pinecone/core-2020-05-10-deduplication huggingface dataset:
Plasmid DNA Vaccines Are Effective in the Absence of IFNγ

AbstractIntramuscular injection of bacterially derived plasmid DNA results in the development of both humoral and cellular immune responses against plasmid-encoded antigens. Immunostimulatory CpG sequences within bacterial DNA are thought to enhance this process by stimulating the secretion of proinflammatory cytokines such as interferon γ (IFNγ) by cells of the innate immune system. Although IFNγ induction by CpG elements within plasmid DNA has been documented in vitro and more recently in vivo, and coimmunization with plasmids expressing IFNγ has been shown to enhance DNA-immunization-induced immune responses, it is unclear if IFNγ is necessary for successful DNA immunization. To address this issue, we compared humoral and cellular immune responses in wild-type and IFNγ-deficient mice vaccinated with a plasmid (pCMVNP) expressing the nucleoprotein gene from the arenavirus lymphocytic choriomeningitis virus (LCMV). IFNγ-positive (BALB/c) and IFNγ-negative (GKO) mice responded to DNA vaccination by the development of antigen-specific CD8+ T cells, which were detectable directly ex vivo by intracellular cytokine staining and comprised 0.7–2.5% of all CD8+ T cells in the vaccinee. DNA vaccines also induced virus-specific cytotoxic T lymphocytes (CTL), even in the absence of IFNγ. DNA vaccination of both mouse strains also was associated with a significant reduction in viral titers after LCMV challenge, indicating that, at least in the presence of other immune effector mechanisms, IFNγ is not required for induction of protective anti-viral immunity by DNA immunization. No quantitative differences were observed in antiviral IgG levels among GKO and BALB/c vaccinees, although GKO mice did exhibit a significant reduction of the IgG2a:IgG1 ratio, in agreement with the previously documented requirement for IFNγ in isotype switching to IgG2a. Immunized BALB/c mice produced similar levels of both IgG1 and IgG2a, indicating a mixed Th1/Th2 response to intramuscular immunization with pCMVNP. These results show that IFNγ induction by bacterially derived plasmid DNA does not contribute to the magnitude of the antibody response and is not required for the induction or short-term maintenance of DNA-induced CTL. However, IFNγ is necessary for the development of IgG2a antibodies that may be crucial for protection against some pathogens
Characterization of Two Chitinase Genes and One Chitosanase Gene Encoded by Chlorella Virus PBCV-1

AbstractChlorella virus PBCV-1 encodes two putative chitinase genes, a181/182r and a260r, and one chitosanase gene, a292l. The three genes were cloned and expressed in Escherichia coli. The recombinant A181/182R protein has endochitinase activity, recombinant A260R has both endochitinase and exochitinase activities, and recombinant A292L has chitosanase activity. Transcription of a181/182r, a260r, and a292l genes begins at 30, 60, and 60 min p.i., respectively; transcription of all three genes continues until the cells lyse. A181/182R, A260R, and A292L proteins are first detected by Western blots at 60, 90, and 120 min p.i., respectively. Therefore, a181/182r is an early gene and a260r and a292l are late genes. All three genes are widespread in chlorella viruses. Phylogenetic analyses indicate that the ancestral condition of the a181/182r gene arose from the most recent common ancestor of a gene found in tobacco, whereas the genealogical position of the a260r gene could not be unambiguously resolved
Similarly Organized Lysogeny Modules in Temperate Siphoviridae from Low GC Content Gram-Positive Bacteria

