pinecone/core-2020-05-10-deduplication huggingface dataset:
Two Domains within the Adenovirus Type 12 E1A UniqueSpacerHave Disparate Effects on the Interaction of E1A with P105-Rb and the Transformation of Primary Mouse Cells

AbstractTransformation of primary rodent cells by functions of the adenovirus type 12 (Ad12) early region 1 (E1) is reduced severalfold compared with transformation by E1 of Ad2. We analyzed whether the uniquespacerregion of Ad12 E1A that borders the conserved region (CR) 2 and represents an oncogenic determinant of Ad12 E1A is involved in this impaired transformation property, putatively by modulating transformation-relevant biological E1A functions. We show that a mutant (E1ASpm1) that lacks 12 amino-terminal residues of thespacerbinds p105-Rb and p130 as Ad12 E1A wild type (E1Awt), whereas a secondspacermutant (E1ASpm2) that lacks an adjacent stretch of six alanines exhibits highly reduced binding to p105-Rb. The binding of this mutant to the p130 pocket protein is, however, little impaired. E1ASpm1diminishes the formation of the p105-Rb-E2F complex more efficiently than E1Awtor, least efficient, E1ASpm2. These properties of thespacermutants to target and to disintegrate the p105-Rb-E2F complex correspond with their ability to transform primary mouse cells in combination with E1B: E1ASpm1(plus Ad12 E1B)-transfected cells could be easily established as cell lines, comparable to Ad12 E1Awt- or Ad2 E1Awt-transfected cells. In contrast, cells transfected with E1ASpm2or Ad12 E1AdelCR2(lacking the entire CR2) died within 6–10 weeks after replating, although foci were formed in all cases. Of note, the E1ASpm1-transformed cells grow as fast as the Ad2 E1Awt-transformed cells, with a doubling rate of 15 h, whereas the doubling of the Ad12 E1Awt-transformed cells takes ∼120 h. Moreover, in the established cell lines, the affinity of E1ASpm1to p105-Rb was higher than with that of E1Awt.Our data suggest the presence of a transformation-suppressing domain within the carboxyl-terminal 12 residues of the Ad12 E1A-uniquespacer, whereas the hydrophobic stretch of six alanines in thespaceris required for stable transformation
Long-Lasting Protection by Live Attenuated Simian Immunodeficiency Virus in Cynomolgus Monkeys: No Detection of Reactivation after Stimulation with a Recall Antigen

AbstractThe infection of cynomolgus monkeys with an attenuated simian immunodeficiency virus (SIV) (C8) carrying a deletion in the nef gene results in a persistent infection associated with an extremely low viral burden in peripheral blood mononuclear cells. The aim of this study was to determine (1) the breadth of the protection after repeated challenges of monkeys with SIV homologous strains of different pathogenicity, (2) the genotypic stability of the live virus vaccine, (3) whether the protection might depend on cellular resistance to superinfection, and (4) whether immunogenic stimuli such as recall antigens could reactivate the replication of the C8 virus. To address these goals, the monkeys were challenged at 40 weeks after C8 infection with 50 MID50 of cloned SIVmac251, BK28 grown on macaque cells. They were protected as indicated by several criteria, including virus isolation, anamnestic serological responses, and viral diagnostic PCR. At 92 weeks after the first challenge, unfractionated peripheral blood mononuclear cells from protected monkeys were susceptible to the in vitro infection with SIVmac32H, spl. At 143 weeks after C8 infection, the four protected monkeys were rechallenged with 50 MID50 of the pathogenic SIVmac32H, spl grown on macaque cells. Once again, they were protected. The C8 virus remained genotypically stable, and depletion of CD4+ cells was not observed during ∼3 years of follow-up. In contrast, it was found that the infection with SIVmac32H, spl induced CD4+ cell depletion in three of three control monkeys. Of importance, stimulation with tetanus toxoid, although capable of inducing specific humoral and T cell proliferative responses, failed to induce a detectable reactivation of C8 virus
Immunogenicity of Herpes Simplex Virus Type 1 Mutants Containing Deletions in One or More α-Genes: ICP4, ICP27, ICP22, and ICP0

