pinecone/core-2020-05-10-deduplication huggingface dataset:
Interaction between Zucchini Yellow Mosaic Potyvirus RNA-Dependent RNA Polymerase and Host Poly-(A) Binding Protein

AbstractViral replication depends on compatible interactions between a virus and its host. For RNA viruses, the viral replicases (RNA-dependent RNA polymerases; RdRps) often associate with components of the host translational apparatus. To date, host factors interacting with potyvirus replicases have not been identified. The Potyviridae, which form the largest and most economically important plant virus family, have numerous similarities with the animal virus family, the Picornaviridae. Potyviruses have a single-stranded, plus sense genome; replication initiates at the viral-encoded, 3′ poly-(A) terminus. The yeast two-hybrid system was used to identify host plant proteins associating with the RdRp of zucchini yellow mosaic potyvirus (ZYMV). Several cDNA clones representing a single copy of a poly-(A) binding protein (PABP) gene were isolated from a cucumber (Cucumis sativus L.) leaf cDNA library. Deletion analysis indicated that the C-terminus of the PABP is necessary and sufficient for interaction with the RdRp. Full-length cucumber PABP cDNA was obtained using 5′ RACE; in vitro and Escherichia coli-expressed PABP bound to poly-(A)–Sepharose and ZYMY RdRp with or without the presence of poly-(A). This is the first report of an interaction between a viral replicase and PABP and may implicate a role for host PABP in the potyviral infection process
The Fitness of Citrus Tristeza Virus Defective RNAs Is Affected by the Lengths of Their 5′- and 3′-Termini and by the Coding Capacity

AbstractPopulations of the Closterovirus Citrus tristeza virus (CTV) generally contain defective RNAs (dRNAs) that vary in size, abundance, and sequence. The variation in abundance of the different dRNAs in a population suggests selection for those of higher fitness. To examine factors affecting fitness of dRNAs, we investigated a series of in vitro constructed dRNAs for their ability to be amplified in protoplasts by an efficiently replicated CTV deletion mutant. The minimal sequences required for accumulation of the dRNAs were within the genomic 5′ proximal ∼1 kb and the 3′ 270 nucleotides. However, other factors were involved, because a dRNA with only the minimal sequences failed to be replicated. Rescue of a nonviable dRNA by insertion of nonviral sequences between the termini suggested that “spacing” between terminal cis-acting signals influenced fitness. A continuous open reading frame (ORF) through most of the sequences derived from the 5′ of the genome was a requirement for dRNA amplification. In general, insertions, deletions, or nucleotide substitutions were tolerated in the dRNAs as long as an ORF was retained, whereas dRNAs with mutations that prematurely terminated the ORF were not viable. To discriminate between a requirement for an essential protein and ribosomal travel, perhaps to present replication signals to the replicase complex, mutations were made to modify the potential protein but still maintain an ORF. Deletions, insertions of nonviral sequences, or switching of reading frames that altered the amino acid sequence of the protein, except the N-terminal 161 amino acids, did not destroy the fitness of the dRNAs. Yet termination of the ORF in the middle of nonviral sequences did destroy the ability of the dRNAs to be amplified. These results suggest that even though a continuous ORF was needed for fitness, its protein product did not affect the amplification of the dRNAs
Mapping of the Tobacco Mosaic Virus Movement Protein and Coat Protein Subgenomic RNA Promoters in Vivo

