pinecone/core-2020-05-10-deduplication huggingface dataset:
Mutational Analysis of the Fusion Peptide of the Human Immunodeficiency Virus Type 1: Identification of Critical Glycine Residues

AbstractThe ability of human immunodeficiency virus type 1 (HIV-1) to fuse its membrane with the membrane of the target cell is a function of a ∼23-amino-acid amino-terminal segment of the gp41 subunit of the envelope glycoprotein complex, known as the fusion peptide. The sequence of the fusion peptide is highly conserved among different variants of HIV-1 and is also very similar to that of HIV-2 and SIV. The fusion peptide is very hydrophobic and has a high content of glycine and alanine residues. Representation of the fusion peptide of HIV-1 as an α-helix predicts that most glycine residues would be found on one face of the α-helix. To assess the importance of the glycine residues for the fusogenic activity of the envelope glycoprotein complex, we mutagenized each glycine residue in the fusion peptide individually to a valine residue. The mutant envelope constructs were tested for their ability to induce syncytia (cell/cell fusion) and to mediate infection (virus/cell fusion) of CD4-positive cells. The results of our analyses show that two glycine residues (G10and G13) located within the sequence FLGFLG in the middle of the fusion peptide are critical for syncytium formation and for the establishment of a productive infection, whereas other glycine residues (G3, G5, and G20) are more permissive to substitutions. Mutation of each of the two phenylalanines (F8and F11) of the FLGFLG sequence to valine also decreased fusion, although to a lesser extent than mutation of G10and G13. These observations demonstrate that G10and G13are critical elements of the fusion peptide and suggest that, in addition to hydrophobicity, the exact amino acid composition and structure of the fusion peptide are critical for function
Analysis of HPV1 E4 Complexes and Their Association with Keratinsin Vivo

AbstractThe HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (>95%) exists as complexes which do not contain keratins. The identification of discrete species of 34K, 58K, 70K, 88K, and 105K suggests that these are simple multimers of the 17K monomer, with very little of the soluble E4 being present in complexes larger than 140K. The truncated 10/11K E4 species, which comprise the C-terminal domain of E4, exist as trimers and dimersin vivo.Less than 5% of the E4 was present as complexes greater than 140K, and these were found to be insoluble. The 34K (dimer) and 58K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105K E4 complex was abolished (and formation of the 58K species enhanced) when keratins were present during E4 synthesisin vitro.We conclude that while E1∧E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 E4 protein in established tumorsin vivois neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers
Transfer of an Esterase-Resistant Receptor Analog to the Surface of Influenza C Virions Results in Reduced Infectivity Due to Aggregate Formation

AbstractA synthetic sialic acid,N-acetyl-9-thioacetamidoneuraminic acid (9-ThioAcNeu5Ac), is recognized by influenza C virus as a receptor determinant but—in contrast to the natural receptor determinant,N-acetyl-9-O-acetylneuraminic acid—is resistant to inactivation by the viral acetylesterase. This sialic acid analog was used to analyze the importance of the receptor-destroying enzyme of influenza C virus in keeping the viral surface free of receptor determinants. Enzymatic transfer of 9-ThioAcNeu5Ac to the surface of influenza C virions resulted in the loss of the hemagglutinating activity. The ability to agglutinate erythrocytes was restored when the synthetic sialic acid was released from the viral surface by neuraminidase treatment. Infectivity of influenza C virus containing surface-bound 9-ThioAcNeu5Ac was reduced about 20-fold. Sedimentation analysis as well as electron microscopy indicated that virions resialylated with the esterase-resistant sialic acid analog formed virus aggregates. These results indicate that the receptor-destroying enzyme of influenza C virus is required to avoid the presence of receptor determinants on the virion surface and thus to prevent aggregate formation and a reduction of the infectious titer
Resistance to V3-Directed Neutralization Caused by an N-Linked Oligosaccharide Depends on the Quaternary Structure of the HIV-1 Envelope Oligomer

AbstractA conserved N-glycan present within the V3 loop of gp120 modulates the sensitivity to neutralization by antibodies directed to the V3 loop. A glycan-deficient mutant of HIVLAI, designated HIVA308, displayed a 100-fold increase in sensitivity to neutralization by anti-V3 MAb NEA-9205 compared to wild-type HIVLAI. This difference in sensitivity was not caused by an alteration of the antibody binding site itself, as NEA-9205 had equal affinity for both wild-type and mutant monomeric gp120. In contrast, virion-associated wild-type gp120 was immunoprecipitated less efficiently with NEA-9205 than virion-associated mutant gp120. This difference was completely abrogated, if immunoprecipitation were carried out in the presence of detergent. Furthermore, treatment of virion preparations with detergent exposed the C-terminal D7324 epitope, which is inaccessible on virion-associated gp120 but readily accessible on monomeric, soluble gp120. Finally, both wild-type and mutant monomeric, soluble gp120 were precipitated equally efficiently by NEA-9205 in the absence of detergent. Thus, the NEA-9205 epitope was readily accessible on monomeric gp120 regardless of the presence of the306N-glycan, and inaccessibility of the NEA-9205 epitope imparted by the306N-glycan was observed only on the intact envelope oligomer
Characterization of a Highly Conserved Baculovirus Structural Protein That Is Specific for Occlusion-Derived Virions