AbstractReplication defective mutants of HSV have been proposed both as vaccine candidates and as vehicles for gene therapy because of their inability to produce infectious progeny. The immunogenicity of these HSV replication mutants, at both qualitative and quantitative levels, will directly determine their effectiveness for either of these applications. We have previously reported (Brehm et al., J. Virol., 71, 3534, 1997) that a replication defective mutant of HSV-1, which expresses a substantial level of viral genes without producing virus particles, is as efficient as wild-type HSV-1 in eliciting an HSV-specific cytotoxic T-lymphocyte (CTL) response. In this report, we have further evaluated the immunogenic potential of HSV-1-derived replication defective mutants by examining the generation of HSV-specific CTL following immunization with viruses that are severely restricted in viral gene expression due to mutations in one or more HSV α genes (ICP4, ICP27, ICP22, and ICP0). To measure the CTL responses induced by the HSV α-mutants, we have targeted two H-2Kb-restricted CTL epitopes: an epitope in a virion protein, gB (498–505), and an epitope in a nonvirion protein, ribonucleotide reductase (RR1 822–829). The HSV mutants used in this study are impaired in their ability to express gB while a majority of them still express RR1. Our findings demonstrate that a single immunization with these mutants is able to generate a strong CTL response not only to RR1 822–829, but also to gB498–505 despite their inability to express wild-type levels of gB. Furthermore, a single immunization with any individual mutant can also provide immune protection against HSV challenge. These results suggest that mutants which are restricted in gene expression may be used as effective immunogens in vivo
Salmon Pancreas Disease Virus, an Alphavirus Infecting Farmed Atlantic Salmon, Salmo salar L.

AbstractA 5.2-kb region at the 3′ terminus of the salmon pancreas disease virus (SPDV) RNA genome has been cloned and sequenced. The nucleotide and predicted amino acid sequences show that SPDV shares considerable organizational and sequence identity to members of the genus alphavirus within the family Togaviridae. The SPDV structural proteins encoded by the 5.2-kb region contain a number of unique features when compared to other sequenced alphaviruses. Based on cleavage site homologies, the predicted sizes of the SPDV envelope glycoproteins E2 (438 aa) and E1 (461 aa) are larger than those of other alphaviruses, while the predicted size of the alphavirus 6K protein is 3.2 K (32 aa) in SPDV. The E2 and E1 proteins each carry one putative N-linked glycosylation site, with the site in E1 being found at a unique position. From amino acid sequence comparisons of the SPDV structural region with sequenced alphaviruses overall homology is uniform, ranging from 32 to 33%. While nucleotide sequence analysis of the 26S RNA junction region shows that SPDV is similar to other alphaviruses, analysis of the 3′-nontranslated region reveals that SPDV shows divergence in this region
Analysis of Human Lymphotropic T-Cell Virus Type II-like Particle Production by Recombinant Baculovirus-Infected Insect Cells

AbstractThe molecular processes involved in retrovirus assembly and budding formation remain poorly understood. The gag-pro-pol genes of human lymphotropic T-cell virus type II (HTLV-II) are translated into Gag, Gag-Pro, or Gag-Pro-Pol by frameshift events. In the present study, we investigated the roles of the gag, pro, and pol regions of HTLV-II in viral particle formation using recombinant baculoviruses. In this study we could successfully produce mature HTLV-II viral particles containing core structures using a construct expressing the entire gag-pro-pol region. We also investigated the role of the pol region in particle formation. Deletion of the pol region affects viral particle assembly or release very little, indicating that the gag-pro region is sufficient for viral particle formation and maturation. Expression of the Gag proteins alone or Gag proteins with inactivated viral proteases (Pro) resulted in the formation of viral particles; however, these particles did not contain core structures. These results suggest the intracellular expression of Gag with Pro of HTLV-II is essential for the production of mature virus particles, whereas that of Pol is not
Phosphorylation of Nonstructural 5A Protein of Hepatitis C Virus: HCV Group-Specific Hyperphosphorylation