AbstractTemperate Siphoviridae from an evolutionarily related branch of low GC content gram-positive bacteria share a common genetic organization of lysogeny-related genes and the predicted proteins are linked by many sequence similarities. Their compact lysogeny modules [integrase/1–2 orfs (phage exclusion? and metalloproteinase motif proteins)/cI-like repressor/cro-like repressor/antirepressor (optional)] differ clearly from that of λ-like and L5-like viruses, the two currently established genera of temperate Siphoviridae, while they resemble those of the P2-like genus of Myoviridae. In all known temperate Siphoviridae from low GC content gram-positive bacteria the lysogeny module is flanked by the lysis module and the DNA replication module. This modular organization is again distinct from that of the known genera of temperate Siphoviridae. On the basis of comparative sequence analysis we propose a new genus of Siphoviridae: “Sfi21-like” phages. With a larger database of phage sequences it might be possible to establish a genomics-based phage taxonomy and to retrace the evolutionary history of selected phage modules or individual phage genes. The antirepressor of Sfi21-like phages has an unusual widespread distribution since proteins with high aa similarity (40%) were found not only in phages from gram-negative bacteria, but also in insect viruses
Double-Layered Membrane Vesicles Released from Mammalian Cells Infected with Sendai Virus Expressing the Matrix Protein of Vesicular Stomatitis Virus

AbstractThe matrix (M) protein of vesicular stomatitis virus (VSV) was reported to form vesicles on the cell surface and subsequently to be released into the cultured medium when expressed from cDNA by virus vectors. To further investigate VSV M activity, we generated a recombinant Sendai virus (SeV) expressing the VSV M protein (SeV-MVSV). When cells were infected with SeV-MVSV, VSV M was found abundantly in the culture medium. Electron microscopy demonstrated the budding of two-membraned vesicles (≥0.8 μm in diameter) from the infected cells. The outer membrane of the vesicle was derived from the plasma membrane and the inner one possibly derived from the membrane of an intracellular vesicle. Immuno-gold labeling showed that VSV M was exclusively located in a double-layered region. The released membranes were divided into three parts: the VSV M vesicles with SeV F and HN glycoproteins, SeV particles, and vesicles associated with the cytosolic components. The last abundantly contained phosphorylated SeV matrix (M) protein, which is not released in a usual SeV infection. Furthermore the VSV M protein expressed without using a virus vector was efficiently released into the culture medium. These results suggest that the VSV M protein has a budding activity per se and that SeV proteins are passively involved in the release of VSV M
Comparison of the Protective Efficacy of Naked DNA, DNA-based Sindbis Replicon, and Packaged Sindbis Replicon Vectors Expressing Hantavirus Structural Genes in Hamsters

AbstractSeoul virus (SEOV) is a member of the Hantavirus genus (family Bunyaviridae) and an etiological agent of hemorrhagic fever with renal syndrome. The medium (M) and small (S) gene segments of SEOV encode the viral envelope glycoproteins and nucleocapsid protein, respectively. We compared the immunogenicity and protective efficacy of naked DNA (pWRG7077), DNA-based Sindbis replicon (pSIN2.5), and packaged Sindbis replicon vectors (pSINrep5), containing either the M or S gene segment of SEOV in Syrian hamsters. All of the vectors elicited an anti-SEOV immune response to the expressed SEOV gene products. Vaccinated hamsters were challenged with SEOV and monitored for evidence of infection. Protection from infection was strongly associated with M-gene vaccination. A small number of S-gene-vaccinated animals also were protected. Hamsters vaccinated with the pWRG7077 vector expressing the M gene demonstrated the most consistent protection from SEOV infection and also were protected from heterologous hantavirus (Hantaan virus) infection
Gene Expression from the ORF50/K8 Region of Kaposi's Sarcoma-Associated Herpesvirus

