pinecone/core-2020-05-10-deduplication huggingface dataset:
Reconstitution of a Functional Bovine Papillomavirus Type 1 Origin of Replication Reveals a Modular Tripartite Replicon with an Essential AT-Rich Element

AbstractA functional replication origin was reconstituted using oligonucleotide cassettes corresponding to three sequence subelements within the Bovine Papillomavirus Type 1 (BPV-1) replication origin: the 23-bp AT-rich region (ATR), the 18-bp binding site for the viral replication initiator protein E1 (E1BS), and a binding site for the viral transcriptional transactivator and replication enhancer protein E2 (E2BS). Replication of the reconstituted origin depended on heterologous expression of both the E1 and E2 proteins and on the presence of both the E1BS and E2BS, indicating that it is functionally analogous to the authentic BPV-1 origin. In addition, pairwise testing of subelement combinations revealed that the ATR was also essential and that a functional origin required at least one copy of all three subelements. While the E1BS and E2BS are sequence-specific elements, the function of the BPV-1 ATR could be at least partially substituted with heterologous AT-rich sequences, suggesting that the role of this element is primarily AT content-dependent rather than sequence-dependent. A stringent requirement for the ATR was also observed in the context of an authentic minimal origin sequence confirming that it is an intrinsic property of the BPV-1 origin and not simply an artifact of the reconstitution system. This study indicates that the minimal functional BPV-1 origin shares the tripartite modular organization characteristic of other simple eukaryotic replication origins. The reconstitution system described now provides a convenient approach to define the physical and functional interrelationships between the three subelements in a systematic fashion
Identification of a MMTV Insertion Mutation within the Coding Region of theFgf-3Protooncogene

AbstractTheFgf-3protooncogene (previously calledint-2) is a target of proviral insertion mutations in mammary tumors induced by the mouse mammary tumor virus (MMTV). These insertion mutations result in the transcriptional activation ofFgf-3,which is not normally expressed in the adult mammary gland. Previous mapping studies of numerousFgf-3insertion mutations have failed to reveal any provirus integrations within the gene coding region. This finding is consistent with the hypothesis that oncogenesis occurs in this system as a consequence of up-regulation ofFgf-3transcription, rather than from alterations of the gene product. During an analysis of a new cohort of tumors from the WXG-2 mouse strain, a breast tumor was identified which had a MMTV provirus integrated 24 bp upstream of theFgf-3stop codon. This insertion mutation generated a fusion transcript which was readily detectable in tumor RNA by RT-PCR. The predicted protein product of this fusion transcript is missing 8 aa of native sequence and contains an additional 8 aa of cryptic MMTV-encoded sequence. These data document the first exception to the generalization that theFgf-3coding region is not disrupted by MMTV insertion mutation
Frequency of Memory Cytotoxic T Lymphocytes to Equine Infectious Anemia Virus Proteins in Blood from Carrier Horses

AbstractHorses with equine infectious anemia virus (EIAV) have episodes of viremia and disease; however, most eventually become inapparent carriers. A possible mechanism of control is cytotoxic T lymphocytes (CTL). To evaluate CTL in inapparent carriers with low viral loads, peripheral blood mononuclear cells (PBMC) were stimulatedin vitrowith autologous EIAV-infected PBMC and human IL-2 to detect memory CTL (CTLm). In initial studies, three carriers had CTLm and one of these had low-level effector CTL (CTLe). The CTLm were restricted by equine lymphocyte alloantigen-A (ELA-A) locus encoded MHC class I molecules on autologous equine kidney (EK) target cells. In addition, EK cells did not express MHC class II molecules. The CTLm frequency in PBMC from five inapparent carriers infected for 22 to 50 months was determined by limiting dilution analysis. PBMC were diluted, stimulated, and tested on EK cell targets infected with EIAV and recombinant vaccinia viruses expressing EIAV Env or Gag/Pr proteins. All five carriers had CTLm to EIAV-infected targets, while four had CTLm to targets expressing Env and four had CTLm to targets expressing Gag/Pr proteins. The CTLm frequency range was 60 to 468 per 106PBMC to EIAV-infected targets, 4 to 286 to Env-expressing targets, and 25 to 190 to Gag/Pr-expressing targets. These results should facilitate the identification of epitopes recognized by predominant CTLm from horses controlling a lentivirus infection
Self-Assembly and Protein–Protein Interactions between the SV40 Capsid Proteins Produced in Insect Cells

AbstractSoluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and VP3, in Sf9 cells using baculovirus expression vectors. Coproduction of the capsid proteins VP1, VP2, and VP3 was achieved by infecting Sf9 cells with the three recombinant baculovirus species at equal multiplicities. All three proteins were found to be localized in the nuclear fraction. Electron microscopy of nuclear extracts of the infected cells showed an abundance of SV40-like capsid structures and heterogeneous aggregates of variable size, mostly 20–45 nm. Under the same staining conditions wild-type SV40 virions are 45 nm. The capsid-like particles sedimented in glycerol gradients similarly to authentic wild-type SV40 virions. Pentamers of the major capsid protein VP1 were also seen. Protein analysis on sucrose gradients demonstrated that the capsid-like particles can be disrupted by treatment with the reducing agent dithiothreitol and the calcium chelator EGTA. The capsid-like particles were found to be significantly less stable than SV40 virions and were partially stabilized by calcium ions. Understanding the complex interactions between the capsid proteins is important for the development of an efficientin vitropackaging system for SV40 virions and pseudovirions
Characterization of New Simian Foamy Viruses from African Nonhuman Primates

AbstractSimian foamy viruses (SFV) are exogenous retroviruses present in most if not all nonhuman primate species. Baboons and other African monkey species are known to harbor SFVs, yet there is presently no data in regard to their genetic relationship. Here we studied SFVs from baboons as compared to other SFVs isolated from a Hamlyn's guenon, a patas monkey, and a vervet. By Western blot analysis, thegagprecursor proteins (p74/p70) were detected from all SFVs. In addition, the envelope glycoproteins from a vervet isolate (SFV-Agm2) were comparable in size to theenvprecursor gp130, the exterior glycoprotein (gp70), and the transmembrane protein (gp48) as detected by lentil lectin binding and radioimmunoprecipitation (RIPA). Molecular comparison of PCR amplified products frompoland LTR regions of each SFV demonstrated a close relationship among baboon SFVs while SFVs from patas, Hamlyn's guenon, and vervet clustered together. The baboon viruses only varied by 4% among each other in the LTR region; however, as much as 26% variation was noted when compared to the other African monkey SFVs. To determine the prevalence rate of SFV-Bab in our baboon colony, we employed both Western blotting and PCR analysis. Antibodies to SFVgagprecursor proteins were seen in 7 of 10 infants; however, none were positive by PCR, suggesting that these infants were virus negative and that their antibodies were maternal in origin. Only one juvenile (1/10) and all adults (38/38) were infected with SFV. Taken together these results suggest that SFVs have arisen and diverged along with the evolution of their natural hosts. Furthermore, the high prevalence rates to SFV seen in adult baboons strongly suggest a sexual or oral routes of transmission
TNFα Cooperates with the Protein Kinase A Pathway to Synergistically Increase HIV-1 LTR Transcription via Downstream TRE-like cAMP Response Elements