AbstractWe previously showed that two proteins with molecular weights of 56 and 58 kDa are produced from nonstructural protein 5A (NS5A) derived from hepatitis C virus (HCV)-1b genotype. The 56-kDa protein is phosphorylated at serine residues in NS5A, including those located in the C-terminal region of NS5A, while the 58-kDa protein, the hyperphosphorylated form of the 56-kDa protein, is phosphorylated at serine residues in the central region. This hyperphosphorylation is dependent on the presence of HCV NS4A protein. To clarify whether NS4A-dependent phosphorylation also occurs in other HCV genotypes, phosphorylation of NS5A was analyzed by two-dimensional gel electrophoresis. Here, we report that NS5A from the HCV-2a genotype was phosphorylated. However, hyperphosphorylation of NS5A occurs in the HCV-1b genotype but not in the -2a genotype. This result suggests that modification of NS5A phosphorylation reflects the virological features of HCV and that there are physiological differences in the roles of differently phosphorylated NS5A between HCV genotypes
High-Frequency Homologous Genetic Recombination of an Arterivirus, Lactate Dehydrogenase-Elevating Virus, in Mice and Evolution of Neuropathogenic Variants

AbstractOn the basis of genome nucleotide differences between a nonneuropathogenic and a neuropathogenic lactate dehydrogenase-elevating virus (LDV) quasispecies (LDV-P and LDV-C, respectively), we have designed sets of primers for polymerase chain reaction (PCR) amplification that can detect recombinants between them in a 1276-nt-long segment ranging from ORF 5 to ORF 7. Mice were infected with large amounts of both LDVs and bled at various times postinfection (p.i.). RNA was extracted from plasma samples and reverse transcribed and the first-strand products were PCR amplified with four sets of sense and antisense primers that discriminate between parental (P/P and C/C) and recombinant (P/C and C/P) genomic segments. Both P/C and C/P recombinants were detected in plasma from six different mice at 1 day p.i. No recombinant products were generated within vitromixtures of LDV-P and LDV-C. End-point dilution experiments indicated that the generation of P/C and C/P recombinants varied between mice but that in some mice the frequency of recombination in the 1276-nt-long genome segment was as high as 5%. Sequence analyses of clones of some recombinants indicated that recombination had occurred at 26- to 43-nt-long stretches of homology between the LDV-P and the LDV-C genomes. Sequence analyses of the 3157-nt-long 3′ end of the genomes of the neuropathogenic LDV-v and of a newly discovered nonneuropathogenic quasispecies, LDV-vx, showed that LDV-v is a natural recombinant of LDV-vx that has specifically acquired by a double recombination about 400 nt of the 5′ end of ORF 5 of the neuropathogenic LDV-C and thereby the unique properties of LDV-C, neuropathogenicity and high sensitivity to antibody neutralization. In dual infections of mice with LDV-P and LDV-C all genetic recombinants, like the LDV-C parent itself, had been lost by 7 days p.i., and only LDV-P persisted. The results further support the view that LDV-P and LDV-vx have evolved to a highly stable relationship with their host, the mouse
The Isolation of the Ectodomain of the Alphavirus E1 Protein as a Soluble Hemagglutinin and Its Crystallization