AbstractThe ORF50 gene of Kaposi's sarcoma (KS)-associated herpesvirus, or human herpesvirus 8 (KSHV), activates viral replication and is weakly homologous to the herpesvirus family of R transactivators; therefore, the transcription and translation events from this region of KSHV are key events in viral reactivation. We demonstrate that ORF50 is expressed in a bicistronic message after induction of the viral lytic cycle. ORF50 migrated as a series of polypeptides: the major ones as 119 and 101 kDa, respectively. Using 3′ rapid amplification of cDNA ends, RT-PCR, and cDNA library screening, we demonstrate that the major ORF50 transcript also encodes K8. The ORF50/K8 transcript was resistant to cyclohexamide, whereas the K8 transcript was only partially resistant to cyclohexamide at early timepoints. Both transcripts showed partial resistance after 12 h of phorbol ester induction. Using a GAL4-ORF50 fusion protein expression vector, we demonstrate that the transactivation domain of ORF50 resides within a 160-amino-acid region of the carboxyl portion of the ORF. Upstream regions of both ORF50 and K8 have basal promoter activity in KSHV-infected cells. K8, which had sequence homology to Bzip proteins, did not activate either promoter. However, both promoters were activated after cotransfection of ORF50 in BCBL-1 cells
Tyrosines 60, 64, and 101 of Epstein–Barr Virus LMP2A Are Not Essential for Blocking B Cell Signal Transduction

AbstractEpstein–Barr virus (EBV) latent membrane protein 2A (LMP2A) is expressed on the membrane of B-lymphocytes and blocks B cell receptor (BCR) signaling in EBV-transformed B-lymphocytes in vitro. The LMP2A amino-terminal domain, which is essential for the LMP2A-mediated block of B cell signal transduction, contains eight tyrosine residues. Three of these tyrosine residues (Y74, Y85, and Y112) have been demonstrated to be essential for the LMP2A-mediated block on protein tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression after BCR activation. To investigate the importance of tyrosines at positions 60, 64, and 101 on B cell signaling, EBV recombinants were constructed containing a tyrosine-to-phenylalanine point mutation at amino acid 60, 64, or 101 within LMP2A. Tyrosine phosphorylation, calcium mobilization, and induction of BZLF1 expression were not affected by any of the tyrosine point mutations after BCR activation. In addition, constitutive phosphorylation of LMP2A was unaffected by any of the tyrosine point mutations. These data indicate that tyrosines 60, 64, and 101 are not essential for the LMP2A-mediated block of B cell signal transduction in transformed cell lines
Requirement of Replication Licensing for the Dyad Symmetry Element-Dependent Replication of the Epstein–Barr Virus oriP Minichromosome

AbstractLatent Epstein–Barr virus genome is maintained in cells by the viral oriP-binding factor EBNA1 and cellular replication factors. EBNA1 binds to the dyad symmetry (DS) element in oriP and initiates DNA replication once in a single S phase, but the mechanism by which this DS-dependent replication is initiated is unknown. Replication licensing of cellular chromatins occurs during early G1 phase. Because licensing is essential for the next round of replication in S phase, it facilitates once-in-a-cell-cycle replication of the cellular genome. Using the transient replication assay with HeLa/EB1 cell, we demonstrate that the oriP plasmid required a cell cycle window including early G1 phase for replication in the next S phase. The plasmid containing only the DS element had a similar requirement of early G1 phase for replication. Analysis using sucrose density gradient centrifugation revealed that the oriP minichromosome existed in two distinct states: one formed at late G1 and the other formed at G2/M. These results suggest that the DS-dependent DNA replication from oriP requires the replication licensing, implying a possible involvement of the cellular licensing factor MCM in the DNA replication from oriP
Lack of Responsiveness of a Nuclear Factor-κB-Regulated Promoter to Transactivation by Human Immunodeficiency Virus 1 Tat in HeLa Cells

AbstractTranscriptional activation by Tat protein is in large part dependent on interactions with the TAR RNA element located in the 5′-untranslated region of all human immunodeficiency virus type 1 (HIV-1) transcripts. In addition, Tat has been shown to induce nuclear translocation of nuclear factor-κB (NF-κB), potentially contributing to gene induction. The NF-κB responsive reporter construct, (PRDII)4-CAT, was used to explore transcription resulting from NF-κB activated by Tat. Tat did not activate (PRDII)4-CAT, whereas (PRDII)4-CAT was highly responsive to either transfected Rel A or to tumor necrosis factor-α (TNF-α). Despite its inability to directly induce, Tat enhanced the responsiveness of (PRDII)4-CAT to either transfected Rel A or to TNF-α by ∼2.5-fold. High levels of CAT activity were seen with HIV-LTR-derived reporters that contained κB and TAR elements in response to transfected Tat in the absence of either transfected Rel A or exogenous TNF-α, and overexpression of IκBα with Tat inhibited CAT activity by 60% to 80%, suggesting that some activation of NF-κB by Tat was occurring. HIV-LTR reporter activities were enhanced three fold to sixfold compared with Tat alone when additional NF-κB was provided by transfection or by activation with TNF-α. These data indicate that Tat is unable to activate some NF-κB-responsive promoters but is able to synergize with NF-κB in the activation of both HIV-derived and non-HIV-derived promoters
Expression of Mouse Mammary Tumor Virus Envelope Protein Does Not Prevent Superinfection In Vivo or In Vitro

