pinecone/core-2020-05-10-deduplication huggingface dataset:
Analysis ofcis-Acting Elements Required for Replication of Barley Stripe Mosaic Virus RNAs

AbstractThe replicative abilities of mutant RNA transcripts derived from barley stripe mosaic virus cDNA clones were investigated in barley protoplasts that had been coinoculated with wild-type RNAα and -γ transcripts. The 5′ and 3′ noncoding regions were required for replication, and lack of a 5′ cap structure (GpppG) reduced the replicative ability substantially. All internal deletions within RNAα abrogated replicationin trans.A 2-base change that produced a truncated αa protein lacking the first 16 amino acids also compromised the ability of RNAα to be replicated. In contrast, RNAβ transcripts containing deletions involving each ORF and the downstream poly(A) tract were effectively amplified by RNAs α and γ, but collective deletion of all four ORFs drastically reduced accumulation. The intergenic region between βa and βb was not absolutely required for replication, but small deletions within this region reduced the abundance of RNAβ by at least 10-fold. Deletions within the first 507 nt of the γa ORF abrogated replication. However, transcripts containing deletions within the central and 3′ regions of the γa ORF, the γa–γb intergenic region, and the γb ORF could be amplifiedin trans.Two mutants containing extensive deletions encompassing the central region of the γa ORF and most of γb behaved like defective interfering RNAs because they multiplied to high levelsin transand caused a pronounced reduction in accumulation of the coinoculated wild-type RNAs α and γ
A Novel Ribozyme Target Site Located in the HIV-1NefOpen Reading Frame

AbstractWe have tested the sequence UUC CAG UCA GAC CU, at position 9016–9029 within the HIV-1SF2nefopen reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate-modified 14-nt DNA oligo (complementary to thenef9016–9029site). A dose-dependent repression of HIV-1SF2growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for thenef9016–9029site (hhRz.nef9016–9029) was constructed and transfected into the human T-cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016–9029were infected with HIV-1SF2. A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (103-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviralpolandgagsequences in hhRz.nef9016–9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since nopolandgagPCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support thenef9016–9029site as a strong new candidate for ribozymal gene therapy against HIV-1 infection
Evolutionary Relationships among Large Double-Stranded DNA Viruses That Infect Microalgae and Other Organisms as Inferred from DNA Polymerase Genes

AbstractIn order to examine genetic relatedness among viruses that infect microalgae, DNA polymerase gene (DNApol) fragments were amplified and sequenced from 13 virus clones that infect three genera of distantly related microalgae (Chlorellastrains NC64A and Pbi,Micromonas pusillaandChrysochromulina spp.). Phylogenetic trees based on DNApolsequences and hybridization of total genomic DNA showed similar branching patterns. Genetic relatedness calculated from the hybridization and sequence data showed good concordance (r= 0.90), indicating that DNApolsequences can be used to determine genetic relatedness and infer phylogenetic relationships among these viruses. The phylogenetic tree inferred from the deduced amino acid sequences of DNApolfrom 24 dsDNA viruses, including phycodnaviruses, herpesviruses, poxviruses, baculoviruses, and African swine fever virus corresponded well with groupings based on the International Committee on Taxonomy of Viruses. Microalgal viruses are more closely related to each other than to the other dsDNA viruses and form a distinct phyletic group, suggesting that they share a common ancestor and belong to the Phycodnaviridae. Moreover, the Phycodnaviridae are more closely related to the Herpesviridae than to other virus families for which DNApolsequences are available
The Low pH-Dependent Entry of Avian Reovirus Is Accompanied by Two Specific Cleavages of the Major Outer Capsid Protein μ2C

