pinecone/core-2020-05-10-deduplication huggingface dataset:
Presentation of the HPV16E7 Protein by Skin Grafts Is Insufficient to Allow Graft Rejection in an E7-Primed Animal

AbstractThe E7 transforming protein of Human Papillomavirus type 16 (HPV16) is expressed in the skin of a line of FVB mice transgenic for the E6 and E7 open reading frames of HPV16 driven from the αA crystallin promoter (FVBαAcryHPV16E6E7). We have transferred skin from FVBαAcryHPV16E6E7 mice to naive or E7-primed syngeneic FVB recipients to assess whether the E7 protein of HPV16 can function as a minor transplantation antigen (MTA) and promote skin graft rejection. FVB mice did not reject E7 expressing tail or flank skin grafts. E7 immunized FVB × C57BL/6J mice recipients of FVBαAcryHPV16E6E7 × C57BL/6J skin generated humoral and DTH responses to E7in vivoand E7-specific CTL precursors in the spleen, but failed to reject E7 expressing tail skin grafts by 100 days posttransfer. Thus although HPV16 E7 + ve mesenchymal and endodermal tumors can be eliminated by an E7-specific immune response, the same protein is unable to act as a MTA and promote graft rejection when expressed in skin cells. Lack of rejection of grafts expressing MTAs such as E7 may be relevant to the immunology of epithelial tumors expressing tumor-specific antigens and to our understanding of the immunology of diseases of the skin
Mutant CD4 Molecules with Improved Binding to HIV Envelope Protein gp120 Selected by Phage Display

AbstractPhage-display methodology has been used to select variant CD4 proteins exhibiting increased binding to the surface envelope glycoprotein, gp120, of Human Immunodeficiency Virus Type-1. To facilitate the selection, a library of mutant CD4 proteins was constructed by cloning a PCR-generated error prone population of the first two domains of CD4 into the phagemid expression vector pHEN1. Phage displaying CD4 in functional form were confirmed by Western blot with CD4-specific antibody and by phage ELISA on immobilized gp120. Biopanning of CD4 phage on immobilized gp120 followed by individual characterization identified five clones with increased binding to gp120. All of the selected variants had one or two amino acid substitutions within the V1 domain of CD4, notably at positions 15, 27, 30, 50, and 66 located in the strands surrounding the main binding loop. Variants which exhibited increased binding to recombinant gp120in vitrowere also shown to have an increased capacity for virus neutralization broadly in line with theirin vitrobinding activity
Disruption of the Varicella–Zoster Virus dUTPase and the Adjacent ORF9A Gene Results in Impaired Growth and Reduced Syncytia Formationin Vitro

AbstractVaricella-zoster virus (VZV) open reading frame 8 (ORF8) is predicted to encode the viral dUTPase and the adjacent gene, ORF9A, is thought to encode a membrane protein homologous to HSV-1 UL49.5. A fusion protein, in which the amino portion of glutathione-S-transferase was fused to amino acids 5 to 396 of VZV ORF8 protein, had dUTPase activityin vitro.Construction of a mutant VZV with stop codons or a deletion in the ORF8 gene resulted in loss of viral dUTPase activity. Antibody to VZV ORF9A protein demonstrated a 7-kDa protein located in the membranes of virus-infected cells. Insertion of stop codons into VZV ORF9A resulted in VZV that produced smaller plaques than parental virus. Inactivation of both VZV ORF8 and ORF9A resulted in a virus that grew to lower titers and was impaired for syncytia formation when compared to parental virus. In contrast, a similar mutation in HSV-1 has no effect on growth of the virusin vitro.These results identify loci in the VZV genome that are required for a syncytial phenotypein vitro
Purification and Characterization of the Protein Kinase Encoded by the UL13 Gene of Herpes Simplex Virus Type 2

