pinecone/core-2020-05-10-deduplication huggingface dataset:
The Alphavirus 3′-Nontranslated Region: Size Heterogeneity and Arrangement of Repeated Sequence Elements

AbstractThe 3′-nontranslated region (NTR) of representative strains of all known alphavirus species was amplified by reverse transcription–polymerase chain reaction. For 23 of them, the 3′-NTR sequence was determined. Together with previously published data, this allowed an analysis of the 3′-NTR of the viruses in the genus Alphavirus. The length of the 3′-NTRs varied from 77 nt for Pixuna virus to 609 nt for Bebaru virus. The 19-nt conserved sequence element directly adjacent to the poly(A) tract was found in all viruses, supporting the hypothesis that this region is acis-acting sequence element during viral replication and essential for virus growthin vitro. Within the 3′-NTR of all alphaviruses, repeated sequence elements of various numbers and lengths were found. Their composition was very consistent in both the Venezuelan equine encephalitis (VEE) and the Sindbis-like viruses, although their number was constant only within the latter group. For the VEE viruses, our data suggested that insertion events rather than deletions from an ancestor with a long 3′-NTR created the various number of repeated sequence elements. Among the remaining viruses, both the number and the composition of repeated sequence elements varied remarkedly
Antigenic Sites of Coxsackievirus A9

AbstractAntigenic analysis of coxsackievirus A9 (CAV9) was carried out by using a peptide scanning method. Immunogenic regions in the capsid proteins VP1, VP2, and VP3 were recognized by antibodies in the sera of virus-immunized rabbits. The peptide sequences were scanned using a 12-amino-acid window and three-residue shift. Three immunogenic regions, located in the N- and C-terminal parts of VP1 and in the N-terminus of VP3, were identified. Trypsin treatment of the virus, known to cleave off the C-terminus of VP1 containing a functional RGD motif, completely abolished the reactivity against this region but did not have any other significant effect on antigenicity. In further studies, it was found that the RGD motif itself was poorly immunogenic whereas antibody-binding sites were located at both sides of the motif. New antigenic sites emerged after heat treatment of CAV9 at 56 or 100°C prior to immunization; in particular, loop structures between β strands in VP2 exhibited increased immunogenicity. New antigenic sites in VP1 and VP3 also appeared after the treatments. In spite of the markedly altered reactivity in peptide scanning, the virus treated at 56°C elicited high titers of neutralizing antibodies. To reveal cross-reactive antigenic sites, antisera raised against coxsackievirus B3 and echovirus 11 were also tested. The cross-reactive antigenic sites were located mainly in the N-terminal parts of VP1 and VP3
Identification of Conserved Hydrophobic C-Terminal Residues of the Human Papillomavirus Type 1 E1∧E4 Protein Necessary for E4 Oligomerisationin Vivo

AbstractPrevious studies have shown that human papillomavirus (HPV) E4 proteins undergo oligomerisation, although the precise sequences involved have not been identified. Using the yeast two-hybrid system we have identified HPV 1 E4 sequences that are critical to multimerisation. Fusion proteins were created by linking wild-type and mutant E4 proteins to a LexA DNA-binding domain or a B42 transactivation domain. HPV 1 E4:E4 interactions were examined by expression of these fusion proteins inSaccharomyces cerevisiae.This assay showed that (1) amino acid residues 95 to 115 at the carboxy-terminus were critical for oligomerisation and (2) hydrophobic residues (isoleucine 107, phenylalanine 114) in this domain are major determinants in the formation of oligomers. Interestingly, the carboxy-terminal domain shares homology with other E4 proteins of cutaneous HPV types and, furthermore, positions 107 and 114 are conserved residues. Substitution of the conserved aspartate amino acids (residues 110 and 112) did not abrogate E4 oligomerisation. Chemical cross-linking of wart and recombinant (baculovirus-expressed) HPV 1 E4 protein indicated that in solution this viral protein forms complexes consistent in size with either trimers or tetramers. These complexes were resistant to urea denaturation and are not dependent on the formation of disulphide linkages. A mutant protein containing a deletion of residues 110 to 115 was unable to form oligomers following cross-linking supporting a role for this region in mediating E4:E4 interactions. We conclude that oligomerisation of the HPV 1 E4 protein is likely to be mediated by carboxy-terminal residues and that conserved hydrophobic residues of this domain play a major role in E4 oligomerisation
Identification of Residues within the Herpes Simplex Virus Type 1 Origin-Binding Protein That Contribute to Sequence-Specific DNA Binding