AbstractAvian reoviruses are capable of inducing rapid and extensive syncytium formation, a process that occurs preferentially under conditions of neutral or alkaline pH. In order to ascertain whether the membrane fusion-inducing capability of avian reovirus confers a pH-independent entry mechanism on the virus, virus entry was investigated using internalization assays and several lysomotropic agents that inhibit endosomal acidification. The ability of avian reovirus to infect cells was severely restricted under all conditions that prevented endosomal acidification. The decreased infection efficiency in the presence of the lysomotropic agents correlated with an inhibition in the proteolytic processing of the major outer capsid protein μ2C. The importance, with respect to virus infection, of the low pH-dependent cleavage of the avian reovirus μ2C protein was confirmed by demonstrating that infectious subviral particles, generated by proteolytic processingin vitro,were capable of efficiently infecting cells in the presence of the lysomotropic agents. These results indicated that avian reovirus entry-specific membrane interactions are largely dependent on an endosome-mediated proteolytic processing of the virus particle, suggesting that the syncytium-inducing property of the σ3 protein is not sufficient to promote virus uptake. Furthermore, avian reovirus internalization was associated with two distinct cleavages of the major outer capsid protein μ2C, unlike the entry-specific processing of the analagous mammalian reovirus major outer capsid protein μ1C. The μ2C cleavages occured sequentially and appeared to involve distinct cleavage specificities. Moreover, the second cleavage event was observed to be both virus strain- and cell type-independent, suggesting that the cleavage is both specific and biologically significant
Preliminary Crystallographic Studies of Bacteriophage T4 Fibritin Confirm a Trimeric Coiled-Coil Structure

AbstractFibritin, a 52-kDa product of genewacof bacteriophage T4, forms fibrous “whiskers” that connect to the phage tail and facilitate the later stages of phage assembly. Preliminary experiments suggest that fibritin is a trimer, and its predominant central part has a parallel α-helical coiled-coil structure. To investigate the oligomerization and function of fibritin, we have designed and studied two related deletion mutants, denoted M and E, that consist of its last 75 and 120 amino acids, respectively. Both proteins contain part of the coiled-coil region and the 29 amino acid carboxy-terminal domain essential for the trimerization of fibritin. The proteins are expressed as a soluble product in anEscherichia colisystem. We have obtained crystals of fibritins M and E. Complete native X-ray diffraction data sets have been collected to 1.85 and 2.7 Å resolution, respectively. The crystals have space groupP3 witha= 44.3 Å,c= 91.3 Å (fibritin M) andR32 witha= 41.2 Å,b= 358.7 Å (fibritin E) in the hexagonal setting. Symmetry and packing considerations show that fibritin is a triple coiled coil
Protection from Pathogenic SIVmac Challenge Following Short-Term Infection with a Nef-Deficient Attenuated Virus

AbstractInfection of rhesus macaques with attenuated SIVmac is, at present, the only strategy which confers significant protection from challenge with wild-type SIVmac grown in monkey PBMC. However, initial results suggest that the protective mechanism does not develop until late after “vaccination” (approx 10 months). As part of a European study using the C8 variant of SIVmac251-32H(containing an in-frame 12-bp deletion in thenefgene), we wished to determine (a) if protection could be achieved against challenge with a “swarm” of SIVmac251-32Hproduced in monkey cells and (b) if protection could be demonstrated after a short period of infection with the attenuated virus. Eight Indian rhesus macaques were infected with C8 and four were challenged after 10 weeks with 50 MID50of an uncloned stock of SIVmac251-32Hgrown in rhesus cells, and the other four were challenged after 20 weeks. Four animals served as naive controls. Three of the four monkeys challenged at 10 weeks and three of those challenged at 20 weeks were protected from productive superinfection. From one monkey in each group it was, however, possible to demonstrate the presence of the wild-type provirus in monkey PBMC by diagnostic PCR and anamnestic immune response. There was no apparent correlation between the levels of binding or neutralizing antibodies on the day of challenge and subsequent protection. Approximately 1 year after infection with the attenuated virus all monkeys were rechallenged with the heterologous SIVsm strain, first with 10–20 MID50and then with 1000 MID50. Although not all of the SIVsm-inoculated naive controls became productively infected, PCR analysis failed to reveal any evidence for infection of the “immunized” monkeys
Perturbation of the Host Cell Cycle and DNA Replication by the Bovine Papillomavirus Replication Protein E1

