pinecone/core-2020-05-10-deduplication huggingface dataset:
Induction of Protective Immunity in Rodents by Vaccination with a Prokaryotically Expressed Recombinant Fusion Protein Containing a Respiratory Syncytial Virus G Protein Fragment

AbstractA subunit approach to the development of a respiratory syncytial virus (RSV) vaccine was investigated. It involved the production, inEscherichia coli,of an RSV (Long) G protein fragment (G2Na) as a C-terminal fusion partner to an albumin binding region (BB) of streptococcal protein G. G2Na incorporated amino acid residues 130–230 and was specifically recognized by murine anti-RSV-A polyclonal serum. In mice, intraperitoneal immunization with BBG2Na induced high anti-RSV-A serum ELISA titers and low to moderate neutralization activity. The immune response induced by BBG2Na demonstrated a potent protective efficacy against upper and lower respiratory tract RSV-A infection. The immunogenicity and protective efficacy of BBG2Na was maintained for at least 47 and 48 weeks, respectively, and was as potent and durable as live RSV-A administered in a similar fashion. Intramuscular immunization of cotton rats with BBG2Na protected lungs from both homologous and heterologous virus challenge. In contrast to mice, however, cotton rat nasal tracts were not protected after BBG2Na immunization. Consistent with antibody-mediated protection, virus was cleared within 24 hr from the lungs of BBG2Na-immunized mice. The anti-RSV-A antibodies induced in mice were exclusively of the IgG1 isotype and were detected in the serum, lungs, and nasal tracts. Passive transfer of these antibodies prevented acute, and eliminated chronic, RSV-A lung infection in normal and immunodeficient mice, respectively, confirming that such antibodies are important and sufficient for BBG2Na-induced pulmonary protection. Our results clearly demonstrate that BBG2Na contains an important immunogenic domain of the RSV G protein. The prokaryotic origin of this protein indicates that glycosylation of the RSV G protein is not necessary for protective efficacy. Thus, BBG2Na has potential as an RSV subunit vaccine
Characterization of Feline Immunodeficiency Virus Integrase and Analysis of Functional Domains

AbstractThe wild-type and mutant derivatives of the integrase protein of feline immunodeficiency virus (FIV) were cloned and expressed inEscherichia coli.The purified proteins were examined using various model DNA substrates for their catalytic activities: 3′-end processing, 3′-end joining, and disintegration. The reactions required the presence of either Mn2+or Mg2+as a divalent cation. The N-terminal and C-subterminal domains (residues 1–52 and 189–235, respectively) were necessary for 3′-end processing and joining reactions but not for disintegration. Substitution of asparagine for the highly conserved aspartic acid at position 118 resulted in a complete loss of all three activities, confirming that the catalytic domain resides in the central core region (residues 53–188) of the protein. Deletion of the C-terminus (residues 236–281) resulted in a FIV integrase mutant that had efficient 3′-end processing and disintegration activities but weak 3′-end joining activity, a finding that has not been reported previously with other retroviral integrases. The result suggests that the C-terminus is the primary binding site for target DNA. Attachment of a histidine-tag at the N-terminus of the wild-type and deletion derivatives increased the binding affinity to the DNA substrate, resulting in altered levels of catalytic activities and selection of integration sites. Similar to other retroviral integrases, certain pairs of mutant derivatives of FIV integrase could complement each other to restitute 3′-end processing and joining activities, suggesting that formation of functional multimers is a general feature of proteins in the integrase family
Increased Frequency of Specific Locus Mutation Following Human Cytomegalovirus Infection