AbstractGene UL9 of herpes simplex virus type 1 encodes an 851-amino-acid protein which is essential for viral DNA synthesis and functions as a sequence-specific origin-binding protein and DNA helicase. We generated monoclonal antibodies against purified UL9 protein and identified one such antibody (MAb 13924) that can block the interaction of the UL9 C-terminal DNA-binding domain (amino acids 534–851) with its recognition sequence. MAb 13924 interacted with immobilized peptides containing residues 780–786 of UL9. Although the corresponding region of the homologous protein encoded by varicella-zoster virus differs at only a single position it was not recognized by MAb 13924. Site-directed mutagenesis experiments confirmed that residues within this region contribute to the epitope recognized by MAb 13924 and may be involved in sequence-specific DNA binding. In addition, all eight lysine residues within the DNA-binding domain were separately changed to alanine and the DNA-binding properties of the mutated proteins were examined. The results showed that lysine residues that are located close to the peptide recognized by MAb 13924 or lie within the region of the DNA-binding domain most highly conserved among homologous alphaherpesvirus proteins play a role in sequence-specific DNA binding. Moreover, alteration of a lysine residue 18 amino acids from the recognized peptide prevented the interaction of MAb 13924 with the UL9 C-terminal DNA-binding domain. Three helical segments are predicted to occur within the region containing mutations that affect sequence-specific binding and interaction with MAb 13924
Identification and Characterization of Human Herpesvirus-8 Lytic Cycle-Associated ORF 59 Protein and the Encoding cDNA by Monoclonal Antibody

AbstractMonoclonal antibody (Mab) 11D1 specific for HHV-8 showed a predominantly nuclear membrane fluorescence with about 30% of phorbol ester (TPA)-induced HHV-8-carrying BCBL-1 cells and with 2–8% of uninduced cells, but not with other herpes viruses infected cells. This Mab immunoprecipitated a 50-kDa polypeptide from BCBL-1 cells. The synthesis of this polypeptide was reduced but not inhibited by phosphonoacetic acid (PAA). A 2.3-kb cDNA insert from a cDNA library of TPA-induced BCBL-1 cells was identified by Mab 11D1. Sequence analysis shows that this cDNA is open at the 5′ end and encodes two ORFs of 396AA (5′ end) and 357AA (3′ end). These ORFs are identical to the published HHV-8 ORFs 59 and 58, respectively.In vitrotranscription and translation of the cDNA resulted in the synthesis of a 50-kDa polypeptide and its partial peptide map was identical to that of the 50-kDa polypeptide detected in the TPA induced BCBL-1 cells. Riboprobe made from the cDNA insert hybridized with several viral specific RNAs from BCBL-1 cells. Levels of these RNA species were reduced, but not inhibited by PAA. These characteristics are similar to other herpes viruses genes encoding the lytic cycle associated early–late class accessory proteins that are essential for viral DNA replication. This Mab 11D1 recognizing the HHV-8 lytic cycle associated ORF 59 protein will be highly useful in monitoring the lytic replicative cycle
Papillomavirus Virus-like Particles Can Deliver Defined CTL Epitopes to the MHC Class I Pathway

AbstractTo evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus of bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses
Characterization ofgagandpolSequences from Long-Term Survivors of Human Immunodeficiency Virus Type 1 Infection