AbstractA stable cell line expressing the bovine papillomavirus E1 protein (C2E1) was compared with an E1 minus control line (CNEO) to study the effects of E1 protein on host cell growth. C2E1 and CNEO cells were synchronized either at mitosis or at the G1/S boundary by the cell cycle inhibitors nocodazole and mimosine, respectively. After release from the drug-induced cell cycle block, the progression through the succeeding stages of the cell cycle was temporally monitored using flow cytometry. In addition, incorporation of bromodeoxyuridine (BrdUrd) was used to determine precisely the time of initiation of DNA synthesis in C2E1 and CNEO cells after release from drug-induced cell cycle arrest. Expression of E1 protein decreased the duration of G1 phase and increased S and G2 phase durations without affecting the overall cell doubling time. In conjunction with the increase in G2 phase duration, histone H1 kinase activity was prolonged during the G2 to M phase transition in C2E1 cells, which suggested that E1 protein may affect the mechanisms which ensure proper timing of kinase inactivation. During the G1 to S phase transition in C2E1 cells, the timing of appearance and abundance of cyclin D1 were altered compared to CNEO cells, while cyclin E levels were unaffected. Consequently, E1 protein may affect G1 phase duration through a cyclin D1-dependent pathway. Finally, a subpopulation of cells with a greater than G2 DNA content (>G2 DNA), and which was still capable of incorporating BrdUrd, was shown to exist only in the E1-expressing cell line. These combined results demonstrate that the viral replication protein E1 has the potential to influence the host cell environment significantly, which may contribute to pathogenesis and viral persistence
A Replication-Defective Human Adenovirus Recombinant Serves as a Highly Efficacious Vaccine Carrier

AbstractIn this manuscript, an E1 and E3 deleted adenoviral recombinant expressing the rabies virus glycoprotein (G protein) under the control of the cytomegalovirus early promoter was tested for induction of a rabies virus-specific immune response in mice. The construct was found to induce neutralizing antibodies and cytolytic T cells to rabies virus. Mice vaccinated with the adenoviral construct either by the systemic route or by application into the airways were protected against a subsequent infection with a virulent strain of rabies virus. The efficacy of the replication-defective construct was far superior to that of a well-characterized vaccinia rabies glycoprotein recombinant
p53 Inhibits JC Virus DNA Replicationin Vivoand Interacts with JC Virus Large T-Antigen

AbstractThe onset of DNA replication is an important step within the life cycle of the human neurotropic polyomavirus JC. In this report, evidence that both the human and the murine tumor suppressor protein p53 strongly inhibit JCV DNA replicationin vivois presented. This inhibition is dose-dependent and not a secondary effect of a decreased expression of JCV large T-antigen in response to p53. Using deletion mutants of murine p53 and tumor-derived point mutations of human p53, the basis of the suppression of JCV DNA replication by p53 was dissected. Deletion of either the amino- or the carboxy-terminal domain of murine p53 did not interfere with the repression of JCV DNA replication. However, deletion of the highly conserved central region of p53 abolished the inhibitory effect on replication. The tumor-derived human mutant p53(His273) inhibited JCV DNA replication significantly, whereas another tumorigenic mutant, p53(His175), had no inhibitory effect. Concomitantly, a direct protein–protein interaction between p53 and JCV large T-antigen was lost in mutants which did not affect JCV DNA replication. These results strongly suggest that p53 inhibits JCV DNA replication by interacting with JCV large T-antigen
Domains of the E1 Protein of Human Papillomavirus Type 33 Involved in Binding to the E2 Protein