AbstractThe proteins encoded by the UL13 genes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) have been predicted to be protein kinases. To identify the UL13 gene product, we have raised a rabbit polyclonal antiserum against a His · Tag-HSV-1 UL13 fusion protein. The antibody specifically reacted with the 60-kDa UL13 fusion protein expressed inEscherichia coliand also recognized 56- to 57-kDa late proteins in nuclear fractions of HSV-1- and HSV-2-infected cells. On the other hand, novel casein kinase activity was induced at the late stage of infection when Vero cells were infected with HSV-1 and HSV-2. The induction of the activity was most prominent in the nuclear fractions of HSV-2-infected cells and therefore we purified the protein kinase (PK) from the nuclear extracts by successive column chromatography (phosphocellulose, DEAE-cellulose, and hydroxyapatite) using casein as an exogenous substrate. The final preparation of the enzyme contained a single major protein with an apparent molecular weight of 56 kDa which was specifically reacted with the UL13 antiserum. The PK activity was optimal in the absence of NaCl and at relatively high pH. Acidic proteins such as casein and phosvitin were efficiently phosphorylated by the PK. A basic protein, protamine, which is the best substrate for the HSV-2 US3 PK, was not detectably phosphorylated but histone was a relatively good substrate for the UL13 PK. Phosphoamino acid analysis revealed that the PK phosphorylated serine and threonine but not tyrosine. Moreover the enzyme was found to be highly resistant to heparin, a potent inhibitor of casein kinase II (CK II) and also resistant to CK I-7, a synthetic inhibitor of CK I, but very sensitive to a bioflavonoid quercetin. These results indicate that the HSV-2 UL13 PK had unique catalytic properties different from those of cellular CK I, CK II, and the viral PK encoded by the US3 gene. We have also determined the complete nucleotide sequence of the HSV-2 UL13 gene. The overall amino acid homology between the HSV-2 and HSV-1 UL13 PKs was 85.9% and the homology was highly conserved in the C-terminal region
The Genome-Linked Protein of Potato Leafroll Virus Is Located Downstream of the Putative Protease Domain of the ORF1 Product

AbstractThe sequence of the 32 N-terminal amino acids of the protein (VPg) which is covalently linked to the RNA of potato leafroll virus has been determined. The obtained VPg sequence mapped to position 400 to 431 of the PLRV ORF1 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Comparison with other viral sequences revealed significant similarities with the ORF1 products of beet western yellows virus, cucurbit aphid-borne yellows virus, and beet mild yellowing virus
Identification of cis-Acting Repressor Activity within Human Immunodeficiency Virus Type 1 Protease Sequences

AbstractHuman immunodeficiency virus type-1 (HIV-1) Rev overcomes negative elements within viral RNAs to allow expression ofgag, pol,andenv.The effect of Rev on protein and RNA expression of HIV-1 protease (PR)-containing constructs was investigated utilizing transient transfection of COS cells. Rev, through the Rev response element (RRE), resulted in a large increase in proteolytic activity and cytoplasmic RNA accumulation. Furthermore, Rev increased the level of total RNA produced by a PR-containing construct. The increase in cytoplasmic RNA accumulation in the presence of Rev indicated the presence of cis-acting repressor sequences (CRS) within the RNA produced by this construct. Therefore, components of the construct were analyzed for CRS activity. PR sequences in both sense and antisense orientations exhibited CRS activity. RRE sequences alone conferred a small CRS effect. Additional CRS activity was present within an unspliced RNA containing onlynefand LTR sequences. These results indicate a novel form of cis-acting repressor activity within HIV-1 PR; this activity is exerted regardless of the orientation of PR and appears to function at the level of cytoplasmic or nuclear RNA stability
The Molecular Epidemiology of Human Immunodeficiency Virus Type 1 in Six Cities in Britain and Ireland

AbstractWe have sequenced the p17 coding regions of thegaggene from 211 patients infected either through injecting drug use (IDU) or by sexual intercourse between men from six cities in Scotland, N. England, N. Ireland, and the Republic of Ireland. All sequences were of subtype B. Phylogenetic analysis revealed substantial heterogeneity in the sequences from homosexual men. In contrast, sequence from over 80% of IDUs formed a relatively tight cluster, distinct both from those of published isolates and of the gay men. There was no large-scale clustering of sequences by city in either risk group, although a number of close associations between pairs of individuals were observed. From the known date of the HIV-1 epidemic among IDUs in Edinburgh, the rate of sequence divergence at synonymous sites is estimated to be about 0.8%. On this basis we estimate the date of divergence of the sequences among homosexual men to be about 1975, which may correspond to the origin of the B subtype epidemic
Gene Transfer Using Replication-Defective Human Foamy Virus Vectors