AbstractWe previously identified a group of 10 long-term survivors (LTS) of human immunodeficiency virus type 1 (HIV-1) infection. Extensive biological analysis revealed that some of these individuals do well, at least in part, because they possess weakened or attenuated viruses. Also, previously, to determine the genotype associated with the attenuated phenotypein vivo,we characterizednef, vif, vpr, vpu, env,and LTR in our cohort of LTS. In this study, we analyzedgagandpolgenes derived from eight individuals in our cohort. For each subject, multiple full-lengthgagandpolclones were obtained for analysis. In most cases, the sequences derived from the LTS had an intact open reading frame. At the protein level, there were no discernible differences between the sequences derived from LTS and those derived from patients with AIDS. Thus, no common defect ingag and polwas found in our cohort. One individual (subject SF), however, had only Gag-defective proviral sequences (10 of 10) in his peripheral blood mononuclear cells. Furthermore, longitudinal studies of the samples collected from SF over a 2-year period showed that all p17gagclones sequenced (24 of 24) were defective due to G-to-A hypermutations. This viral defect in Gag may provide the molecular basis for this individual's extremely low viral load and long-term asymptomatic state. These results, together with previous findings in our LTS cohort, reinforce the notion that it is unlikely that a single common viral genetic determinant accounts for the lack of disease progression in all cases. Multiple host and viral factors undoubtedly contribute to the well-being of LTS of HIV-1 infection
Comparison of Sindbis Virus-Induced Pathology in Mosquito and Vertebrate Cell Cultures

AbstractWe have compared Sindbis virus-induced cytopathology in vertebrate and mosquito (Aedes albopictus) cell cultures. It has been shown that vertebrate cells undergo apoptosis when infected by Sindbis virus and this was confirmed here using hamster cells (BHK). The occurrence of cell death in Sindbis virus-infectedA. albopictuscells is a cell clone-specific phenomenon and, unlike in BHK cell cultures, mosquito cell death does not correlate with a large induction of apoptosis, as determined by assays testing for DNA fragmentation or reduced cellular DNA content. Cell cycle distribution changes were observed in Sindbis virus-infected BHK and C7-10 cell cultures, and the changes are distinct, both in the time of induction and the types of perturbations. In Sindbis virus-infected BHK cells, the major cell cycle profile change is the early accumulation of cells with sub-G1 DNA content and a corresponding reduction in the proportion of cells in G1 and G2/M. For Sindbis virus-infected C7-10 cells, the major perturbations are an increased proportion of cells showing G2/M or polyploid DNA content and a reduction in the proportion of G1 and S phase cells. These data suggest that the pathology induced in mosquito cell cultures by Sindbis virus infection may be distinct from the pathology which appears in vertebrate cell cultures
The M1 and M2 Proteins of Influenza A Virus Are Important Determinants in Filamentous Particle Formation

AbstractInfluenza A virus is highly pleomorphic with particles exhibiting either spherical or filamentous morphology. The mechanisms behind this pleomorphism and its importance in viral pathogenesis are not clearly understood. We have observed that budding of filamentous influenza A/Udorn virus particles can be readily visualized by immunofluorescence microscopy. Filamentous particle formation was inhibited by treatment of cells with the anti-M2 14C2 antibody, but was not inhibited with the isotype identical 5C4 anti-M2 antibody or by anti-neuraminidase antibody. To further explore the viral determinants of filamentous particle formation, we investigated the morphology and growth characteristics of three variants of A/Udorn/72 virus, which had previously been selected for their resistance to growth inhibition by the 14C2 anti-M2 monoclonal antibody. Two of the variant viruses, 5A and 10A, contain single amino acid substitutions in the cytoplasmic domain of the M2 protein, whereas the 1A variant contains a single amino acid substitution in the viral matrix protein, M1. Variants 5A and 10A both were found to retain the filamentous particle phenotype found in the parental strain A/Udorn/72, and the production of filamentous virions by both variants was resistant to inhibition by the 14C2 antibody. However, immunofluorescence and electron microscopy revealed that the variant 1A was composed almost exclusively of spherical particles. The 1A variant displayed higher viral yields and a larger plaque size than the filamentous viruses. In addition, we separated distinct populations highly enriched in spherical or filamentous particles by velocity gradient centrifugation. Analysis of the protein compositions of these particles revealed that the NP:M1 or NP:HA ratios in filamentous particles were significantly lower than in spherical particles, but the filaments have higher levels of NP per particle. The spherical and filamentous particles were found to have similar specific infectivity. These results indicate that the filamentous morphology of the A/Udorn virus depends upon the matrix (M1) and/or M2 proteins
Proper Spacing between Heptad Repeat B and the Transmembrane Domain Boundary of the Paramyxovirus SV5 F Protein Is Critical for Biological Activity