AbstractActivating protein-1 (AP-1) binding TPA responsive elements (TRE) are located downstream of the transcription initiation site in the U5 region of the HIV-1 long terminal repeat (LTR). These downstream sequence elements, termed DSE, can bind both AP-1 and CREB/ATF transcription factors. Recently, we demonstrated that the DSE are also cAMP-responsive elements (CRE), since they mediated activation signals elicited by cholera toxin (Ctx), a potent activator of the cAMP-dependent protein kinase A (PKA) signal transduction pathway. In the present study, we demonstrate that the HIV-1 DSE can mediate the transcriptional synergy elicited by the combination of Ctx and TNFα. Ctx combined with TNFα or IL-1β to produce a synergistic increase in p24 antigen production in U1 promonocytic cells. Transfection studies of LTR reporter constructs indicated that mutation of the DSE sites abrogated the LTR-mediated synergy induced by Ctx and TNFα, whereas the synergy induced by Ctx and IL-1β was unaffected, suggesting TNFα and IL-1β cooperate differently with the cAMP/PKA activation pathway to induce HIV-1 expression in U1 cells. Because the DSE are also TRE sites, we assessed the effect of the agonist combinations on AP-1-dependent transcription. TNFα as well as IL-1β cooperated with Ctx to produce a synergistic activation of AP-1-mediated transcription. These data indicate that the TRE-like cAMP-responsive DSE sites within the 5′-untranslated leader can mediate the transcriptional cooperativity between TNFα and the cAMP/PKA pathway. Since the DSE and TRE sites cannot bind CREB/ATF homodimers, we propose a mechanism in which the HIV-1 DSE bind heterodimers composed of both AP-1 and CREB/ATF proteins
Membrane Fusion Activity of Semliki Forest Virus in a Liposomal Model System: Specific Inhibition by Zn2+Ions

AbstractSemliki Forest virus (SFV) has been shown previously to fuse efficiently with cholesterol- and sphingolipid-containing liposomal model membranes in a low-pH-dependent manner. Several steps can be distinguished in this process, including low-pH-induced irreversible binding of the virus to the liposomes, facilitated by target membrane cholesterol, and subsequent fusion of the viral membrane with the liposomal bilayer, specifically catalyzed by target membrane sphingolipid. Binding and fusion are mediated by the heterodimeric viral envelope glycoprotein E2/E1. At low pH the heterodimer dissociates, and the E1 monomers convert to a homotrimeric structure, the presumed fusion-active conformation of the viral spike. In this paper, we demonstrate that SFV–liposome fusion is specifically inhibited by Zn2+ions. The inhibition is at the level of the fusion reaction itself, since virus–liposome binding was found to be unaffected. Zn2+did not inhibit E2/E1 dissociation, but severely inhibited exposure of an acid-specific epitope on E1, E1 homotrimer formation, and acquisition of trypsin-resistance. It is concluded that virus–liposome binding solely requires low-pH-induced E2/E1 heterodimer dissociation, while fusion depends on further rearrangements in the E1 spike protein. As these rearrangements occur subsequent to the binding step, their precise course, including the formation of a fusion complex, may be influenced by interaction of E1 with target membrane lipids
North American and French Caprine Arthritis-Encephalitis Viruses Emerge from Ovine Maedi-Visna Viruses

AbstractThe full extent of genetic diversity among small ruminant lentiviruses (SRLVs), i.e., caprine arthritis encephalitis viruses (CAEVs) and maedi-visna viruses (MVVs), remains unknown. This is due in part to the fact that few sequences of CAEV are available. To contribute to this knowledge, gag, pol, and env nucleotide sequences from an SRLV named CA680 originating from a goat from western France were determined. This analysis revealed that this virus is closely related to the Cork and 63 CAEV American isolates. Mismatched amino acids between the CA680 virus and prototype CAEVs ranged from 6.7, 0.7, and 17.5% for gag, pol, and SU sequences, respectively. The differences between the CA680 virus and MVV prototypes ranged from 16.5, 12.5, and 32.3% for the protein sequences, respectively. A screening using a heteroduplex mobility assay (HMA) adapted to SRLVs revealed that 6 of 10 caprine virus field isolates were closely related to CA680, indicating that this latter isolate was a prototype of CAEVs common in the west of France. Phylogenetic trees drawn using CA, RT, or SU sequences of numerous SRLVs and rooted with EIAV sequences revealed that CA680 and CAEV prototypes, all infectious for goat, clustered in one group. From these HMA and phylogenetic analyses, it appears that U.S. and French caprine SRLVs form a clade that had emerged from a much more diverse group containing all SRLVs infectious for sheep. These ovine SRLVs form a more ancient group in which the EIAV is rooted
Recovery of Infectious SV5 from Cloned DNA and Expression of a Foreign Gene