AbstractReplication-defective vectors based on an infectious molecular clone of human foamy virus (HFV) were constructed by deletion and replacement of the accessory genes with expression cassettes for puromycin-resistance and β-glucouronidase. Cell lines which produced in excess of 105helper virus-free transducing units/ml were generated bytrans-complementation of the replication defect using a BHK-21-derived cell line expressing the Bel-1 transactivator. Vectors based on the HFV genome may provide useful alternatives to existing retroviral vectors
Suppression of Potyvirus Infection by Coexpressed Closterovirus Protein

AbstractA tobacco etch virus (TEV)-based expression vector has been used for insertion of several ORFs derived from the unrelated beet yellows virus (BYV). Hybrid TEV variants expressing the BYV capsid protein, 20-kDa protein, or HSP70 homolog systemically infectedNicotiana tabacumand stably retained BYV sequences. In contrast, insertion of the ORF encoding BYV leader proteinase (L-Pro) resulted in severely impaired systemic transport and accumulation of recombinant TEV. Progeny of this virus underwent various deletions affecting the L-Pro sequence and mitigating the defects in virus spread. Model experiments involving several spontaneous and engineered mutants indicated that the central domain of BYV L-Pro was responsible for the defect in hybrid virus accumulation, whereas full-size L-Pro was required for maximal debilitation of systemic transport. Strikingly, BYV L-Pro expression did not debilitate systemic infection of hybrid TEV inNicotiana benthamianaplants. No major defects in replication or encapsidation of recombinant RNA were revealed inN. tabacumprotoplasts. These results indicated that BYV L-Pro specifically interfered with TEV systemic transport and accumulation in a host-dependent manner and suggested a potential utility of closterovirus L-Pro as an inhibitor of potyvirus infection. In addition, it was demonstrated that the 107-amino-acid-residues-long N-terminal part of the TEV helper component proteinase is not essential for systemic infection
Evidence for Induction of Interferon-α and Interferon-β in Retinal Glial Cells of Müller

AbstractStudies were performed to determine if retinal glial cells of Müller transcribe the genes for interferon-α (IFNα) or IFNβ upon exposure to virus. Responses to herpes simplex virus type 1 (HSV-1) were tested with cultured murine Müller cells and,in vivo,with retinas obtained after bilateral injection of either HSV-1 or buffer into the anterior chamber of the eyes of BALB/c mice. Induction of IFN transcription and relative temporal changes in transcript levels occurred over time after eitherin vitroorin vivoexposure to HSV-1. Transcription of both IFN genes was induced in cultured glia within 1 hr after exposure to virus. IFN transcripts were detected in retinas by 24 hr postinfection and these were maximal at 3 days. Byin situhybridization (ISH), IFNα2mRNA localized to focal areas in the intact retinas of virus-injected eyes and was consistent with our previous report of a transient, focal appearance of viral antigens in those retinas. Uninfected cells and ocular tissues were negative for IFN transcripts. Combined ISH and immunohistochemistry on retinal impression smears confirmed that glial fibrillary acidic protein-positive Müller cells are an intraretinal source of IFNα and IFNβ transcripts after ocular exposure to HSV-1. Our results support a role for Müller cells as participants in intraretinal antiviral or immunomodulatory responses via type 1 IFN production and may have implications for future therapeutic interventions
Infected Macaques That Controlled Replication of SIVmacor Nonpathogenic SHIV Developed Sterilizing Resistance against Pathogenic SHIVKU-1