AbstractInbred mice expressing endogenous mouse mammary tumor virus envelope proteins can be infected with exogenous virus, and the mammary tumors that develop in these mice usually have many proviruses integrated in their genomes, indicating that this virus is not subject to receptor interference. We show here that transgenic mice expressing an exogenous mouse mammary tumor virus (C3H) envelope protein can still be infected with this virus. Moreover, cultured mammary gland cells expressing the mouse mammary tumor virus (C3H) envelope protein can be superinfected with pseudotyped viruses bearing that same protein. Thus cellular expression of the mouse mammary tumor virus envelope protein does not block superinfection in vivo or in vitro
Crystal Structure of an Inhibitor Complex of the 3C Proteinase from Hepatitis A Virus (HAV) and Implications for the Polyprotein Processing in HAV

AbstractThe proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the Sγ atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide were found to be monoclinic, space group P21, having 4 molecules in the asymmetric unit and diffracting to 1.9-Å resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-Å resolution and the corresponding Rfree is 29.8%. The dipeptide inhibitor is bound to the S1′ and S2′ specificity subsites of the proteinase. The crystal structure reveals that the HAV 3C proteinase possesses a well-defined S2′ specificity pocket and suggests that the P2′ residue could be an important determinant for the selection of the primary cleavage site during the polyprotein processing in HAV
Identification of a Protein That Recognizes a Distal Negative Regulatory Element within the Mouse Mammary Tumor Virus Long Terminal Repeat

AbstractThe mouse mammary tumor virus (MMTV) long terminal repeat contains a distal negative regulatory element (dNRE) that selectively represses activity of the proviral promoter in the absence of steroid hormone receptor-mediated activation. A protein, termed MMTV NRE-binding protein 1 (MNBP-1), that recognizes long terminal repeat sequences between −433 and −418 was identified by gel electrophoresis mobility shift assays and methylation interference footprinting in nuclear extracts of HeLa and Ltk− cells. Mutations within the defined binding site affect dNRE-mediated promoter repression in vivo. MNBP-1 has an apparent molecular mass of approximately 100 kDa as determined by gel filtration chromatography
An Amino Acid Change in the Exodomain of the E2 Protein of Sindbis Virus, Which Impairs the Release of Virus from Chicken Cells but Not From Mosquito Cells

AbstractIn order to obtain a mutant of Sindbis virus (SV) with a low methionine-resistant (LMR) phenotype, i.e., able to replicate in methionine-deprived Aedes albopictus mosquito cells, standard SV (SVSTD) was passaged 17 times in mosquito cells maintained in a low methionine medium and then plaque-purified, also in mosquito cells. Although the virus obtained by this procedure, SVLM17, did have the desired LMR phenotype, it also appeared to have acquired a host-range phenotype. We have now characterized the host-range phenotype of SVLM17 in greater detail. In yield assays, the titer of SVLM17 produced by chick embryo fibroblasts (CEF) was 100- to 1000-fold lower than that from mosquito cells. SVSTD, in contrast, produced a similar titer of virus from the two cell types. On the other hand, when SVLM17 was assayed directly by plaque formation on CEF and on mosquito cell monolayers, no host restriction in CEF was observed. When CEF were infected with SVLM17, viral proteins were synthesized normally, pE2 was processed to E2, and E2 was demonstrated by the fluorescent antibody method to reach the cell surface. However, electron microscopy of SVLM17-infected cells revealed an absence of extracellular virions and of budding particles; also, nucleocapsids were not aligned beneath the plasma membrane. By sequence determination and by site-directed mutagenesis, it was determined that the host restriction of SVLM17 was due to a change from Ala to Val at position 251 of the E2 protein. Substitution of Gly or Leu at this position also resulted in the same host range phenotype
Chlorella Virus PBCV-1 Encodes a Functional Homospermidine Synthase

