pinecone/core-2020-05-10-deduplication huggingface dataset:
The p20 Gene Product of Citrus Tristeza Virus Accumulates in the Amorphous Inclusion Bodies

AbstractCitrus tristeza virus (CTV) has 10 3′ open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3′ most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of ∼22–23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3′ end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20–GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein–p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells
Full-Length Tobacco Mosaic Virus RNAs and Defective RNAs Have Different 3′ Replication Signals

AbstractThe viral replicase complex of positive-stranded RNA viruses interacts with cis-acting elements that are usually located at the termini of the viral RNAs. On comparison of the replication requirement of a tobacco mosaic virus (TMV)-based defective RNA (dRNA) and its helper virus, we found different requirements for replication of TMV RNAs in cis and in trans. The level of replication of full-length TMV RNA decreased substantially in the absence of pseudoknot (pk) 1 and/or 2, whereas identical deletions in dRNAs did not affect their replication. However, pk3 was required for replication of both full-length TMV RNAs and dRNAs. The requirements for homologous sequences were greater for dRNA replication than for replication of full-length TMV RNAs. Defective RNAs with heterologous 3′ nontranslated regions (NTRs) failed to be replicated or replicated minimally, whereas replication of similarly mutated full-length RNAs was much less affected. Increasing amounts of contiguous heterologous sequences in the dRNAs compensated for the impaired interactions between the replicase and 3′ NTR. The precision requirement appeared to involve the terminal 28 nucleotides, specifically the pseudoknot in the aminoacyl acceptor arm of the tRNA like structure, which was important in replication of both dRNAs and full-length TMV RNAs
Strain-Specific Interaction of the Tobacco Etch Virus NIa Protein with the Translation Initiation Factor eIF4E in the Yeast Two-Hybrid System

AbstractThe NIa protein of potyviruses provides VPg and proteolytic functions during virus replication. It has also been shown to confer host genotype-specific movement functions in plants. Specifically, NIa from tobacco etch virus (TEV)-Oxnard, but not from most other strains, confers the ability to move long distances in Nicotiana tabacum cultivar “V-20.” This led to the hypothesis that all or part of NIa may interact with one or more cellular factors. To identify cellular proteins that interact with NIa in a host- or strain-specific manner, a yeast two-hybrid search of a tomato cDNA library was done. Ten proteins that interacted with NIa were recovered, with translation initiation factor eIF4E being by far the most common protein identified. Interaction of eIF4E with NIa was shown to be TEV strain-specific. eIF4E from both tomato and tobacco interacted well with NIa from the HAT strain, but not from the Oxnard strain. However, using chimeric NIa proteins, the determinant for systemic infection of V20 plants was found to be genetically distinct from the determinant controlling eIF4E interaction. In TEV–eIF4E coexpression experiments, evidence suggesting that eIF4E provides a positive effect on genome amplification was obtained
Role of the DExH Motif of the Japanese Encephalitis Virus and Hepatitis C Virus NS3 Proteins in the ATPase and RNA Helicase Activities

AbstractThe role of the conserved DExH motif of the Japanese encephalitis virus (JEV) NS3 protein in the ATPase and RNA helicase activities was compared with that of the hepatitis C virus (HCV) NS3 protein. In the DExH motif of JEV NS3, Asp-285 and Glu-286 were essential for both ATPase and RNA helicase activities. Cys-287 was critical for the RNA helicase activity of JEV NS3 but not for ATPase activity. A His-288-to-Ala substitution in the DExH motif of HCV NS3 resulted in an increase in ATPase activity which was suppressed by poly(U). In contrast, alanine substitution at the same site in JEV NS3 did not increase basal ATPase activity which remained to be stimulated by poly(U). Thus, the mutational effect at His in motif II was different in the HCV and JEV NS3 proteins. Mutagenesis at His-288 of JEV NS3 revealed that His was the most preferable amino acid for ATPase activity and Ala, Gly, Asn, Gln, Ser, or Arg could partly substitute for it. However, any other mutation at His-288 completely disrupted the RNA helicase activity of JEV NS3. The results suggest that Cys-287 and His-288 are essential residues especially for the RNA helicase activity of JEV NS3 and the ATPase and helicase activities are separable enzymatic functions
Expression of the Human Poliovirus Receptor/CD155 Gene during Development of the Central Nervous System: Implications for the Pathogenesis of Poliomyelitis