AbstractA highly conserved baculovirus late gene calledodvp-6ewas shown to be a structural protein that is specific for occlusion-derived virus (ODV) envelopes. The complete sequence of this gene is presented for bothOrgyia pseudotsugatanuclear polyhedrosis virus (OpMNPV) andCydia pomonellagranulosis virus (CpGV). The predicted sizes of the OpMNPV and CpGV ODVP-6E are 40, 241, and 38,655 respectively. The OpMNPVodvp-6egene was transcriptionally mapped and was shown to initiate from a consensus late gene motif, TTAAG, and is expressed from 18–120 hr postinfection. Polyclonal antiserum was generated against a bacterial fusion protein and used to analyze the cellular steady-state levels of ODVP-6E and to determine if this protein was a component of either budded virus (BV) or ODV. Western blots showed that ODVP-6E is a component of the ODV but not BV. This was confirmed by immunoelectron microscopy of ODV fromAutographa californicaNPV (AcMNPV) which localized ODVP-6E to the ODV envelope. The sequences of theodvp-6egene from the baculovirusesChoristoneura fumiferanaNPV (CfMNPV), AcMNPV, andHelicoverpa zeaNPV (HzSNPV) were obtained from GenBank. Comparisons of the predicted amino acid sequences of OpMNPV, CpGV, AcMNPV, CfMNPV, and HzSNPV show that there are two possible membrane-spanning domains and a cysteine-rich domain that are conserved in all of the proteins
Vpx Association with Mature Core Structures of HIV-2

AbstractVpx is a virion-incorporated protein encoded by HIV-2 and certain strains of SIV which is necessary for efficient infection of lymphocytes and macrophages. To determine the location of Vpx within the HIV-2 particle, core structures were obtained from mature HIV-2 virions. Analysis of purified cores reveal the presence of monomeric Vpx as well as a nondenaturable and nonreducible higher order Vpx complex. Characterization of the core-associated Vpx complex suggests it to be a Vpx homodimer, indicative of high concentrations of Vpx. The incorporation and concentration of Vpx in HIV-2 cores support the hypothesis that it functions within the viral preintegration complex
Use of Lysolecithin-Permeabilized Infected-Cell Extracts to Investigate thein VitroBiochemical Phenotypes of Poxvirus ts Mutations Altered in Viral Transcription Activity

AbstractLysolecithin permeabilization of vaccinia virus-infected cells was employed to prepare extracts that support faithful transcription initiationin vitroon plasmids possessing early, intermediate, and late viral gene promoters. Conditions which optimize transcription from each promoter were defined. Thein vitrosystem was used to investigate the multifunctional viral mRNA capping enzyme, which also functions as the viral early gene transcription termination factor (VTF) and a viral intermediate gene transcription initiation factor. A low level of signal-dependent termination of early gene transcription was observedin vitrowhich could be elevated by the addition of pure mRNA capping enzyme. VTF-dependent transcription termination was found to be restricted to templates that possessed an early promoter. This restriction mimics that observedin vivoand demonstrates that transcription termination is limited to RNA polymerase molecules that recognize early rather than intermediate or late gene promoters. Extracts prepared from cells infected at the nonpermissive temperature with a virus containing a ts mutation in gene D12L, which encodes the small subunit of VTF, are incapable of supporting both early gene transcription termination and intermediate gene transcription initiation. Both activities are restored upon addition of the purified wild-type mRNA capping enzyme
Inhibition of HIV Infection by Pseudopeptides Blocking Viral Envelope Glycoprotein-Mediated Membrane Fusion and Cell Death

AbstractThe RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as “template assembled synthetic peptide” (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1–5 μM,respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement ofL-amino acid residues byD-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-μM,concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients
Regulation of Neighboring Gene Expression by the Herpes Simplex Virus Type 1 Thymidine Kinase Gene