AbstractPapillomavirus E1 and E2 proteins are essential for the initiation of viral DNA replication. We have now analyzed the interaction of E1 and E2 of human papillomavirus type 33, which is associated with cervical carcinoma. When synthesized in insect cells using the baculovirus expression system, the E1 and E2 proteins interacted efficiently at 4°. A monoclonal antibody recognizing E1 amino acids 584–600 inhibited the binding of E2 and vice versa, indicating that these amino acids are involved in E2 binding. To confirm this result, a mutational analysis of E1 was performed. The E2 binding activity of E1 deletion and point mutant proteins was assayed using glutathione S-transferase E1 fusion proteins andin vitrotranslated proteins. At 4°, the C-terminal portion of E1 including amino acids 312–644 was sufficient for E2 binding. Introduction of C-terminal deletions or a point mutation at position 586 (Pro → Glu) resulted in the loss of the E2 binding activity. A second more N-terminally located binding domain (E1 amino acids 312–450) became active when the assays were performed at 22°. The monoclonal antibody still inhibited E2 binding at this temperature, indicating that both E2 binding domains are engaged in the context of the full-length protein
Genetic Control of Infection of Primary Macrophages with T-Cell-Tropic Strains of HIV-1

AbstractHuman immunodeficiency virus type 1 (HIV-1) NDK, a Zairian subtype D virus highly cytopathic for CD4-positive lymphocytes, and the prototype subtype B virus HIV-1 LAV are about 104and 105times more infectious, respectively, for T lymphocytes than for blood-derived macrophages (BDM). Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control and the step of the virus/cell cycle responsible for infection of BDM with T-cell-tropic viruses. We found that recombinants bearing the envelope glycoprotein of HIV-1 NDK are able to enter more efficiently into BDM than recombinants with HIV-1 LAV envelope glycoprotein. We also found that a genetic region outside of theenvgene is responsible for production of HIV-1 NDK infectious progeny from BDM. This region consists of thevifgene and the C- and N-terminal portions ofpolandvprgenes, respectively. Our results suggest that productive infection of primary macrophages with T-cell-tropic strains of HIV-1 is determined by two different genetic mechanisms: one effective at the virus/cell entry, controlled by theenvgene, and the second after entry, controlled by genesvifandvpr.In comparison with HIV-1 LAV, HIV-1 NDK has been able to more easily overcome both restriction mechanisms
Heat-Resistant Factors in Human Erythrocyte Membranes Mediate CD4-Dependent Fusion with Cells Expressing HIV-1 Envelope Glycoproteins

AbstractIt has been shown that human CD4 expressed in nonhuman cells does not support HIV-1 entry into those cells and that components from human cells in addition to CD4 are required to overcome the block. We have used human red blood cells (huRBC) as a source for the accessory components since their membrane composition is less complex than that of nucleated cells and they are well characterized. Components were transferred by fusion of huRBC to nonhuman CD4+cells mediated by influenza hemagglutinin or polyethylene glycol. The RBC-modified nonhuman CD4+cells were labeled with fluorescent markers and incubated with gp120–gp41-expressing cells labeled with a different fluorescent probe. Fusion between RBC-modified nonhuman CD4+cells and gp120–gp41-expressing cells was quantified by fluorescence video microscopy. Human erythrocyte components transferred to nonhuman CD4+cells conferred HIV-1 envelope glycoprotein-mediated fusion susceptibility to those cells. The fusion was enhanced by pretreatment of the erythrocytes for 10 min at 56°. No gp120–gp41-mediated fusion was observed when components from nonhuman RBC were transferred to nonhuman CD4+cells. Human cell lines with pre-RBC characteristics (K562-CD4) also supported HIV-1 envelope glycoprotein-mediated fusion
Expression of the 69K Movement Protein of Turnip Yellow Mosaic Virus in Insect Cells

AbstractThe nonstructural 69-kilodalton (K) protein of turnip yellow mosaic virus is necessary for systemic spread of the virus within the plant. To examine the behavior of the 69K proteinin vivo,antibodies were raised against the carboxy-terminal region of this protein. The full-length 69K protein was also expressed in insect cells using a recombinant baculovirus. Studies on the posttranslational modifications of the 69K protein in insect cells revealed that the protein is phosphorylated but not glycosylated. Further experiments of subcellular fractionation and indirect immunolocalization in insect cells showed that the 69K protein is localized in the cytoplasm and/or in the plasma membrane
Deletion of Glycoprotein gE Reduces the Propagation of Pseudorabies Virus in the Nervous System of Mice after Intranasal Inoculation