AbstractTwenty macaques were used to evaluate the ability of nonpathogenic SIVmacor nonpathogenic chimeric SIV-HIV (SHIV) to induce protection in macaques against superinfection with a pathogenic variant of SHIV (SHIVKU-1) originally containing thetat, rev, vpu,andenvof HIV-1 (strain HXB2) in a genetic background of SIVmac239. Specifically, three macaques inoculated with molecularly cloned, macrophage-tropic SIVmacLG1 developed an early systemic infection but recovered with only traces of SIVmacDNA in visceral lymphoid tissues. These animals were then inoculated parenterally with pathogenic SHIVKU-1. All three animals resisted infection with SHIVKU-1, as indicated by lack of virus recovery and absence of SHIV-specificenvandvpusequences in the visceral lymphoid tissues and multiple regions in the CNS. We also examined the ability of five macaques that had been inoculated with nonpathogenic SHIV (NP-SHIV) to withstand challenge with the pathogenic SHIVKU-1. Like the SIVmacLG1-inoculated macaques, these animals also resisted SHIVKU-1challenge as judged by the inability to recover infectious virus, normal CD4+T cell counts, and the absence of SHIVKU-1signature sequences in the lymph node tissue. Thus, eight of eight animals that developed control over primary lentivirus infections had also developed resistance to infection with pathogenic SHIVKU-1. Three groups of macaques were used as controls for this study. The first group consisted of six macaques inoculated with SHIVKU-1alone. All animals developed viremia, showed severe loss of CD4+T cells within 4 weeks, and succumbed to AIDS within 6 months. The second group of three macaques was inoculated first with SHIVKU-1and inoculated later with uncloned, neurovirulent SIVmac7F-Lu. A third group of three macaques was inoculated with SIVmac7F-Lu followed by inoculation with SHIVKU-1. PCR analyses using oligonucleotide primers specific for the SIV or HIVenvrevealed that macaques from the last two groups had widespread infection with both SHIVKU-1and SIVmac, indicating that animals that failed to control productive replication of either SHIVKU-1or SIVmac7F-Lu could not resist superinfection with the other virus. These data indicate that sterilizing immunity against the virulent SHIV could be induced in animals that had experienced an immunizing infection. Moreover, the divergence of the envelope glycoprotein of the protective avirulent and virulent challenge virus suggests that a single vaccine could protect against infection with a virus containing a different envelope glycoprotein
Persistence and Evolution of Feline Coronavirus in a Closed Cat-Breeding Colony

AbstractFeline coronavirus (FCoV) persistence and evolution were studied in a closed cat-breeding facility with an endemic serotype I FCoV infection. Viral RNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in the feces and/or plasma of 36 of 42 cats (86%) tested. Of 5 cats, identified as FCoV shedders during the initial survey, 4 had detectable viral RNA in the feces when tested 111 days later. To determine whether this was due to continuous reinfection or to viral persistence, 2 cats were placed in strict isolation and virus shedding in the feces was monitored every 2–4 days. In 1 of the cats, virus shedding continued for up to 7 months. The other animal was sacrificed after 124 days of continuous virus shedding in order to identify the sites of viral replication. Viral mRNA was detected only in the ileum, colon, and rectum. Also in these tissues, FCoV-infected cells were identified by immunohistochemistry. These findings provide the first formal evidence that FCoV causes chronic enteric infections. To assess FCoV heterogeneity in the breeding facility and to study viral evolution during chronic infection, FCoV quasispecies sampled from individual cats were characterized by RT-PCR amplification of selected regions of the viral genome followed by sequence analysis. Phylogenetic comparison of nucleotides 7–146 of ORF7b to corresponding sequences obtained for independent European and American isolates indicated that the viruses in the breeding facility form a clade and are likely to have originated from a single founder infection. Comparative consensus sequence analysis of the more variable region formed by residues 79–478 of the S gene revealed that each cat harbored a distinct FCoV quasispecies. Moreover, FCoV appeared to be subject to immune selection during chronic infection. The combined data support a model in which the endemic infection is maintained by chronically infected carriers. Virtually every cat born to the breeding facility becomes infected, indicating that FCoV is spread very efficiently. FCoV-infected cats, however, appear to resist superinfection by closely related FCoVs
Double-Stranded RNA-Dependent Protein Kinase (PKR) Is Regulated by Reovirus Structural Proteins

AbstractReovirus σ3 is a virion outer shell protein that also binds dsRNA and stimulates translation by blocking activation of the dsRNA-dependent protein kinase, PKR. Purified σ3 was shown by gel shift assay to bind specifically to RNA duplexes of minimal length 32–45 base pairs. PKR binding to dsRNA was prevented by σ3, and translation inhibition of luciferase reporter by PKR expression in transfected cells was reversed by σ3. Association of σ3 with its outer capsid partner μ1/μ1C eliminated dsRNA binding and prevented restoration of protein synthesis. Analyses of σ3 mutants demonstrated a direct correlation between dsRNA binding and reversal of the down-regulation of translation by PKR. In infected cells, σ3 was stable but dsRNA binding decreased, presumably due to μ1/μ1C complex formation. The results suggest a functional transition from early inhibition of PKR activation by σ3 to its association with μ1/μ1C in capsid structures
In VivoSelection of Duck Hepatitis B Virus Pre-S Variants Which Escape from Neutralization