AbstractThe herpes simplex virus type 1 thymidine kinase (tk) gene (UL23) lies upstream of thegH(UL22) gene with its 3′ end overlapping thegHpromoter, and it overlaps theUL24gene's regulatory and coding sequences at its 5′ end in a head-to-head orientation. Thus,tkexpression could affectgHexpression by promoter occlusion andUL24expression by transcriptional or posttranscriptional mechanisms. To investigate these possibilities, we analyzed the effects oftkpromoter mutations that reducetkexpression ongHandUL24expression. ForgH,tkpromoter mutations did not increase the accumulation ofgHmRNA or the rate ofgHtranscription. Thus,tkexpression does not appear to down-regulategHexpression. In contrast, we found that decreasedtkexpression correlated with increased accumulation ofUL24mRNA, particularly a 1.4-kb transcript, at early times postinfection during peak expression oftk, but not at late times whentkmRNA levels have fallen. Results from viral co-infection experiments indicated that down-regulation ofUL24mRNA accumulation requirestkexpressionin cis. Nuclear run-off experiments did not detect differences inUL24transcription rates in the mutant viruses. Although we cannot completely exclude a transcriptional mechanism for this down-regulation, these results can be explained by an antisense RNA mechanism acting preferentiallyin cis
Temporal Regulation of Herpes Simplex Virus Type 1UL24mRNA Expression via Differential Polyadenylation

AbstractUsing Northern blot, primer extension, and S1 nuclease analyses of wild-type and deletion-containing herpes simplex type 1 viruses, we found thatUL24sequences are contained in six different transcripts that originate from three previously identified mRNA start sites. Thus, the sixUL24transcripts represent three different pairs of 5′ coterminal mRNAs. Each transcript pair consists of a short species whose 3′ end corresponds to a polyadenylation signal located just downstream of theUL24open reading frame, and a longer species whose 3′ end corresponds to a polyadenylation signal located downstream of theUL26gene. Maximal accumulation of the shortUL24transcripts was at early times during infection, while accumulation of the longer species did not decrease at late times. Consistent with early kinetics, the short transcripts were less sensitive to drugs that inhibited viral DNA replication than the longer transcripts which exhibited leaky–late kinetics. Quantitative S1 nuclease analysis indicated that 3′ ends corresponding to theUL24polyadenylation site were significantly more abundant at early times than at late times. Thus, differential polyadenylation determines the kinetics of accumulation of differentUL24transcripts
Complete Sequencing of a Gibbon Hepatitis B Virus Genome Reveals a Unique Genotype Distantly Related to the Chimpanzee Hepatitis B Virus

AbstractWe have sequenced the complete genome of a hepatitis B virus (HBV) strain that was transmitted from a gibbon with chronic hepatitis B to a chimpanzee that subsequently developed acute hepatitis B. The genome was 3,182 nucleotides long and had a genetic organization identical to and including the characteristics of other mammalian hepadnaviruses. Thus, the regulatory elements, the direct repeats, and the four open reading frames (ORFs) of this virus were all maintained, although there were amino acid substitutions affecting all the ORFs. Within the S gene encoding for the hepatitis B surface antigen (HBsAg), the subtype could be deduced asayw3in accordance with previous serological results. There were 25 amino acid substitutions affecting the P gene, 12 of which were within the spacer region. This region, which was the most divergent part of the genome compared to other HBV strains, also encodes for the pre-S proteins. A comparison with sequences of other hepadnaviruses revealed that the genome of gibbon HBV was unique as compared to previously described HBV genotypes. It was most similar to the chimpanzee HBV strain with which it shared 90.3% nucleic acid homology at the level of the complete genome and 96.3% homology at the level of the S-gene region corresponding to HBsAg, although being a distinct genotype as compared to the latter virus. Analyses performed using five different algorithms for phylogenetic tree construction showed more than 99% bootstrap support for the gibbon and the chimpanzee HBV to be grouped within the human HBV strains and that they represented later offshoots than the HBV strains of genotype F. However, in most of the dendrograms both the gibbon and the chimpanzee strains represented early lineages, indicating that these viruses are indigenous to their respective hosts and not recent acquisitions from man
Expression of the Myxoma Virus Tumor Necrosis Factor Receptor Homologue and M11L Genes Is Required to Prevent Virus-Induced Apoptosis in Infected Rabbit T Lymphocytes