AbstractThe Tobacco mosaic virus movement protein (MP) and coat protein (CP) are expressed from 3′-coterminal subgenomic RNAs (sgRNAs). The transcription start site of the MP sgRNA, previously mapped to positions 4838 (Y. Watanabe, T. Meshi, and Y. Okada (1984), FEBS Lett. 173, 247–250) and 4828 (K. Lehto, G. L. Grantham, and W. O. Dawson (1990), Virology 174, 145–157) for the TMV OM and U1 strains, respectively, has been reexamined and mapped to position 4838 for strain U1. Sequences of the MP and CP sgRNA promoters were delineated by deletion analysis. The boundaries for minimal and full MP sgRNA promoter activity were localized between −35 and +10 and −95 and +40, respectively, relative to the transcription start site. The minimal CP sgRNA promoter was mapped between −69 and +12, whereas the boundaries of the fully active promoter were between −157 and +54. Computer analysis predicted two stem–loop structures (SL1 and SL2) upstream of the MP sgRNA transcription start site. Deletion analysis and site-directed mutagenesis suggested that SL1 secondary structure, but not its sequence, was required for MP sgRNA promoter activity, whereas a 39-nt deletion removing most of the SL2 region increased MP sgRNA accumulation fourfold. Computer-predicted folding of the fully active CP sgRNA promoter revealed one long stem–loop structure. Deletion analysis suggested that the upper part of this stem–loop, located upstream of the transcription start site, was essential for transcription and that the lower part of the stem had an enhancing role
Soluble, Template-Dependent Extracts from Nicotiana benthamiana Plants Infected with Potato Virus X Transcribe both Plus- and Minus-Strand RNA Templates

AbstractWe have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3′ terminal region were not copied, suggesting that elements in the 3′ region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis
Molecular Studies on Bromovirus Capsid Protein VII. Selective Packaging of BMV RNA4 by Specific N-Terminal Arginine Residues

AbstractAn arginine-rich RNA-binding motif (ARM) found at the N-proximal region of Brome mosaic virus (BMV) coat protein (CP) adopts α-helical conformation and shares homology with CPs of plant and insect RNA viruses, HIV-Rev and Tat proteins, bacterial antiterminators, and ribosomal splicing factors. The ARM of BMV CP, consisting of amino acids 9 through 21 with six arginine residues, is essential for RNA binding and subsequent packaging. In this study analysis of the α-helical contents of wild-type and mutant peptides by circular dichroism spectra identified protein determinants required for such conformation. Electrophoretic mobility-shift assays between viral RNA and BMV CP peptides with either proline or alanine substitutions revealed that the interaction is nonspecific. Expression in vivo of mature full-length BMV CP subunits, having the same substitutions for each arginine within the ARM, derived from biologically active clones was found to be competent to assemble into infectious virions and cause visible symptom phenotypes in whole plants. However, analysis of virion progeny RNA profiles of CP variants and subsequent in vitro reassembly assays between mutant CP and four BMV RNAs unveiled the ability of arginine residues at positions 10, 13, or 14 of the ARM to confer selective packaging of BMV RNA4. Thus, BMV CP contains determinants that specifically interact with RNA4 to ensure selective packaging
Differential Phosphorylation of the HPV-16 E7 Oncoprotein during the Cell Cycle

AbstractThe human papillomavirus type 16 encodes two principal oncoproteins, E6 and E7. The E7 protein has been shown to deregulate the cell cycle through interactions with a variety of proteins involved in cell cycle control and transcriptional regulation. These activities result in E7 being able to cooperate with activated oncogenes in the transformation of primary rodent cells, and with the viral E6 protein during human keratinocyte immortalization. Although a large number of activities have been ascribed to the E7 protein, little is known about its regulation during the cell cycle. We have performed a series of studies to investigate potential changes in E7 phosphorylation during the cell cycle and we show that E7 is indeed differentially phosphorylated. Casein kinase II is the principal kinase during the early part of the cell cycle, but this activity decreases rapidly as cells progress toward S phase. In addition, E7 is transiently phosphorylated at Ser71 in S phase by another, as yet unknown, kinase. These results demonstrate differential regulation of the E7 protein during the cell cycle that most likely represents a means of providing specificity to E7's activities
A New Vector System with Inducible E2a Cell Line for Production of Higher Titer and Safer Adenoviral Vectors