AbstractA pseudorabies virus (PrV) mutant, deficient in the nonessential glycoprotein E (gE) and expressing theLacZgene (gE−βgal+PrV), and its rescued virus were inoculated intranasally in mice. The median lethal dose of gE−βgal+PrV was similar to that of the parental Kaplan strain, but mice survived longer and did not develop symptoms of pseudorabies. In the nasal mucosa, gE−βgal+PrV replicated less efficiently than rescued virus. gE−βgal+PrV could infect first-order trigeminal and sympathetic neurons innervating the nasal mucosa. However, transneuronal transfer to second-order cells groups did not occur in trigeminal pathways and was severely reduced in sympathetic pathways. The mutant was also unable to propagate in the parasympathetic system. In contrast, gE-rescued virus was transferred transneuronally in trigeminal, sympathetic, and parasympathetic pathways, like wild-type PrV. These findings provide further evidence that deletion of gE specifically affects transneuronal transfer of PrV more than penetration and multiplication of the virus in first-order neurons
The Phylogeny of New World (Tacaribe Complex) Arenaviruses

AbstractSeveral New World (Tacaribe complex) arenaviruses (Arenaviridae) are known to cause severe hemorrhagic disease in humans. Phylogenetic reconstruction of the Tacaribe complex arenaviruses previously has been limited by the relative scarcity of sequence data for arenavirus genomes. In the present study, oligonucleotide primers were designed based on conserved regions of the nucleocapsid (N) protein gene and then used to amplify, by reverse transcription–polymerase chain reaction, a 613- to 649-nucleotide region of the N gene of all known Tacaribe complex arenaviruses. This has allowed completion of the first detailed genetic characterization and phylogenetic analysis of all known members of the Tacaribe complex. These viruses formed three lineages. Lineage A contained Flexal, Parana, Pichinde, and Tamiami viruses; lineage B contained Amapari, Guanarito (GUA), Junin (JUN), Machupo (MAC), Sabia (SAB), and Tacaribe viruses. Latino and Oliveros viruses occupied lineage C. The highly pathogenic Tacaribe complex arenaviruses (GUA, JUN, MAC, SAB) were all members of lineage B, suggesting the possibility that the highly pathogenic phenotype is the result of evolutionary radiation from a common ancestor. The approach described here provides a rapid method for characterization of novel Tacaribe complex arenaviruses and may provide clues as to their potential public health importance
HSV-1-Mediated Modulation of Cytokine Gene Expression in a Permissive Cell Line: Selective Upregulation of IL-6 Gene Expression

AbstractPathological effects of herpes simplex virus (HSV) can result due to a combination of direct viropathic effects and immunological reactions to viral antigens. The immunological reactions are orchestrated by a variety of cytokines and chemokines released by the host cells. Therefore, the cytokine gene expression in response to HSV-1 infection in a permissive murine cell line was investigated. The data demonstrate that HSV-1 induced a selective activation of IL-6 gene expression at the mRNA and protein levels, in the permissive cell line. The cell line used was capable of expressing IL-1, IL-7, and IL-10 in addition to IL-6, upon lipopolysaccharide stimulation. UV or heat-inactivated viruses were unable to upregulate IL-6 expression. However, mutant HSV-1 strains lacking fully functional ICP0, ICP4, ICP8, or ICP27 genes, thereby rendering them replication incompetent or impaired inin vitrocell growth (ICP0), enhanced IL-6 expression selectively. Considering the role of IL-6 in inflammation, immune response, and its known association with increased levels of MyD116 and GADD 34 mRNAs (genes involved in the prevention of apoptotic death of cells), the present data may have relevance to HSV-1-mediated diseases as well as to the prevalence of HSV-1 in the host