ATP a significant fraction of cells could reconstitute IV assemblyin vitro.Under no conditions were we able to reconstitute any later stages of assembly. The potential of this system for thein vitroreconstitution of viral assembly in general is discussed
Novobiocin Inhibits Vaccinia Virus Replication by Blocking Virus Assembly

AbstractNovobiocin inhibits the replication of vaccinia virus in cultured BSC40 cells. All classes of viral proteins were synthesized during synchronous infection in the presence of drug. The onset of DNA replication was delayed slightly, yet the extent of DNA replication in the presence of novobiocin was comparable to that of a control infection. A delay in the temporal transition to late viral protein synthesis was in keeping with the effects on DNA replication. Although the precursor forms of the major viral structural proteins were synthesized normally at late times, the proteolytic processing of these polypeptides was inhibited, which suggested an impediment to virus assembly. Electron microscopy revealed that novobiocin blocked virus morphogenesis at an early stage. Conversion of the concatameric DNA replication intermediates into hairpin telomeres occurred in the presence of novobiocin, confirming that telomere resolution was not coupled to virus assembly. Novobiocin is the latest addition to a class of antipoxviral agents, which includes rifampin and IMCBH, that arrest morphogenesis
Structure of Broadhaven Virus by Cryoelectron Microscopy: Correlation of Structural and Antigenic Properties of Broadhaven Virus and Bluetongue Virus Outer Capsid Proteins

AbstractThe three-dimensional structure of Broadhaven virus (BRDV) has been determined to 23 Å resolution by cryoelectron microscopy and image processing. As predicted from sequence homology, the BRDV structure resembles that of bluetongue virus (BTV) with the notable exception of one of the outer shell proteins. The cores of BRDV and BTV are identical at medium resolution; they have a diameter of 710 Å and the VP7 trimers are arranged on a T = 13 icosahedral lattice. The outer shell proteins, VP5 of BRDV and BTV, have roughly the same molecular weight while VP4 of BRDV is only half the molecular weight of the corresponding VP2 of BTV. This size difference allows unambiguous determination of the identity of the triskelion shape as trimers of VP4 of BRDV (VP2 of BTV). The VP4 of BRDV sits on the VP7 trimers and projects outwards 40 Å, giving the capsid an overall diameter of 790 Å. This contrasts with VP2 of BTV, which projects outwards 95 Å to give the capsid a diameter of 900 Å. The difference in accessibility of the outer shell proteins of BRDV and BTV correlates with the difference in antigenic properties of these viral proteins. The shape of the BRDV VP5 indicates that it too is a trimer, thus implying that there are 360 copies of VP5 and 180 copies of VP4 per virion