AbstractTo better understand the role of specific residues within the duck hepatitis B virus (DHBV) pre-S protein in neutralization and infectivity, we have selected and identified pre-S variants which escape neutralization. A highly neutralizing monoclonal antibody (Mab 900) which recognizes an epitope83IPQPQWTP90localized previously on the DHBV pre-S protein, within a region suspected to mediate the virus interaction with hepatocytes, was used as immune pressure. After only twoin vivoneutralization rounds with Mab 900, five different pre-S mutant genomes were identified, which harbored point mutations affecting only proline residues located at position 90 within this epitope (83IPQPQWTP90) and/or at a distance at position 5. We have shown that a single (P5L) or double proline (P5L + P90H) substitution affect neither virus replication capacity norin vivoinfectivity. However, the P5 mutation reduces mutant recognition by Mab 900 twofold, while the substitution of both prolines 5 and 90 almost completely abolishes mutant P5L + P90H reactivity with this Mab and leads to a decrease of neutralization. Therefore we describe here an experimental system which allows rapidin vivoselection and identification of DHBV pre-S variants and provide evidence that residues within and at a distance from the neutralization epitope are important in DHBV neutralization but do not affect its replication capacity and infectivity
Mapping and Expression of Southern Bean Mosaic Virus Genomic and Subgenomic RNAs

AbstractThe coat protein of the cowpea strain of southern bean mosaic sobemovirus (SBMV-C) is translated from a subgenomic RNA (sgRNA) that is synthesized in the virus-infected cell. Like the SBMV-C genomic RNA, the sgRNA has a viral protein (VPg) covalently bound to its 5′ end. The mechanism(s) by which ribosomes initiate translation on the SBMV-C RNAs is not known. To begin to characterize the translation of the sgRNA it was first necessary to precisely map its 5′ end. Primer extension was used to identify SBMV-C nucleotide (nt) 3241 as the transcription start site. As a control, the 5′ end of the genomic RNA was also mapped. Surprisingly, the 5′ terminal nt of this RNA was identified as SBMV-C nt 2. The primary structure of the 5′ ends of these two RNAs is therefore expected to be VPg-ACAAAA. Precise mapping of the 5′ end of the sgRNA of the bean strain of SBMV (SBMV-B) demonstrated that it has these same elements. Translation of coat protein from the SBMV-C sgRNA and p21 from the SBMV-C genomic RNA was compared using a cell-free system. The results of these experiments were consistent with translation of these proteins by a 5′ end-dependent scanning mechanism rather than by internal ribosome binding
Recombinant Human Immunodeficiency Pr55gagVirus-like Particles Presenting Chimeric Envelope Glycoproteins Induce Cytotoxic T-Cells and Neutralizing Antibodies

AbstractVery recently, we demonstrated that the replacement of the human immunodeficiency virus type-1 (HIV-1) gp41 transmembrane protein by an Epstein–Barr virus gp220/350-derived membrane anchor resulted in the incorporation of chimeric envelope (Env) oligomers into Pr55gagvirus-like particles (VLPs), exceeding that of wild-type gp160 by a factor of 10. In this study, we examined the immunostimulatory properties of Pr55gagVLPs to both (i) chimeric HIV-1 gp120 external envelope proteins and (ii) full-length gp160 presented on the outer surface of the particles. Immunization studies carried out with VLPs presenting different derivatives of the chimeric and wild-type Env proteins elicited a consistent anti-Pr55gagas well as anti-Env antibody response in complete absence of additional adjuvants. In both cases, the immune sera exhibited anin vitroneutralizing activity against homologous HIV-1 infection in MT4 cells. Noteworthy, these VLPs were also capable of inducing a strong CD8+cytotoxic T-cell (CTL) response in immunized BALB/c mice that was directed toward a known CTL epitope in the third variable domain V3 of the gp120 external glycoprotein. However, the induction of V3-loop-specific CTLs critically depended on the amounts of Env proteins that were presented by the Pr55gagVLPs. Moreover, the CD8+CTL response was not significantly altered by adsorbing the VLPs to alum or by repeated booster immunizations. These results illustrate that Pr55gagVLPs provide a safe and effective means of enhancing neutralizing humoral responses to particle-entrapped gp120 proteins and are also capable of delivering these proteins to the MHC class I antigen processing and presentation pathway. Therefore, antigenically expanded Pr55gagVLPs represent an attractive approach in the design of vaccines for which specific stimulation of neutralizing antibodies and cytotoxic effector functions to complex glycoproteins is desired