pinecone/core-2020-05-10-deduplication huggingface dataset:
Nitric Oxide and Viral Infection: No Antiviral Activity against a Flavivirusin Vitro,and Evidence for Contribution to Pathogenesis in Experimental Infectionin Vivo

AbstractUpon stimulation murine macrophages produce high levels of nitric oxide (NO), a potent microbicidal and tumoricidal agent recently also implicated as a mediator of antiviral defense. As dysregulated production of NO may lead to extensive tissue damage, the production of this powerful mediator is tightly regulated. Viral infection, however, may alter the regulation of certain macrophage functions, and recent work from our group demonstrated that viral infection—via induction of interferon-αβ synthesis—may either prime for or down-modulate NO production. In light of antiviral activities of NO, down-modulation of NO production in viral infection would seem contradictory to antiviral defense. As others, however, have provided evidence that NO production may contribute to pathogenesis of infection with several neurotropic viruses, the role of NO production was investigatedin vitroandin vivoin murine macrophages and in BALB/c mice infected with tick-borne encephalitis virus (TBE-V), a flavivirus. Macrophages from TBE-V-infected mice, but not from control mice, spontaneously produced NO upon culturein vitro.In contrast to the inhibitory effect of NO on replication of several poxviruses and herpes simplex virus, high levels of NO production did not display an inhibitory influence on TBE-V replicationin vitro.And finally,in vivoadministration of a competitive inhibitor of NO production, aminoguanidine, to TBE-V-infected mice significantly increased their mean survival time. Our results thus demonstrate that the antiviral activity of NOin vitromay be confined to certain viruses, whereas others remain unaffected. Furthermore, we provide evidence that NO production may even contribute to pathogenesis of viral infectionin vivo
Proteolytic Activity of Human Immunodeficiency Virus Vpr- and Vpx-Protease Fusion Proteins

AbstractIn addition to Gag, Pol, and Env, primate lentiviruses encode other virion-associated proteins, including Vpr, Vpx, and Vif. Vpr- and Vpx-staphylococcal nuclease and chloramphenicol acetyltransferase fusion proteins incorporate into human immunodeficiency virus (HIV) virions and retain enzyme activity when expressedin transwith HIV proviruses (Wuet al., J. Virol.69, 3389, 1995). To explore whether the viral protease (PR) could be expressed as a proteolytically active fusion protein, the HIVPRcoding region was fused in-frame with the HIV-2vpxand HIV-1vprgenes. Using a vaccinia virus-T7 expression system, the Vpx-PR fusion protein was expressed and formed homodimers. Coexpression with Pr55Gagdemonstrated that Vpx-PR possessed Gag-specific proteolytic activity and inhibited the production of Gag virus-like particles.Trans-expression of a PR-Vpr fusion protein with HIV-1 provirus caused a profound reduction in viral protein expression and virion production. Importantly, the PR-Vpr fusion protein caused a similar level of inhibition and intracellular cleavage of Pr55Gagprecursor protein when coexpressed with protease defective HIV-1 provirus. The inhibitory effect of PR-Vpr expression on virion production was markedly greater than that of PR alone. These results indicate that Vpr augments the intracellular proteolytic activity of PR when expressed as a fusion protein and thus may be relevant for the expression of PR in intracellular immunization strategies against HIV infection. Moreover, the ability to express and package enzymatically active PR-Vpr fusion protein, independent of Gag/Pol, may provide a novel means to study enzyme function
Complete Sequence of Venezuelan Equine Encephalitis Virus Subtype IE Reveals Conserved and Hypervariable Domains within the C Terminus of nsP3