AbstractThe gene for the human poliovirus receptor (hPVR/CD155) is the founding member of a new family of genes encoding proteins belonging to the immunoglobulin superfamily. To determine whether CD155 is expressed during mammalian development, we have made use of the previously characterized promoter of the CD155 gene and generated mice transgenic for a CD155 promoter-driven β-galactosidase reporter gene. Expression of the reporter gene in transgenic embryos was observed during midgestation in anterior midline structures of the developing central nervous system and in the neuroretina. During that period, reporter gene expression appeared within the notochord and floor plate along the entire spinal cord reaching into the caudal diencephalon. In addition, transgene expression was observed in axonal projections emanating from retinal ganglion cells forming the optic nerve to reach the future region of the optic chiasm. Analysis of expression of CD155 during human embryonic development confirmed the distribution of reporter gene expression specified by CD155 promoter activity. The anatomical distribution of CD155 promoter activity during embryogenesis matches that of transacting factors previously identified to regulate transcription of the CD155 gene. Expression of CD155 within embryonic structures giving rise to spinal cord anterior horn motor neurons may explain the restrictive host cell tropism of poliovirus for this cellular compartment of the CNS
Characterization of Modified Hepatitis C Virus E2 Proteins Expressed on the Cell Surface

AbstractThe envelope proteins of hepatitis C virus (HCV) are the likely targets of neutralizing antibodies and their molecular and functional characterization is relevant for vaccine development. We previously showed that surface-expressed E2 is a better immunogen than intracellular E2 and, therefore, we were interested in exploring more efficient ways to present E2 protein on the cell surface. We found that E2 targeted to the cell surface by replacement of its transmembrane domain did not bring E1 to the surface although E1 could be expressed independently on the cell surface if its transmembrane domain was similarly replaced. FACS analysis suggested that E2 expressed on the cell surface acquired its native conformation more efficiently when truncated at aa 661 than when truncated at aa 715. The shorter form of truncated E2 better retained the ability to bind the second extracellular loop (EC2) of CD81, the putative HCV receptor. Interestingly, deletion of the hypervariable region 1 (HVR1) did not perceptibly alter E2 structure; cell-surface forms of E2 lacking the HVR1 remained reactive with conformation-sensitive MAbs and were able to bind recombinant EC2 of CD81
Identification of Efficiently Cleaved Substrates for HIV-1 Protease Using a Phage Display Library and Use in Inhibitor Development

AbstractThe recognition sequences for substrate cleavage by aspartic protease of HIV-1 are diverse and cleavage specificities are controlled by complex interactions between at least six amino acids around the cleavage site. We have identified 45 efficiently cleaved peptide substrates of HIV-1 protease (PR) using substrate phage display, an approach that can elucidate both context-dependent and context-independent preferences at individual subsites of a protease substrate. Many of the selected peptides were cleaved more efficiently and had lower Km values than physiologically relevant substrates of HIV-1 PR. Therefore, mutations occurring in the cleavage sites of the Gag and Gag-pol polyproteins of HIV-1 could significantly lower the Km values to better compete against drugs for protease binding while maintaining cleavage rates necessary for viral replication. The most efficiently cleaved peptide substrate derived from these phage, Ac-GSGIF*LETSL-NH2, was cleaved 60 times more efficiently and had a Km approximately 260 times lower than a nine-amino-acid peptide based on the natural reverse transcriptase/integrase cleavage site when assayed at pH 5.6, 0.2 M NaCl. The peptide substrates selected served as frameworks for synthesis of tight binding reduced amide inhibitors of HIV-1 PR. The results show that the most efficiently cleaved substrates serve as the best templates for synthesis of the tightest binding inhibitors. Thus, defining changes in substrate preferences for drug-resistant proteases may aid in the development of more efficacious inhibitors
Mutational Analysis of the 5′ Leader Region of Simian Foamy Virus Type 1

AbstractThe sequence within the 5′ untranslated region of the retroviral genome contains important cis elements for many steps in viral replication. There is limited information available on the role of this region in foamy virus replication. Similar to other retroviruses, the 5′ untranslated region of foamy viruses predicts extensive RNA secondary structure. Serial mutations that could change parts of the predicted secondary structure were introduced in the 5′ leader sequence including the R-U5 region of simian foamy virus type 1 (SFV-1) to investigate their role in virus genome packaging and virus replication. Point mutations in the R-U5 regions at nucleotide positions 7–12 (I), 241–243 (B), and 256–257 (D) had no effect on virus replication. Base substitution mutation at positions 193–195 (C), however, severely impaired virus replication. Deletion of sequences in the leader region, between the primer-binding site and the gag gene, at positions 364–399 (d1), 397–435 (d2), or 364–435 (d3), which included sequences for RNA genome dimerization, also blocked SFV-1 replication. Interestingly, none of these mutations affected genome packaging or the synthesis of viral transcripts, suggesting that a step(s) of virus replication following packaging is affected. The region between the primer-binding site and the gag gene, therefore, is not essential for foamy virus genome packaging. Furthermore, the cis-acting elements for genome dimerization and packaging appear to be localized in separate regions for foamy viruses
Selection of a Subgroup A Avian Leukosis Virus [ALV(A)] Envelope Resistant to Soluble ALV(A) Surface Glycoprotein