AbstractThe effect of human cytomegalovirus (HCMV) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus was studied in Chinese hamster lung V79 cells. When V79 cells were infected with HCMV (strain AD169) at multiplicities of 0.1 to 50 plaque forming units (PFU) per cell the presumptive mutation frequency, as determined by the number of 6-thioguanine-resistant (TGr) colonies, was increased up to 16.8-fold (P< 0.005), depending on the multiplicity of infection. Increases in the mutation frequency at thehprtlocus were also observed for other laboratory-adapted HCMV strains (C-87, Davis) and for low passage clinical isolates (82-1, 84-2). The expression time required for the maximum increase in TGrcolonies was 3 days and was consistent among the HCMV strains evaluated in this study. UV-irradiation of HCMV stock up to a dose of 9.6 × 104ergs/mm2increased the mutation frequency, but further exposure to UV light or to heat (56° for 30 min) significantly decreased the frequency of TGr-resistant colonies, suggesting that expression of HCMV genes was involved in the mutation process. HCMV-induced TGrcells demonstrated substantially reduced (>96%) incorporation of [3H]hypoxanthine. PCR analysis of thehprtlocus demonstrated deletions in 9 of 19 HCMV-induced TGrcolonies randomly selected for further study, while 2 of 17 spontaneously developed TGrcolonies demonstrated deletions. Although insertions were not detected in spontaneously developed clones, 3 of 19 HCMV-induced TGrclones had insertions in thehprtgene. Neither HCMV-specific DNA sequences nor HCMV-specific proteins were detected in the TGrclones obtained after HCMV infection. Infection of V79 cells with HCMV also increased their sensitivity to mutation withN-methyl-N′-nitro-N-nitrosoguanidine, giving a synergistic enhancement of the mutation frequency. These results indicate that HCMV infection has the capacity to induce mutations in the cellular genome and increase the sensitivity of infected cells to mutation by genotoxic chemicals. Although inactivated HCMV particles are responsible for a modest increase in the mutation frequency, expression of HCMV genes is associated with a substantial enhancement of the mutation frequency
Characterization of Cucumber Mosaic Virus

AbstractCucumber mosaic virus (CMV), generated from biologically active cDNA clones of Fny-CMV RNA 1 plus 2 and Sny-CMV RNA 3, derived from the Fny-and Sny-strains of CMV, was able to infect tobacco but not squash plants systemically. In squash, viral RNA, movement protein, and coat protein all accumulated in the inoculated cotyledons. The lack of systemic infection was associated with a reduced rate of cell-to-cell movement within the cotyledons. The restricted movement mapped to two sequence changes in the codons of amino acids 51 and 240 of the Sny-CMV 3a gene. These same sequence changes previously were shown to be associated with high levels of 3a protein accumulation and chronic vs acute, cyclic infection typical of Sny-CMV vs Fny-CMV [Gal-Onet al.(1996).Virology226, 354–361]. Fny-CMV, mutated in the codons of 3a gene amino acids 51 and 240, was still able to infect several solanaceous hosts (tobacco, tomato, and pepper) systemically, but did not elicit a typical CMV systemic infection on any of several cucurbit hosts (cucumber, melon, or squash). The significance of the location of amino acid positions 51 and 240 in the 3a movement protein is discussed
Loss of the SIVsmmPBj14 Phenotype andnefGenotype during Long-Term Survival of Macaques Infected by Mucosal Routes

AbstractThe ability of the simian immunodeficiency virus SIVsmmPBj14 (SIV-PBj14) to activate and induce proliferation of quiescent peripheral blood lymphocytes from macaques is anin vitrocorrelate of its acutely lethalin vivophenotype. SIV-PBj14 differs from other SIV strains by encoding tyrosine at amino acid 17 (Y17) in Nef, which generates an activation motif important for signal transduction. Although intravenous inoculation of pig-tailed macaques with SIV-PBj14 uniformly leads to death within 2 weeks, inoculation by mucosal routes results in persistent infections that progress to AIDS. In the present study, we determined whether viruses in long-term survivors retained not only the Nef Y17 residue but also the biologic properties associated with rapid disease and death. Viruses reisolated at early and late times after mucosal infection of macaques with SIV-PBj14 were testedin vivofor acute lethality andin vitrofor the ability to replicate in and induce activation and proliferation of quiescent macaque lymphocytes. In addition, the coding sequence for the first 55 amino acids in Nef was amplified from proviral DNA or plasma virion RNA by PCR or RT-PCR, respectively, and nucleotide sequences were obtained. The results showed that the majority of the quasispecies that persisted as disease progressed not only lost biological properties unique to SIV-PBj14, but also lost through mutation either Y17 or Y28 in Nef, which together were part of the activation motif. In the case of Y17, these mutations were stepwise to histidine then arginine, the amino acid encoded in this position in other SIV strains. We conclude, therefore, that replicative properties of the acutely lethal virus provide no selective advantage during long-term infections with SIV-PBj14 and that disruption of the activation motif in Nef is associated with loss of the acutely lethal phenotype
The Epstein–Barr Virus (EBV) DNA Polymerase Accessory Protein, BMRF1, Activates the Essential Downstream Component of the EBV oriLyt