AbstractThe complete nucleotide and predicted amino acid sequences of Venezuelan equine encephalitis (VEE) virus subtype IE (isolate 68U201) were determined and compared to those of other antigenic variants within the VEE complex, strains IAB-TrD, IC-P676, ID-3880, IE-Menall, and II-Fe3-7c. The 68U201 structural proteins were most closely related to their Menall counterparts (97–100% identity) and more distantly related to VEE strains of other antigenic varieties (83–93% identity). With the exception of nsP3, the 68U201 nonstructural proteins were 94–95% identical to those of TrD, P676, and 3880 (nonstructural gene sequences are not available for Menall and Fe3-7c). The amino-terminal region of nsP3 (aa 1–329), which is highly conserved among all alphaviruses, was 93–94% identical for all VEE strains. The nsP3 carboxyl region is highly divergent among alphaviruses in general, but well conserved among previously sequenced VEE strains (>90% identity). Surprisingly, the carboxyl region of 68U201 nsP3 (aa 330–563) was only 59–61% identical to that of subtype IAB, IC, and ID viruses, with large insertions and deletions in addition to numerous substitutions. The differences between the 68U201 and other VEE nsP3 carboxyl regions were not randomly distributed, as there were four domains of high similarity within the nonconserved region. To examine this divergence more closely, we sequenced a portion of the Menall ns3 gene. The 68U201 and Menall nsP3 nonconserved regions were 85.3% identical and had the same basic domain structure, which was distinct from the IAB, IC, and ID nsP3 proteins, suggesting that the domain structure of nsP3 may be subtype/variety-specific. VEE nsP3 sequence diversity may reflect ecological differences such as adaptation to different mosquito vectors or vertebrate hosts
Simian Virus 40 Large-T Bypasses the Translational Block Imposed by the Phosphorylation of eIF-2α

AbstractOne of the cellular defense mechanisms against virus infection is mediated by activating the interferon-induced, double-stranded-RNA-activated protein kinase, PKR. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor, eIF-2, leading to cessation of protein synthesis. Viruses have evolved diverse strategies to counteract this cellular antiviral response. A majority of these strategies target PKR to prevent its activation. Recently, we showed that simian virus 40 (SV40) large-T antigen reverses PKR-mediated translational inhibition at a step downstream of PKR activation (Rajanet al., J. Virol.69, 785–795, 1995). In this paper, we present evidence showing that SV40 can restore efficient translation in cells despite the elevated levels of phosphorylated eIF-2α resulting from PKR activation. Thus, SV40 large-T-mediated translational rescue occurs at a step downstream of eIF-2α phosphorylation
The Two Nonstructural Proteins from Wheat Dwarf Virus Involved in Viral Gene Expression and Replication Are Retinoblastoma-Binding Proteins

AbstractTumor-inducing viruses like simian virus 40 or the human adenovirus produce oncoproteins which interfere with the cellular retinoblastoma (Rb) tumor-suppressor protein to create an appropriate molecular environment in the nucleus for viral transcription and replication. Such a strategy has been considered to be restricted to animal viruses. Here we demonstrate that plant viruses may use similar mechanisms for recruiting host factors. Wheat dwarf virus (WDV) encodes two potential nonstructural proteins, C1 and C1:C2, both containing the consensus Rb-binding motif LeuXCysXGlu that allows the oncoproteins from animal viruses to inactivate Rb. C1:C2 is a key determinant of viral replication and V(virion)-sense expression. Using a yeast two-hybrid protein assay, we demonstrate for the first time that the C1:C2 protein from WDV interacts with a retinoblastoma protein, providing an explanation for the previously observed dependence of viral replication on an intact Rb-binding motif. We also show that C1, for which no function had been demonstrated, is required for V-sense gene expression. This suggests that V-sense expression might be dependent on the interaction of C1 with Rb. Our findings provide further evidence for the presence of transforming-like proteins in a plant virus and will help to explain the production of symptoms in a plant viral infection through a mechanism mediated by a key regulator of cell cycle and differentiation
Resistance to Pepper Mild Mottle Tobamovirus Conferred by the 54-kDa Gene Sequence in Transgenic Plants Does Not Require Expression of the Wild-Type 54-kDa Protein

AbstractWe previously reported thatNicotiana benthamianaplants transformed with the wild-type 54-kDa region of the pepper mild mottle tobamovirus, S strain (PMMoV-S), displayed two different resistance responses against PMMoV infection. Some of the transgenic plants exhibited a complete and highly resistant phenotype while the remaining plants showed a delayed resistance (Tenlladoet al.,1995,Virology211, 170–183). Here we show that some of theN. benthamianaplants transformed with a construct expressing a PMMoV-S truncated 54-kDa protein coding sequence also displayed a complete and highly resistant phenotype similar to that shown by the wild-type 54-kDa transgenic plants. This result indicates that the wild-type, full-length 54-kDa protein is not required in mediating the complete resistance phenotype against PMMoV. The remaining truncated 54-kDa transgenic plants were susceptible to PMMoV infection but showed a variable delay in the appearance of symptoms. Unlike the wild-type 54-kDa transgenic plants, which were initially susceptible to the infection but recovered later, the truncated 54-kDa transgenic plants never exhibited this delayed resistance phenotype. However, they displayed a new type of altered symptomatic phenotype. The truncated 54-kDa transgenic lines also exhibited a lower level of transgenic transcripts compared to the wild-type 54-kDa transgenic lines which could account for the absence of the delayed resistance phenotype
When Two Strands Are Better Than One: The Mediators and Modulators of the Cellular Responses to Double-Stranded RNA