AbstractThe host developing resistance to retroviral infection is believed to be a major force in the evolution of multiple receptor usage by retroviruses. The avian leukosis–sarcoma virus (ALV) group of retroviruses provides a powerful system for studying the envelope–receptor interactions involved in retrovirus entry; different members of this group of closely related viruses use distinct cellular receptors. Analysis of the ALV envelope subgroups suggests that the different ALVs evolved from a common ancestor by mutations in the env gene. Cells and animals that express subgroup A ALV envelope glycoproteins are highly resistant to ALV(A) infection due to receptor interference. In this study, we tested whether expression of a soluble form of subgroup A surface glycoprotein (SU) would result in receptor interference and whether this interference would select for resistant viruses with altered receptor usage. Chicken cells expressing the secreted ALV(A) SU immunoadhesin SU(A)-rIgG, which contains the subgroup A SU domain fused to the constant region of a rabbit immunoglobulin (IgG) heavy chain, showed significant receptor interference. A variant virus resistant to SU(A)-rIgG receptor interference was obtained. This virus had a six-amino-acid deletion in the subgroup A hr1 that altered receptor usage. This approach may identify regions of SU that play a critical role in receptor specificity
NS3 Serine Protease of Bovine Viral Diarrhea Virus: Characterization of Active Site Residues, NS4A Cofactor Domain, and Protease–Cofactor Interactions

AbstractThe gene expression of bovine viral diarrhea virus (BVDV), a pestivirus, occurs via translation of a hypothetical polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. A protease located in the N-terminal region of nonstructural (NS) protein NS3 catalyzes the cleavages, leading to the release of NS4A, NS4B, NS5A, and NS5B. Our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute together with the previously identified serine at position 1752 (S1752) the catalytic triad of the pestiviral NS3 serine protease. Interestingly, a mutant protease encompassing an exchange of the active site S1752 to threonine still showed residual activity. This finding links the NS3 protease of pestiviruses to the capsid protease of Sindbis virus. Furthermore, we observed that the minimal protease domain of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitive to N-terminal truncation because a deletion of 6 amino acids significantly reduced the cleavage efficiency at the NS4A/4B site. Larger N-terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to a different degree at each site. The NS3 protease of BVDV has previously been shown to depend on NS4A as cofactor. We demonstrate here that the central region of NS4A represents the cofactor domain. Furthermore, coprecipitation studies strongly suggest an interaction between NS4A and the N-terminal region of NS3. Besides the remarkable similarities observed between the pestiviral NS3 protease and the corresponding enzyme of hepatitis C virus (HCV), our results suggest a common ancestry between these enzymes and the capsid protease of Sindbis virus
Chimeric Langat/Dengue Viruses Protect Mice from Heterologous Challenge with the Highly Virulent Strains of Tick-Borne Encephalitis Virus

AbstractLangat virus (LGT), a tick-borne flavivirus, is naturally attenuated for humans but it is very virulent in SCID mice. In contrast, viable recombinant chimeras of LGT (preM and E genes) and dengue type 4 virus (all other sequences) recovered in mosquito cell culture were completely attenuated in SCID mice but still capable of providing protection against LGT. To develop the chimeras into vaccine candidates, we adapted them to replicate efficiently in simian Vero cells, a satisfactory substrate for human vaccines. The adapted chimeras remained completely attenuated for SCID mice and, significantly, provided protection in immunocompetent mice against tick-borne encephalitis virus, the most virulent of the tick-borne flaviviruses
Early Alteration of Nucleocytoplasmic Traffic Induced by Some RNA Viruses

AbstractA HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine · HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins
Mapping of Domains on the Human Parainfluenza Type 2 Virus P and NP Proteins That Are Involved in the Interaction with the L Protein

AbstractEleven monoclonal antibodies (MAbs) directed against the large (L) protein of human parainfluenza type 2 virus (hPIV-2) were prepared to examine the interactions of the L protein with other viral proteins. Coimmunoprecipitation assays using these MAbs revealed that the L protein directly interacted with the phospho- (P) and nucleocapsid (NP) proteins in vivo and in vitro. Mutational analysis of the P or NP protein was performed to identify the region(s) on these proteins interacting with L protein, indicating that amino acids 278–353 on the P protein and amino acids 403–494 on the NP protein are essential for the binding to the L protein
Tobacco Mosaic Virus Replicase-Mediated Cross-Protection: Contributions of RNA and Protein-Derived Mechanisms