AbstractAlphaviruses are isometric enveloped viruses approximately 70 nm in diameter. The viral surface contains 80 glycoprotein spikes arranged in aT= 4 lattice. Each of these spikes consists of three heterodimers of the viral membrane proteins E1 (approximately 49 kDa) and E2 (approximately 51 kDa). Cryoelectron microscopic analyses have shown that the spikes form a protein shell on the viral surface. We have made an attempt to isolate biologically active protein fragments from this surface and to grow crystals from such fragments. To this end membrane proteins were extracted with Nonidet-P40 from the Semliki Forest alphavirus and the proteins were separated from detergent by centrifugation. A protein complex containing the E1 and E2 molecules in quantitative yield was obtained by this procedure. This complex has the following properties: It sediments at approximately 30S, it chromatographs with an apparent molecular mass of approximately 580,000 Da during gel filtration, it cannot be dissociated by either nonionic detergents or 6 M urea, and at acid pH it is a highly active hemagglutinin. The data indicate that this 30S hemagglutinin complex, which has not been hitherto described for alphaviruses, may represent a variant form of the protein lattice present on the alphavirus surface. Cleavage of this complex by subtilisin selectively removes carboxy-terminal sequences from the E1 and E2 proteins, which contain the cytoplasmic and transmembrane segments of the proteins and a small part of their ectodomain. The remaining ectodomains are called E1ΔS and E2ΔS. This proteolysis also leads to dissociation of the 30S complex. The cleavage products accumulate in the form of a heterodimer of the E1ΔS and E2ΔS proteins. Treatment of the heterodimer with PNGase F leads to rapid removal of carbohydrate from the E2ΔS protein and a dissociation of the complex into the constituent molecules, which can be separated by chromatography. The finding that the heterodimer and the purified E1ΔS protein both function as hemagglutinin at acid pH indicates that the E1 protein represents the alphavirus hemagglutinin. We have obtained crystals of the E1ΔS protein and are currently in the process of determining the atomic structure of this protein by the isomorphous replacement method
Role of Interferon and Interferon Regulatory Factors in Early Protection against Venezuelan Equine Encephalitis Virus Infection

AbstractTo investigate the role of type I interferon (IFN) and its regulatory transacting proteins, interferon regulatory factors (IRF-1 and IRF-2), in early protection against infection with virulent Venezuelan equine encephalitis virus (VEE), we utilized mice with targeted mutations in the IFN-α/β receptor, IRF-1, or IRF-2 genes. IFN-α/β-receptor knockout mice are highly susceptible to peripheral infection with virulent or attenuated VEE, resulting in their death within 24 and 48 h, respectively. Treatment of normal macrophages with anti-IFN-α/β antibody prior to and during infection with molecularly cloned virulent VEE resulted in increased VEE replication. However, treatment with high doses of IFN or IFN-inducing agents failed to alter percentage mortality or average survival times in mice challenged with a low dose of virulent VEE. In IRF-1 and IRF-2 knockout mice (IRF-1−/−and IRF-2−/−), the 100% protection against virulent VEE that is conferred by attenuated VEE within 24 h in control C57BL/6 mice was completely absent in IRF-2−/−mice, whereas 50% of IRF-1−/−mice were protected. IRF-2−/−mice were deficient in clearing VEE virus from the spleen and the brain compared to the heterozygous IRF-2+/−knockout or C57BL/6 (+/+) mice. Furthermore, a distinct pattern of histopathological changes was observed in brains of IRF-2−/−mice after VEE exposure. Taken together, these findings imply that the altered immune response in IRF-1 and IRF-2 knockout mice results in altered virus dissemination, altered virus clearance, and altered virus-induced pathology. Thus, type I interferon, as well as IRF-1 and IRF-2, appears to play an important and necessary role in the pathogenesis of, and protection against, VEE infection
Adenovirus E4 Open Reading Frame 4-Induced Dephosphorylation Inhibits E1A Activation of the E2 Promoter and E2F-1-Mediated Transactivation Independently of the Retinoblastoma Tumor Suppressor Protein