AbstractThe EBV DNA polymerase accessory protein, BMRF1, is an essential component of the viral DNA polymerase and is required for lytic EBV replication. In addition to its polymerase accessory protein function, we have recently reported that BMRF1 is a transcriptional activator, inducing expression of the essential oriLyt promoter, BHLF1. Here we have precisely mapped the BMRF1-response element in the BHLF1 promoter. We demonstrate that a region of oriLyt (the “downstream component”), previously shown to be one of two domains absolutely essential for oriLyt replication, is required for BMRF1-induced activation of the BHLF1 promoter. Furthermore, the downstream component of oriLyt is sufficient to confer BMRF1-responsiveness to a heterologous promoter. The downstream component contains Sp1 binding sites, and confers Sp1-responsiveness to a heterologous promoter. A series of plasmids containing various portions of the oriLyt downstream component were constructed and analyzed for their ability to respond to the BMRF1 versus Sp1 transactivators. Although the BMRF1-responsive region of the downstream component overlaps the Sp1-responsive element, certain oriLyt sequences required for maximal BMRF1-responsiveness were not required for maximal Sp1-responsiveness. In particular, a site-directed mutation altering the downstream component sequence GATGG (located from −588 to −592 relative to the BHLF1 transcription initiation site) did not affect Sp1-responsiveness, but reduced BMRF1-responsiveness by 75% and abolished oriLyt replication. Although BMRF1 possesses nonspecific DNA binding activity, we were unable to demonstrate specific BMRF1 binding to the downstream component of oriLyt. Our results suggest that BMRF1-induced activation of the essential downstream component of oriLyt may play an important role in oriLyt replication
Down-regulation of Groundnut Rosette Virus Replication by a Variant Satellite RNA

AbstractSymptom production in groundnut plants infected with groundnut rosette virus (GRV) depends on the presence of satellite RNA (sat-RNA) in the GRV culture, and sat-RNA variants that induce only mild symptoms are known. One such variant drastically diminished the replication of GRV genomic RNA in infectedNicotiana benthamianaplants. This down-regulating ability did not involve either of the two open reading frames in the sat-RNA but was controlled by a region near its 5′ end, which is required for sat-RNA replication. WhenN. benthamianaplants were inoculated with GRV and the mild satellite and challenged by inoculation with a GRV isolate (YB) containing a sat-RNA that induces yellow blotch symptoms, no symptoms appeared and little GRV genomic RNA or sat-RNA was detected in the plants, provided the two inoculations were no more than 2 days apart. A GRV isolate containing a sat-RNA that neither induces symptoms inN. benthamiananor affects genomic RNA accumulation also provided protection against yellow blotch symptom production if inoculated before or up to 2 days after isolate YB. However, in this case protection was incomplete and both GRV RNA and sat-RNA accumulated to normal levels. It is suggested that sequences from the mild sat-RNA may provide a novel source of resistance against rosette disease
Nontranslatability and Dissimilar Behavior in Plants and Protoplasts of Viral RNA and Movement Protein Complexes Formedin Vitro