AbstractSequence analysis of the 330-kb genome of chlorella virus Paramecium bursaria chlorella virus 1 (PBCV-1) revealed an open reading frame, A237R, that encodes a protein with 34% amino acid identity to homospermidine synthase from Rhodopseudomonas viridis. Expression of the a237r gene product in Escherichia coli established that the recombinant enzyme catalyzes the NAD+-dependent formation of homospermidine from two molecules of putrescine. The a237r gene is expressed late in PBCV-1 infection. Both uninfected and PBCV-1-infected chlorella, as well as PBCV-1 virions, contain homospermidine, along with the more common polyamines putrescine, spermidine, and cadaverine. The total number of polyamine molecules per virion (∼539) is too small to significantly neutralize the virus double-stranded DNA (>660,000 nucleotides). Consequently, the biological significance of the homospermidine synthase gene is unknown. However, the gene is widespread among the chlorella viruses. To our knowledge, this is the first report of a virus encoding an enzyme involved in polyamine biosynthesis
Recombination between Genomic RNAs of Two Cucumoviruses under Conditions of Minimal Selection Pressure

AbstractRecombination is considered to play a key role in RNA virus evolution; however, little is known about its occurrence under natural conditions. We inoculated tobacco plants with wild-type strains of two closely related cucumovirus species: cucumber mosaic virus (CMV) and tomato aspermy virus (TAV). RNA from the inoculated leaves of doubly-infected plants was tested for the presence of recombination events in an 0.8-kb central portion of the viral RNA3. Using a sensitive and specific RT-PCR procedure, we amplified recombinant segments of RNA3 in 3 of 82 tobacco plants infected with both viruses. In each plant in which recombinant segments were amplified, several different crossover sites were observed, all of which were located within a short stretch of high sequence similarity. Two plants had both CMV-TAV and TAV-CMV recombinants. In all cases, precise homologous recombination had occurred. To the best of our knowledge, this is the first report of interspecific recombination between wild-type plant RNA viruses under conditions of minimal selection pressure in favor of the recombinants
The FeLV-945 LTR Confers a Replicative Advantage Dependent on the Presence of a Tandem Triplication

AbstractFeline leukemia virus (FeLV), like other naturally occurring retroviruses, is characterized by a high degree of genetic diversity. FeLV-945 is a natural isolate derived from non-B-cell non-T-cell lymphomas classified anatomically as multicentric. FeLV-945 exhibits a unique structural motif in the LTR composed of a 21-bp tandem triplication downstream of a single copy of enhancer. The unique FeLV-945 LTR is precisely conserved among eight independent multicentric lymphomas collected in a geographic cluster. Previous studies using reporter gene constructs predict that the FeLV-945 LTR would confer a replicative advantage on the virus that contains it, particularly in primitive hematopoietic cells. Such an advantage may account for the precise conservation of the unique LTR sequence. To test that prediction, a set of recombinant, infectious FeLVs was developed that are isogenic other than the presence of the FeLV-945 LTR or mutations of it. Replication assays show that the FeLV-945 LTR confers a distinct growth advantage in K-562, FEA, and 3201 cells and implicate the 21-bp triplication in that function. Replacement of two copies of the triplicated element with random sequence greatly diminished the replicative capacity, thus implicating the triplicated sequence itself in LTR function. The 21-bp triplication was shown to contain specific nuclear protein binding sites, which may account for the selective pressure to conserve the sequence