AbstractA complete cDNA clone of the genome (15,246 nucleotides) of the paramyxovirus SV5 was constructed from cDNAs such that an anti-genome RNA could be transcribed by T7 RNA polymerase and the correct 3′ end generated by cleavage using hepatitis delta virus ribozyme. The plasmid encoding the antigenome sequence was transfected into cells previously infected with recombinant vaccinia virus that expressed T7 RNA polymerase, together with helper plasmids that expressed the viral replication proteins, NP, P, and L, under the control of the T7 polymerase promoter. Rescue of the RNA genome from DNA was demonstrated by recovering SV5 with the tag restriction sites introduced into the DNA clone, using RT-PCR of the genome RNA and nucleotide sequencing. Rescue of SV5 from DNA did not require expression of the viral V protein as a helper plasmid, suggesting that V protein is not essential for initial replication. The infectious cDNA of SV5 was also manipulated to express green fluorescent protein (GFP) under the control of SV5 transcriptional start and stop signals introduced between the HN and L genes. The amount of GFP that was expressed varied depending on the nature of the newly introduced transcription signals
RNA Binding Activity of NIa Proteinase of Tobacco Etch Potyvirus

AbstractThe C-terminal domain of NIa protein (NIaPro) from tobacco etch potyvirus (TEV) is a sequence-specific proteinase required for processing of the viral polyprotein. This proteinase also interacts with NIb, the TEV RNA-dependent RNA polymerase. NIaPro and two NIaPro-containing polyproteins (NIa and 6/NIa) were analyzed from extracts of recombinantEscherichia coli.Using RNA–protein blot and UV-crosslinking assays, NIaPro and the NIaPro-containing polyproteins were shown to possess RNA-binding activity. NIaPro bound nonspecifically to several RNAs, including plus- and minus-strands of the TEV 5′ and 3′ noncoding regions. Saturation binding data obtained using the UV-crosslinking assay were consistent with a possible cooperative RNA-binding activity of NIaPro. In addition, the RNA-binding activities of NIaPro and full-length NIa protein were similar. Based on its RNA-binding activity and other known functions, NIaPro or a NIaPro-containing polyprotein is proposed to serve one or more direct roles during TEV RNA synthesis
Productive Penetration of Rotavirus in Cultured Cells Induces Coentry of the Translation Inhibitor α-Sarcin

AbstractInternalization of rotavirus in MA104 cells was found to induce coentry of α-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration
Characterization of Cucumber Mosaic Virus

AbstractcDNA clones of cucumber mosaic virus (CMV) RNA 3 were modified to express the jellyfish green fluorescent protein (GFP) in place of the 3a movement protein (MP) or coat protein (CP), as fusions to the N (GFP–3a) or C (3a–GFP) terminus of the MP or from a separate open reading frame as part of tricistronic RNAs 3. CMV RNA transcripts containing the individual modified RNAs 3 were unable to infect eitherNicotiana tabacumorNicotiana benthamianasystemically. Infection, as measured by confocal microscopy of GFP fluorescence, generally was limited to one to three epidermal cells at each inoculation site. Limited cell-to-cell movement, but not systemic movement, could be detected by complementation involving expression of MP and CP from two different RNA 3 constructs, each also expressing GFP. Infection involving RNA 3 expressing the GFP–3a fusion showed bright granules of variable size distributed predominantly and nonuniformly throughout the cytoplasm and, to a lesser extent, associated with the cell wall in single fluorescent cells, while infections expressing the 3a–GFP fusion showed bright, punctate fluorescence associated only with the cell wall. Infected cells expressing either 3a–GFP or free GFP showed a halo of less bright, fluorescent, neighboring cells, indicating limited movement of GFP. The initially infected cells also allowed movement of 10-kDa fluorescent dextran to the neighboring halo cells, while infection did not spread, suggesting different requirements for movement of either MP or dextran versus RNA
Analysis of 74 kb of DNA Located at the Right End of the 330-kb Chlorella Virus PBCV-1 Genome