AbstractIt was found that the fusion (His)6-movement proteins (MPs) of two tobamoviruses (TMV UI and a crucifer-infecting tobamovirus, crTMV) were efficient nonspecific translational repressors. Thein vitrotranslation of viral RNAs was blocked by incomplete 30K MP–RNA complexes formed at the MP:RNA molar ratios of 100–150:1. Similar results were obtained with the barley stripe mosaic hordeivirus (BSMV)-encoded 58K MP; however, the translation inhibiting activity of the 58K MP was manifested only in the presence of magnesium. By contrast, the 25K MP of potato virus X (PVX) was incapable of forming MP–RNA complexes under experimental conditions used and did not inhibitin vitrotranslation. The translation repressing ability correlated with the level of MP affinity to RNA. The complexes of the 30K MP and 58K MP with TMV RNA were not infectious in isolated protoplasts; however, they were infectious in indicator plants. Reduction of MP affinity to RNA resulted in translatability of MP–TMV RNA complexes that apparently was due to their destabilization. Thus, the deletion mutant DEL4 MP formed MP–TMV RNA complexes that were translatablein vitro,infectious to protoplasts and plants. In contrast to this, the complexes of TMV RNA with the mammalian RNA-binding protein p50 were nontranslatable and noninfectious to either protoplasts or intact plants. These results implied that nontranslatable MP–RNA complexes which could not replicate in the primary infected cells were converted into a translatable and replicatable form in the course of passage through plasmodesmatain planta
The Location and Sequence Composition of the Murine Cytomegalovirus Replicator (oriLyt)

AbstractDNA replication during human or simian cytomegalovirus (CMV) infection has been shown to be under control of a replicator region referred to asoriLyt. The murine CMVoriLyt has been mapped to a region of the genome located upstream of the gene encoding the herpesvirus-conserved single-stranded DNA binding protein, analogous to human and simian CMVoriLyts. A minimaloriLyt of approximately 1.7 kbp has been identified using a transient replication system. Like occurs with human and simian CMV counterparts, addition of flanking sequences to this minimal origin-stimulated replication efficiency. Analysis of the DNA sequence in this region shows that murine CMVoriLyt is complex and exhibits an asymmetric distribution of nucleotides as well as many repeat sequence elements, including distinct AT- and GC-rich regions and region with arrays of closely spaced direct repeats. Despite similarities in organization of all three CMVoriLyts, no sequence identity and only limited DNA sequence similarity was detectable. Consistent with this sequence divergence, the human and murine CMVoriLyts were unable to substitute for one another in transient replication assays
Characterization of Chimeric Turnip Yellow Mosaic Virus Genomes That Are Infectious in the Absence of Aminoacylation

AbstractPrevious experiments have characterized the chimeric genome TYMC-TMVPSK, in which the 3′-tRNA-like structure of turnip yellow mosaic virus (TYMV) was replaced by 3′ sequences from tobacco mosaic virus. This genome accumulated in turnip protoplasts to a level about 3% of wild type, but was not infectious on plants. In the present study, TYMV sequences introduced into the anticodon loop and amino acid acceptor arm of the 3′ region of this chimera led to three- to fourfold increases in viral accumulation. Two such modified chimeric genomes gave rise to stable infections in plants. After further passaging in plants and the accumulation of minor sequence changes in the 3′ terminal region, the resultant viruses, TYMC-XX and TYMC-YY, were highly infectious. Viral accumulations in protoplasts were about 40% of wild type on the basis of coat protein levels, and virion yields in plants were about 0.1 mg/g leaf. Extensive assays failed to detect aminoacylation of these genomic RNAsin vitro,but they were active substrates for wheat germ CCA nucleotidyltransferase. In separate experiments, the 3′-tRNA-like structure of TYMV RNA was replaced by the 3′ terminal 96 nucleotides from erysimum latent tymovirus RNA, resulting in a genome that was infectious to plants (isolate TYMC-H). This chimeric virus produced similar symptoms and virion yield in plants as TYMC-XX and -YY, although accumulations of coat protein in protoplasts were 13% of wild type. The viral RNA was a poor substrate for CCA nucleotidyltransferase and could not be aminoacylated. TYMC-XX, -YY, and -H are the first TYMV replicons known to amplify efficiently and infect plants in the absence of aminoacylation. Their viability suggests that other properties can compensate for the absence of aminoacylation
Effects of Mutations in the Beet Western Yellows Virus Readthrough Protein on Its Expression and Packaging and on Virus Accumulation, Symptoms, and Aphid Transmission