AbstractThe paramyxovirus, simian virus 5, fusion (F) protein contains seven amino acids between heptad repeat B (a domain required for a biologically active fusion protein) and the presumptive boundary of the transmembrane (TM) domain. The role of the seven membrane proximal residues in stability and fusion promotion was examined by construction of a series of insertion, substitution, and deletion mutants, as manipulation of this region to enable proteolytic cleavage would facilitate production of a soluble F protein. The majority of the mutant F proteins both oligomerized and had kinetics of intracellular transport similar to those of wild-type (wt) F protein. All mutant F proteins were expressed at the cell surface at or near the same level as the wt F protein. However, by using both a qualitative lipid mixing assay and a quantitative content mixing assay for membrane fusion, it was found that mutant F proteins containing insertions in the region between heptad repeat B and the TM domain were unable to induce fusion, whereas the mutant F proteins containing substitutions in this region, together with three of the four mutants with deletions in this region, could induce fusion. Four of the F protein mutants contained a Factor Xa cleavage site, IEGR; however, Factor Xa treatment of cell surfaces released either none or only very small amounts (<1% of total protein) of the soluble heterodimer F1+ F2. As an alternative method of generating soluble F protein, a glycosyl phosphatidylinositol (GPI) anchor was added to the F protein at three membrane-proximal positions. The highest level of surface expression was observed when the final molecule did not contain a significant insertion of amino acids into the membrane proximal region. Two F-GPI mutants reached the surface at approximately 20% of the levels seen with the wt F protein, and approximately 25% of the cell surface population of these mutants could be cleaved with phosphatidylinositol phospholipase C (PI-PLC) to yield soluble F protein. However, all the F-GPI mutants oligomerized aberrantly and failed to promote fusion. Taken together, these data indicate that the spacing of the region immediately adjacent to the presumptive boundary of the TM domain is extremely important for the fusogenic activity of the SV5 F protein
The Bovine Herpesvirus Type 1 UL3.5 Open Reading Frame Encodes a Virion Structural Protein

AbstractThe bovine herpesvirus type 1 (BHV-1) open reading frame (ORF) UL3.5 is similar to ORFs found in pseudorabies virus, infectious laryngotracheitis virus, equine herpesvirus type 1, and varicella zoster virus, but clearly absent from herpes simplex virus. The published sequence for this ORF predicts a 126-amino-acid (13.2 kDa) protein product with an isoelectric point of 12.3. We confirmed the UL3.5 sequence, expressed the ORF as a glutathione-S-transferase fusion protein, and made rabbit antibodies against the purified fusion protein. The antiserum detected a 13-kDa protein in Western blots of MDBK cells infected with BHV-1, but not with other herpesviruses or uninfected cells. The BHV-1 UL3.5 protein was characterized as a component of the virion envelope or tegument because it was expressed as a late protein, it was present in the cytoplasm but not the nucleus of infected cells, and it was removed from purified virions by detergent extraction
Substitution of Leucine 8 in the Simian Immunodeficiency Virus Matrix Protein Impairs Particle Formation without Affecting N-Myristylation of the Gag Precursor