AbstractPrevious studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression during virus growth. E4-ORF4 has previously been shown to bind to and activate the cellular protein phosphatase 2A. The inhibitory effect of E4-ORF4 was relieved by okadaic acid, which inhibits protein phosphatase 2A activity, suggesting that E4-ORF4 represses E2 transcription by inducing transcription factor dephosphorylation. Interestingly, E4-ORF4 did not inhibit the transactivation capacity of a Gal4-E2F hybrid protein. Instead, E4-ORF4 expression appears to result in reduced stability of E2F/DNA complexes
A 110-kDa Nuclear Shuttle Protein, Nucleolin, Specifically Binds to Adeno-Associated Virus Type 2 (AAV-2) Capsid

AbstractA 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed thein vitrobinding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin bothin vitroandin vivosuggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly
DNA Replication Specificity and Functional E2 Interaction of the E1 Proteins of Human Papillomavirus Types 1a and 18 Are Determined by Their Carboxyl-Terminal Halves

AbstractReplication of most papillomaviruses (PVs) requires the viral-encoded E1 and E2 proteins that bind to the origin of replication (ori) containing the E1- and E2-binding sites and help recruit host replication factors during the initiation of DNA replication. We studied the ability of heterologous E1 and E2 proteins to interact in vivo and support replication, using the human papillomavirus (HPV) types 1a and 18 as model systems. The E1 protein of HPV-1a in combination with HPV-18 E2 supported high-level replication of various ori plasmids. In contrast, the HPV-18 E1 protein interacted weakly with HPV-1a E2 during the replication of ori plasmids. We have previously shown that the E1 protein of HPV-1a alone is sufficient for replication of HPV-1a ori plasmids, whereas HPV-18 replication requires both the E1 and E2 proteins. However, in the latter case, E2-binding sites alone in the absence of the E1-binding site can function as the minimal ori. Based on the above observations, we generated hybrids between HPV-1a and HPV-18 E1 proteins in an effort to identify their “replication specificity” domains using a transient replication assay. These hybrids were also used to localize the domains in the E1 proteins that are involved in their functional interaction with the E2 protein during replication. Our results suggest that the “replication specificity” and functional E2 interaction domains of the HPV-1a and HPV-18 E1 proteins are located in their carboxyl-terminal halves
Studies on Antibody Responses Following Neonatal Immunization with Influenza Hemagglutinin DNA or Protein

AbstractNeonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 response following immunization with many vaccine antigens. These studies were undertaken to determine whether 1-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (H1) by either intramuscular (im) or gene gun (gg) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. The newborn mice developed stable, long-lived, protective anti-H1-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult im and gg DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1, IgG2a, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased but did not change these antibody profiles. In contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age
Engineering of Noninfectious HIV-1-like Particles Containing Mutant gp41 Glycoproteins as Vaccine Candidates That Allow Vaccinees to Be Distinguished from HIV-1 Infectees

AbstractMany AIDS vaccine candidates under development may elicit immune responses similar to those observed in and used to screen human immunodeficiency virus type 1 (HIV-1)-infected individuals. Therefore, it is important to develop vaccine candidates that incorporate antigenic markers and allow vaccinees to be distinguished from HIV-1 infectees. To this end, we introduced a series of mutations into and in the vicinity of the major immunodominant region (MIR) of gp41 (residues 598–609), a domain recognized by almost all HIV-1 infectees, and evaluated whether HIV-1-like particles incorporating such mutant glycoproteins could be expressed in mammalian cells. Results indicated that although up to three consecutive amino acids could be replaced within MIR without significantly affecting particle formation or gp160 processing, deletions within MIR impaired envelope processing. Replacement of HIV-1 MIR by part or most of the corresponding domain from other lentiviruses markedly decreased or abolished gp160 processing. Synthetic peptides corresponding to a mutated MIR incorporating three amino acid replacements were not recognized by a panel of sera from HIV-1 infectees, suggesting that HIV-1-like particles with this type of mutation represent potential candidate vaccines that could allow vaccinees to be distinguished from HIV-1 infectees