AbstractVirions of beet western yellows luteovirus contain a major capsid protein (P22.5) and a minor readthrough protein (P74), produced by translational readthrough of the major capsid protein sequence into the neighboring open reading frame, which encodes the readthrough domain (RTD). The RTD contains determinants required for efficient virus accumulation in agroinfected plants and for aphid transmission. The C-terminal halves of the RTD are not well conserved among luteoviruses but the N-terminal halves contain many conserved sequence motifs, including a proline-rich sequence separating the rest of the RTD from the sequence corresponding to the major coat protein. To map different biological functions to these regions, short in-frame deletions were introduced at different sites in the RTD and the mutant genomes were transmitted to protoplasts as transcripts and toNicotiana clevelandiiby agroinfection. Deletions in the nonconserved portion of the RTD did not block aphid transmission but had a moderate inhibitory effect on virus accumulation in plants and abolished symptoms. Deletion of the proline tract and the junction between the conserved and nonconserved regions inhibited readthrough protein accumulation in protoplasts by at least 10-fold. The mutants accumulated small amounts of virus in plants, did not induce symptoms, and were nontransmissible by aphids using agroinfected plants, extracts of infected protoplasts, or purified virus as a source of inoculum. Other deletions in the conserved portion of the RTD did not markedly diminish readthrough protein accumulation but abolished its incorporation into virions. These mutants accumulated to low levels in agroinfected plants and elicited symptoms, but could not be aphid-transmitted. A preliminary map has been produced mapping these functions to different parts of the RTD
Structural and Antigenic Identification of the ORF12 Protein (αTIF) of Equine Herpesvirus 1

AbstractThe equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, αTIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDain vitrotranscription/translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase–ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 αTIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif
Screening of HIV-1 Env Glycoproteins for the Ability to Raise Neutralizing Antibody Using DNA Immunization and Recombinant Vaccinia Virus Boosting

AbstractHIV-1 envelopes from two series of primary isolates (from Swedish patients 5 and 6), from JR-FL and BaL (prototypic monocyte/macrophage tropic viruses) and from HXB-2 (a prototypic T-cell-line-adapted virus), have been screened for their ability to elicit neutralizing antibody to HIV-1. Rabbits were primed by gene gun inoculation with plasmids expressing secreted monomeric (gp120) and oligomeric (gp140) forms of each Env. After four to six DNA immunizations administered over a 1-year period, rabbits were boosted with 108plaque-forming units of a mixture of seven recombinant vaccinia viruses which express chimeric gp140 Envs (primary clade B sequences in a IIIb-related BH10 backbone). Neutralizing antibodies were assayed against two T-cell-line-adapted viruses (MN and IIIb), two non-syncytium-inducing (NSI) and two syncytium-inducing (SI) primary isolates, and two HIV-1-NL4-3-recombinants with patient 5 or 6 Envs (NL4-3/5A, NL4-3/6C). The DNA priming and recombinant vaccinia virus boosting raised low titers of neutralizing antibody in 10 of 19 rabbits. The highest titers of neutralizing activity (∼1:150 for MN) were raised in rabbits DNA primed with Envs from Swedish patient 5. These sera cross-neutralized IIIb and MN but did not neutralize the primary isolates or the NL4-3 recombinant with the homologous 5A Env. Sera from rabbits primed with the HXB-2 Env DNA were, for the most part, type-specific for neutralization of IIIb. In one of three assays, sera from rabbits primed with plasmids expressing the JR-FL and BaL Envs had possible low titer neutralizing activity for two NSI, but not two SI, primary isolates. Our results highlight the low immunogenic potential of the HIV-1 Env and demonstrate that different Envs have different potentials to raise low titer neutralizing antibody