AbstractDouble-stranded RNA is a potent inducer of interferon, a modulator of the expression of a number of other genes involved in the response of cells to virus infection, an activator of the interferon-induced antiviral state, and may be involved in differentiation, induction of apoptosis, and control of oncogenic transformation. This review will attempt to summarize what is known about the cellular proteins that act to mediate the response of cells to double-stranded RNA and the viral and cellular macromolecules that may be able to modulate these responses
Homologous and Heterologous Complementation of HBV and WHV Capsid and Polymerase Functions in RNA Encapsidation

AbstractSuccessful encapsidation of hepadnaviral pregenomic RNA requires the orchestrated interaction of the viral capsid and polymerase proteins with each other and with the RNA packaging substrate. The early steps of this process involve binding of the polymerase to the encapsidation signal, ϵ, and are already understood in some detail. However, the underlying macromolecular interactions resulting in the subsequent encapsidation of this polymerase–ϵ complex by capsid proteins are less clearly defined. To approach this issue we have examined the ability of two different hepadnaviruses to encapsidate each other's pregenomic RNA. H. Okamotoet al.((1990)J. Gen. Virol.71, 959–963) have previously demonstrated that WHV polymerase could encapsidate an HBV pregenome, but the origin of the capsid proteins (i.e., HBV- or WHV-derived) required for this reaction was not clear; some evidence suggested that heterologous capsid and polymerase proteins might not be capable of interaction. To clarify this, we analyzed encapsidated RNA isolated from cytoplasmic cores produced following transient transfection of HepG2 cells with different combinations of plasmids encoding HBV or WHV core and polymerase genes. We found that (i) the essential encapsidation signal of WHV is comprised of a short region including ϵ, as in HBV; (ii) HBV and WHV polymerases are each competent to recognize both HBV and WHV packaging signals; therefore the encapsidation signals are functionally interchangeable; and (iii) HBV capsids encapsidate a WHV polymerase–ϵ complex, and vice versa, although the efficiency of heterologous packaging is slightly lower than that of homologous encapsidation. Our results underscore the close relationship of these two mammalian hepadnaviruses
Importance of Localized Skin Infection in Tick-Borne Encephalitis Virus Transmission

AbstractArboviruses are transmitted to vertebrates by the ”bite“ of infected arthropods. Events at the site of virus deposition are largely unknown despite increasing evidence that blood-sucking arthropods immunomodulate their skin site of feeding. This question is particularly relevant for ixodid ticks that feed for several days. To examine events under conditions mimicking tick-borne encephalitis (TBE) virus transmission in nature (i.e., infected and uninfectedIxodes ricinusticks feeding on the same animal), infected adult and uninfected nymphal ticks were placed in one retaining chamber (skin site A) and uninfected nymphs were placed within a second chamber posteriorly (skin site B) on two natural host species, yellow-necked field mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus). Virus transmission from infected to uninfected cofeeding ticks was correlated with infection in the skin site of tick feeding. Furthermore, virus was recruited preferentially to the site in which ticks were feeding compared with uninfested skin sites. Viremia did not correspond with a generalized infection of the skin; virus was not detected in an uninfested skin site (C) of 12/13 natural hosts that had viremia levels ≥2.0 log10ic mouse LD50/0.02 ml blood. To characterize infected cells, laboratory mouse strains were infested with infected ticks and then explants were removed from selected skin sites and floated on culture medium. Numerous leukocytes were found to migrate from the skin explants of tick feeding sites. Two-color immunocytochemistry revealed viral antigen in both migratory Langerhans cells and neutrophils; in addition, the migratory monocyte/macrophages were shown to produce infectious virus. The results indicate that the local skin site of tick feeding is an important focus of viral replication early after TBE virus transmission by ticks. Cellular infiltration of tick feeding sites, and the migration of cells from such sites, may provide a vehicle for transmission between infected and uninfected cofeeding ticks that is independent of a patent viremia. The data support the hypothesis that viremia is a product, rather than a prerequisite, of tick-borne virus transmission
Stabilization of Vesicular Stomatitis Virus L Polymerase Protein by P Protein Binding: A Small Deletion in the C-Terminal Domain of L Abrogates Binding