AbstractSpecific sequences of the tobacco mosaic virus (TMV) RNA-dependent RNA-polymerase (RdRp) gene were investigated for their ability to confer cross-protection. Nine overlapping segments ranging from 713 to 1070 nucleotides in length and covering the methyltransferase, helicase, and polymerase (POL) domains of the TMV RdRp open reading frame were systemically expressed in Nicotiana benthamiana using a potato X virus (PVX) vector [Chapman, S., Kavanagh, T., and Baulcombe, D. C. (1992). Plant J., 1, 549–557]. PVX-infected plants were subsequently challenge inoculated with 10 μg of wild-type TMV and monitored for TMV accumulation. Mock inoculated plants and plants preinfected with the unmodified PVX vector rapidly accumulated high levels of challenge virus. In contrast, plants preinfected with PVX vectors expressing segments of the TMV RdRp open reading frame displayed either high or low levels of protection. High protection levels were observed for PVX constructs expressing segments of the TMV POL domain, whereas low protection levels were observed for PVX constructs expressing segments covering the methyltransferase and helicase domains. Frameshift mutations that blocked protein expression from RdRp segments disrupted only the high levels of protection derived from POL segments and not the low levels derived from the other segments. However, all RdRp segments conferred similarly high levels of protection against a TMV construct with restricted local movement. Thus both RNA and protein sequences in conjunction with the speed of the infecting challenge virus can affect the protection derived from the TMV RdRp gene
LuSIV Cells: A Reporter Cell Line for the Detection and Quantitation of a Single Cycle of HIV and SIV Replication

AbstractA single cycle of viral replication is the time required for a virus to enter the host cell, replicate its genome, and produce infectious progeny virions. The primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), require on average 24 h to complete one cycle of replication. We have now developed and characterized a reporter assay system in CEMx174 cells for the quantitative measurement of HIV/SIV infection within a single replication cycle. The SIVmac239 LTR (−225 → +149) was cloned upstream of the firefly luciferase reporter gene and this reporter plasmid is maintained in CEMx174 cells under stable selection. This cell line, designated LuSIV, is highly sensitive to infection by primary and laboratory strains of HIV/SIV, resulting in Tat-mediated expression of luciferase, which correlates with viral infectivity. Furthermore, manipulation of LuSIV cells for the detection of luciferase activity is easy to perform and requires a minimal amount of time as compared to current HIV/SIV detection systems. The LuSIV system is a powerful tool for the analysis of HIV/SIV infection that provides a unique assay system that can detect virus replication prior to 24 h and does not require virus to spread from cell to cell. Thus these cells can be used for the study of replication-deficient viruses and the high throughput screening of antivirals, or other inhibitors of infection
Enhanced Replication Contributes to Enrichment of Hepatitis B Virus with a Deletion in the Core Gene

AbstractAccumulation in immunosuppressed patients of hepatitis B virus (HBV) with a deletion in the C gene is associated with severe liver disease. The aim of this study was to determine the phenotype of such genomes in vitro. Four C gene fragments with different types of deletions were inserted in the context of a wild-type genome and tested by transfection into HuH7 cells. The deletions did not influence mRNA and surface protein levels. Truncated C gene translation products were expressed only from variants with in-frame deletions, whereas full-length polymerase was expressed from all variants at a similar or higher level than in wild-type virus. None of the variants was competent for autonomous replication; however, they produced 2- to 4.5-fold more progeny DNA than wild-type HBV when sufficiently complemented with wild-type core protein. Similarly, when variant and wild-type DNA were cotransfected in different ratios, the variants produced 2- to 5-fold more progeny DNA relative to the wild-type; this enrichment required the expression of the viral polymerase in cis. The mechanism of enrichment depended on the percentage of variant in the transfected DNA mixture. When the transfected DNA contained a small percentage of variant, enhanced replication of the variant accompanied by no or little suppression of wild-type replication was seen. Accordingly, overall production of progeny virus was slightly increased. At a high percentage of variant DNA, replication of both variant and wild-type decreased, probably due to a shortage of wild-type core protein. In conclusion, emergence of C gene deletion variants in vivo may be due to enhanced replication mediated at the level of encapsidation or reverse transcription. If the variants constitute a small part of the ccc DNA, they can be fully trans-complemented by wild-type virus which may increase the overall virus production