AbstractMyxoma virus is a leporipoxvirus that causes a highly lethal virulent disease known as myxomatosis in the European rabbit. An important aspect of myxoma virus pathogenesis is the ability of the virus to productively infect lymphocytes and spread to secondary sites via lymphatic channels. We investigated the infection of the CD4+T lymphoma cell line RL-5 with myxoma virus and Shope fibroma virus, a related but benign leporipoxvirus, and observed that myxoma virus, but not Shope fibroma virus, was able to productively infect RL-5 cells. We also discovered that infection of RL-5 cells with Shope fibroma virus or attenuated myxoma virus mutants containing disruptions in either the T2 or the M11L gene resulted in the rapid induction of DNA fragmentation, followed by morphological changes and loss in cell integrity characteristic of cell death by apoptosis. Purified exogenous T2 protein was unable to prevent apoptosis, suggesting that T2 functions intracellularly. Thus, myxoma virus T2, originally described as a secreted homologue of the tumor necrosis factor receptor, and M11L, a novel transmembrane species with no known cellular homologue, function to extend virus host range for replication in rabbit T lymphocytes through the inhibition of apoptosis in infected T lymphocytes
Genotypic and Phenotypic Characterization of a Neutralization-Resistant Breakthrough Population of HIV-1

AbstractCertain antibody neutralization escape mutants of HIV-1 map outside of the antibody recognition epitope, thereby suggesting the presence of nonlinear conformational domains. In an effort to begin to define the interacting regions of the HIV envelope proteins, a neutralization-sensitive clone of HIV-1, HXB2/BH10Sal-Bam, was passaged in the presence of the V3-specific monoclonal antibody 0.5β. DNA sequence analysis of the V3 domain of the breakthrough viral populations revealed one population that retained the parental V3 genotype. Quantitative DNA sequence analysis of this breakthrough population revealed the presence of mutational “hotspots” in several envelope domains that are noncontiguous with V3. Mutations were seen throughout gp41 and the C1, V1/V2, C2, and C5 domains of gp120. In contrast, other regions of gp120, C3, V4, C4, and V5 remained totally unchanged. Within V1, three residues within a 14-amino acid stretch experienced five substitutions and in C5 three residues within a 7-amino acid stretch experienced four substitutions. This finding, that certain residues clustered within particular domains (V1/V2, C5, and gp41) experienced multiple substitutions under a defined environmental stressor, suggests that the degree of adaptive plasticity exhibited by the HIV envelope is limited. Based on this observation it may be possible, using a set of antibodies to various envelope epitopes, to discern a set of rules which explain the interactions of the various envelope domains with each other and with their environment. The insight gained into the physiologic constraints that the envelope proteins are subject to may be useful in developing therapeutic and vaccination strategies
Raccoon Poxvirus Feline Panleukopenia Virus VP2 Recombinant Protects Cats against FPV Challenge

AbstractAn infectious raccoon poxvirus (RCNV) was used to express the feline panleukopenia virus (FPV) open reading frame VP2. The recombinant, RCNV/FPV, was constructed by homologous recombination with a chimeric plasmid for inserting the expression cassette into the thymidine kinase (TK) locus of RCNV. Expression of the VP2 DNA was regulated by the vaccinia virus late promoter P11. Southern blot and polymerase chain reaction (PCR) analyses confirmed the cassette was in the TK gene of the RCNV genome. An immunofluorescent antibody assay using feline anti-FPV polyclonal serum showed the expressed viral antigen in the cytoplasm of infected cells. Radioimmunoprecipitation with the same antiserum detected a 67-kDa VP2 protein which exactly matched the migration of the authentic FPV VP2 protein by SDS–polyacrylamide gel electrophoresis. Nine five-month-old cats were vaccinated and 21 days later were boosted with the recombinant virus. Peroral FPV challenge 2 weeks after the booster showed that the cats were fully protected as measured by examining clinical signs and total white blood cell counts in peripheral blood. Cats not immunized developed low to very low leukocyte counts following peroral FPV challenge. The nine vaccinated cats showed high FPV neutralization antibody prior to challenge, whereas nonvaccinated cats formed anti-FPV antibodies only after challenge
Different Hemagglutinin Cleavage Site Variants of H7N7 in an Influenza Outbreak in Chickens in Leipzig, Germany

AbstractThe hemagglutinin (HA) genes from four avian H7N7 influenza A isolates, from a single outbreak, were shown to possess different cleavage sites that contain varying numbers of basic amino acid residues (KKKKR, KRKKR, KKRKKR, KKKKKKR). All four variants are highly pathogenic in chickens and share an immediate common ancestral HA with A/tern/Potsdam/342-6/79 (H7N7) and A/swan/Potsdam/63-6/81 (H7N7). These viruses are nonpathogenic and contain no extra basic amino acids at the cleavage site of their HA. During evolution a common precursor virus acquired different sequences at the cleavage site of the HA and became highly pathogenic in chickens.In vitroassays revealed that the HA from A/chicken/Leipzig/79 with KKKKR at the cleavage site was only partially cleaved (41%), compared to 93–100% cleavage of the other HAs. Since all four viruses were highly pathogenic in chickens, these findings confirm that the degree of pathogenicityin vivois not exclusively determined by the degree of HA cleavability