AbstractWe showed previously that cells expressing the vesicular stomatitis virus (VSV) L polymerase gene via the vaccinia–T7 RNA polymerase system accumulated 2- to 5-fold more L protein when the P protein was coexpressed (Canteret al.,1993,Virology194, 518–529). The results presented here provide an explanation for this phenomenon. Pulse–chase analysis revealed that L was unstable with a half-life of 3 to 6 hr if expressed in the absence of P protein, but was stable for at least 16 hr when coexpressed with a 10- to 15-fold molar excess of P. The P protein, in contrast, was stable under both conditions. Stabilization correlated with formation of a P:L polymerase complex evidenced both by coimmunoprecipitation and by glycerol gradient sedimentation analyses. A mutant L protein, lacking amino acids 1638 to 1673, was not stabilized by coexpression and showed no binding to P protein. Its anomalous sedimentation, however, suggested misfolding and/or aggregation as the cause for the failure to bind P. Transcription reconstitutionin vitro,using extracts from cells expressing excess of P over L protein, strongly depended on coexpression of the proteins for optimal activity. We propose that the coexpression dependence for polymerase reconstitution documented here for VSV, as well as that reported previously for the Sendai paramyxovirus, reflects the protective effect of P protein on L protein stability
Analysis of the Nucleotide Sequence of the Treehopper-Transmitted Geminivirus, Tomato Pseudo-Curly Top Virus, Suggests a Recombinant Origin

AbstractThe genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced. TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies). Infectivity of the cloned viral genome was demonstrated byAgrobacterium-mediated inoculation of several host species. Progeny virus was transmissible by the treehopper vector of TPCTV,Micrutalis malleifera(Fowler). The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae. The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whiteflyBemisia tabaciGenn. The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus
Characterization of DNA-Binding Proteins and Protein Kinase Activities inChlorellaVirus CVK2

AbstractSeveral capsid proteins were selectively released from the viral core ofChlorellavirus CVK2 by treatment with 4M urea. Among the viral core proteins, seven species (Vp154, Vp73, Vp63, Vp52, Vp48, Vp42, and Vp25) were shown to have DNA-binding activities by Southwestern blot analysis. Except for Vp154 and Vp25, these DNA-binding proteins showed a specific affinity for the viral genomic DNA. The viral core also contained three proteins with protein kinase activity (Vp73, Vp60, and Vp37); Vp73 seemed to have both DNA-binding and protein kinase activities. Antisera raised against Vp73 were used to screen a lambda-CVK2 expression library for the gene encoding Vp73. Three different clones (Vp73-3, Vp73-29,andVp73-42) were obtained and analyzed. ORFs found in these clones all contained characteristic proline-rich motifs. The Vp73-42 ORF showed a strong similarity with histone H1 of various organisms and the Vp73-29 ORF contained two regions with leucine-zipper motifs. All three genes were expressed late in infection
Natural Variation in HIV-1 Protease, Gag p7 and p6, and Protease Cleavage Sites within Gag/Pol Polyproteins: Amino Acid Substitutions in the Absence of Protease Inhibitors in Mothers and Children Infected by Human Immunodeficiency Virus Type 1