AbstractIn the late stages of replication of the simian immunodeficiency viruses (SIV), the matrix protein (MA) plays a central role in the transport of Pr55gagto the plasma membrane, assembly of virus particles, and incorporation of the envelope glycoprotein into particles. Targeting of Pr55gagto the plasma membrane is mediated by two motifs within the MA protein: the N-terminal myristate and a cluster of positively charged amino acids. In this report, we characterized the assembly phenotype of an SIV Gag mutant (L8Q) carrying the single amino acid substitution of glutamine for leucine at position 8 in the MA domain. The hydropathic profile of the mutated MA protein indicated that the L8Q amino acid change disrupts the hydrophobic character of the region comprising the first 10 residues of the protein. Expression of mutant L8Q Gag protein in CV-1 cells, by means of the vaccinia virus vector system, resulted in efficient synthesis and N-myristylation of Pr55gag. However, this mutation severely impaired particle production, as inferred from both biochemical and electron microscopy analyses. Cellular fractionation assays revealed that in cells expressing mutant L8Q, the proportion of cytosol-associated Pr55gagwas significantly increased compared to that observed upon expression of wild-type Gag. Furthermore, mutant L8Q Gag partitioned onto cytosol and membrane fractions in a manner similar to nonmyristylated Gag polyprotein. Taken together, these results indicate that the L8Q mutation reduces the membrane-binding capacity of the Gag precursor. It is therefore likely that in the SIV MA, in addition to the N-myristate group, the hydrophobicity of the neighboring region is important for efficient association of Pr55gagwith the plasma membrane
Attenuation of Recombinant Vesicular Stomatitis Viruses Encoding Mutant Glycoproteins Demonstrate a Critical Role for Maintaining a High pH Threshold for Membrane Fusion in Viral Fitness

AbstractA plasmid-based recovery system was used to generate four unique vesicular stomatitis virus (VSV) mutants that encode glycoproteins (G proteins) with single or double amino acid substitutions in two conserved acidic residues adjacent to the putative G protein fusion domain. Previously we demonstrated that three of the mutant G proteins (D137-L, E139-L, and DE-SS) have slightly reduced pH thresholds for membrane fusion activity. In this report we show that even though the viruses encoding D137-L, E139-L, and DE-SS were recovered with high efficiency, these mutants were attenuated for growth in cell culture. Plaque formation was significantly delayed with these mutants and the plaques were smaller and more diffuse than those produced by wild-type VSV. In addition, cells infected with these mutants produced approximately 5- to 10-fold less infectious virus than cells infected with a similarly recovered VSV encoding the wild-type G protein. Using R18-labeled virus we found that the mutant G proteins had approximately 50% of the fusion activity of wild-type G at pH 6.3 and only 75% activity at pH 5.8. We also show that the mutant viruses were more sensitive to chloroquine inhibition of infection than either wild-type VSV or the mutant E139-T, which has a fusion phenotype similar to wild-type G protein. Reduced fusion activity and attenuation of infectivity was not due to differences in the amount of G protein incorporated into virions, nor to differences in the amount of virus binding to cells at physiological pH. Although infectivity was assayed at neutral pH, we observed an increase in virus binding with both mutant and wild-type virions as the pH was lowered, and the increase in binding occurred near the pH threshold for membrane fusion activity. From these data we propose a model in which VSV entry involves an increase in virus binding to the inner leaflet of the endosomal membrane during endosome acidification. Concomitant with this higher affinity binding, G protein becomes primed to initiate fusion of the viral envelope with the endosomal membrane. Viruses with mutations that delay the onset of increased binding and fusion lag behind wild-type VSV in their ability to initiate a productive infection, potentially because the location within the cytoplasm where these viruses ultimately fuse is not optimal for either virus uncoating or replication of the viral genome