AbstractThis report completes a preliminary analysis of the sequence of the 330,740-bp chlorella virus PBCV-1 genome, the largest virus genome to be sequenced to date. The PBCV-1 genome is 57% the size of the genome from the smallest self-replicating organism,Mycoplasma genitalium.Analysis of 74 kb of newly sequenced DNA, from the right terminus of the PBCV-1 genome, revealed 153 open reading frames (ORFs) of 65 codons or longer. Eighty-five of these ORFs, which are evenly distributed on both strands of the DNA, were considered major ORFs. Fifty-nine of the major ORFs were separated by less than 100 bp. The largest intergenic distance was 729 bp, which occurred between two ORFs located in the 2.2-kb inverted terminal repeat region of the PBCV-1 genome. Twenty-seven of the 85 major ORFs resemble proteins in databases, including the large subunit of ribonucleotide diphosphate reductase, ATP-dependent DNA ligase, type II DNA topoisomerase, a helicase, histidine decarboxylase, dCMP deaminase, dUTP pyrophosphatase, proliferating cell nuclear antigen, a transposase, fungal translation elongation factor 3 (EF-3), UDP glucose dehydrogenase, a protein kinase, and an adenine DNA methyltransferase and its corresponding DNA site-specific endonuclease. Seventeen of the 153 ORFs resembled other PBCV-1 ORFs, suggesting that they represent either gene duplications or gene families
Acyclovir Blocks Cytokine Gene Expression in Trigeminal Ganglia Latently Infected with Herpes Simplex Virus Type 1

AbstractWe have previously found that interleukin (IL)-2, IL-10, interferon (IFN)-γ, RANTES, and tumor necrosis factor (TNF)-α mRNA transcription remain elevated in the trigeminal ganglia (TG) of herpes simplex virus type 1 (HSV-1) latently infected mice up to 120 days postinoculation (p.i.). To determine if this phenomenon was dependent on HSV-1 DNA replication after the establishment of latency (i.e., reactivation), cytokine gene expression was compared in TG of acyclovir-treated and untreated latently infected mice. Oral acyclovir treatment (begun 16 days p.i.) had no effect on serum levels of total anti-HSV-1 antibodies. However, there was a significant reduction in the titer of antibody specific for glycoprotein D and glycoprotein B but not glycoprotein H/L 120 days PI in the acyclovir-treated compared to vehicle-treated mice. These differences were not significant at earlier time points (i.e., days 34 and 60 p.i.). Consistent with these findings, acyclovir had no effect on cytokine gene expression in latently infected TG 35 and 60 days p.i. However, 120 days p.i., IFN-γ and TNF-α mRNA were approaching baseline levels in TG of acyclovir-treated mice, but remained significantly elevated in untreated controls (i.e., IFN-γ mRNA levels were sixfold higher in TG of untreated mice). Therefore, viral DNA replication appears to provide an antigenic stimulus for persistent cytokine gene expression in latently infected TG
Crystals of Rous Sarcoma Virus Capsid Protein Show a Helical Arrangement of Protein Subunits

AbstractCrystals of Rous sarcoma virus (RSV) capsid protein diffract X rays to 3.5 Å resolution and belong to the monoclinic space groupC2 with unit cell parametersa= 374.4 Å,b= 128.1 Å,c= 200.2 Å, and β = 121.8°. One asymmetric unit of the crystal may contain between 28 and 35 molecules, based on reasonable crystal density assumptions. A self-rotation function and Patterson synthesis suggest that RSV capsid protein crystallizes as a helical array. The determinants of the viral particle morphology are not encoded in the capsid alone. The assembly of a helical array in the crystal reflects the absence of any conformational switching. However, it is expected that the subunit interactions seen in the crystal will be preferred and will relate to those found in the immature or mature virion