AbstractReduced sensitivity of human immunodeficiency virus type 1 (HIV-1) to protease inhibitors is associated with multiple amino acid substitutions in the virus-encoded protease. The combination of changes that contribute to drug resistance is dependent in part upon the amino acid residues comprising protease alleles prior to drug therapy. We analyzed within peripheral blood mononuclear cells from HIV-1-infected mothers and their children viralgag/polregions, which included p7, transframe p6/p6*, and protease coding sequences, as well as six protease cleavage sites. Sixty protease alleles from 12 individuals differed by at least 3 to as many as 10 amino acids from proteases encoded by molecular clones of HIV-1, indicating that there is no prototype or consensus wild-type HIV-1 protease sequence. Protease variants with a proline at position 63, a substitution associated with resistance to protease inhibitors, appeared in the absence of antiprotease therapy in 7 patients and were transmitted by 2 mothers to their infants. Gag p7 and p6 regions were significantly more variable than protease. The p6/p6* region contained length variants and amino acid repeats in both reading frames. Five protease cleavage sites (B, D′, D, E, and F) contained highly conserved amino acid sequences in individuals infected by epidemiologically distinct viruses. In contrast, C cleavage sites, localized between Gag p2 and Gag p7, displayed considerable amino acid variability, were unique among groups of infected individuals, and appeared to be related to particular protease alleles. Genetic variabilityin vivoin protease, in cleavage sites, and in proteins upstream of protease provides the potential to modulate enzyme activity and susceptibility to protease inhibitors
Multiple Virus Genes Involved in the Nematode Transmission of Pea Early Browning Virus

AbstractMutations were introduced into four genes encoded by RNA2 of pea early browning virus (PEBV) to determine their possible involvement in the transmission of this virus by nematodes. Deletion of 28 amino acids from the C-terminus of the coat protein abolished the formation of virus particles. Deletion of 15 amino acids at the C-terminus did not affect particle formation but did abolish nematode transmission. In contrast, deletion of 13 amino acids immediately preceding the 16 C-terminal residues did not affect particle formation and decreased rather than abolished nematode transmission. A deletion in the gene encoding a 29-kDa protein and a frameshift mutation in the gene encoding a 23-kDa protein both abolished transmission without affecting virus particle formation. Mutations in an ORF encoding a 9-kDa protein, which is located on the genome between the coat protein gene and 29K gene, gave conflicting results. Removal of the AUG initiation codon from the 9K ORF had no effect on transmission, whereas the introduction of a frameshift mutation, which would prematurely terminate expression of the putative 9-kDa protein, decreased the frequency of transmission. The results show that the coat protein and probably all three of the other RNA2-encoded proteins play a role in the transmission of PEBV by vector nematodes
Differential Effects of Intronic and Exonic Locations of the Human Immunodeficiency Virus Type 1 Rev-Responsive Element

AbstractThe influence of the location of the Rev-response element (RRE) on human immunodeficiency virus type 1 (HIV-1) protein and RNA expression in COS cells was assessed. The RRE was placed intonefwhere it would be present in all HIV-1 RNAs. At this location, Gag and Env proteins were produced and the unsplicedgag/poland partially splicedenv/vpuRNAs were able to accumulate in the cytoplasm. The RRE was also relocated from its normal location in theenvexon to theenvintron. In this way, the RRE would be present in the nuclearenvpre-mRNA, but not in the splicedenvmRNA. Gag, but not Env protein production was detected. The presence of the RRE in theenvpre-mRNA allowed the cytoplasmic accumulation of the splicedenvmRNA, which lacked the RRE. However, this mRNA accumulated at a reduced level relative to that produced by constructs containing the RRE within theenvmRNA. The cytoplasmic accumulation of this mRNA was dependent on the presence of Rev and the RRE. These results demonstrate that the location of the RRE can have differential effects on the fate of HIV-1 RNAs
Mutational Analysis of the Bacteriophage P4 Capsid-Size-Determining Gene

AbstractSatellite phage P4 (11,624 bp) depends on the morphopoietic genes (capsid, tail) and lysis genes of its helper phage P2 (33.5 kb) for its lytic development. In the morphopoietic process, P4 redirects the assembly pathway of large, P2 size, capsids (diameter = 60 nm) to yield smaller, P4 size, capsids (diameter = 45 nm), 1/3 in volume of that of its helper. The P4-specified capsid size determination is dependent on the function of the 27-kDa gpSid. To study the capsid size-determining function, we carried out a mutational analysis of the P4sidgene. Use of a P4-derived genome of 29.1 kb (P461), which can be packaged only into large, P2 size, capsids allowed us to select P4 Sid−mutants. By DNA sequencing we characterized 25 P4 Sid−mutants, of which 10 contain base pair substitutions and 15 contain deletions. Both types of mutations are clustered in separate locations within thesidgene. Our results suggest that the Sid polypeptide contains three distinct functional domains