AbstractAdenoviral vectors have been used in gene therapy and for vaccination. The major concerns with using adenoviral vectors are the pathogenic potential of the virus backbone and the generation of replication-competent adenovirus that may replicate in an uncontrolled manner, especially in immunocompromised patients. It is important to develop new vectors that are safer for clinical trials while maintaining high titer and efficient transduction. A new adenovirus vector production system was developed, which includes several vector backbone plasmids deleted for E2a and a new cell line expressing both E1 and E2a. The new cell line with the tTA-inducible E2a expression cassette can significantly increase the titer of E1/E2a-deleted vectors by four to five orders of magnitude upon withdrawal of tetracycline. Furthermore, there is no sequence overlap between the vector and the cellular DNA within the E2a open reading frame and downstream, making the generation of virus with wild-type E2a through homologous recombination substantially less likely. The new vector system may improve the safety of vectors for vaccination and cancer therapy and may also provide safer backbones for further vector development, such as helper-dependent and hybrid vectors
Identification and Elimination of an Aberrant Splice Product from cDNAs Encoding the Human Adenovirus Type 5 E4orf6 Protein

AbstractGrowing awareness of the central role of the E4orf6 protein in regulating the infectious cycle of human adenoviruses has led to greatly intensified efforts to define its functions and mechanisms of action. Many workers employ cDNAs to generate plasmid or viral vectors to express E4orf6 in the absence of other viral products. In addition to the normal 34-kDa product, such vectors consistently produce a polypeptide of about 8 kDa. In the present report we show that this protein is produced by an aberrant mRNA utilizing the 5′ splice donor site used normally by the virus to produce the E4orf6/7 product, which shares 58 residues with E4orf6. This amino terminal coding sequence is linked to a 3′ sequence via a novel splice acceptor site in an alternative reading frame of the E4orf6 cDNA. The 5′ donor site was altered by PCR-directed mutagenesis to yield a construct that produces high levels of E4orf6 in the absence of the 8-kDa polypeptide. This construct should eliminate some of the problems encountered previously using the wild-type E4orf6 coding sequence
Site-Specific Single Amino Acid Changes to Lys or Arg in the Central Region of the Movement Protein of a Hybrid Bromovirus Are Required for Adaptation to a Nonhost

AbstractA hybrid Cowpea chlorotic mottle virus (CCMV) [CCMV(B3a)] in which the CCMV 3a movement protein gene is replaced by the 3a (B3a) gene of Brome mosaic virus cannot infect cowpea systemically. Previously, analysis of RNA3 cDNA clones constructed from cowpea-adapted mutants derived from CCMV(B3a) revealed that a single codon change in the B3a gene allowed CCMV(B3a) to infect cowpea systemically. In this study, to extend the analysis of the CCMV(B3a) adaptation mechanism, we directly sequenced B3a gene RT-PCR products prepared from 28 cowpea plants in which cowpea-adapted mutants appeared, and found seven patterns of a codon change localized at five specific positions in the central region (Ser118, Glu132, Glu138, Gln178, and Ser180). All of the patterns involved an amino acid change to Lys or Arg. Mutational analysis of the B3a gene demonstrated that a single codon change resulting in either Lys or Arg at any of the five positions was sufficient for the adaptation of CCMV(B3a) to cowpea. In contrast, CCMV(B3a) variants with a codon change resulting in Lys or Arg at three other positions (137, 155, and 161) in the B3a gene not only showed lack of systemic infection of cowpea but also showed weakened initial cell-to-cell movement in the inoculated leaves and diminished B3a accumulation in protoplasts. These results suggest that adaptive changes in the B3a gene are site-specifically selected in cowpea plants
Modulation of Moloney Leukemia Virus Long Terminal Repeat Transcriptional Activity by the Murine CD4 Silencer in Retroviral Vectors

AbstractWe investigated whether CD4 gene regulatory sequences might be useful for developing transcriptionally targeted Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors for gene expression specifically in CD4+ cells. We could modulate Mo-MLV long terminal repeat (LTR) activity by inserting a 438-bp-long fragment containing the murine CD4 silencer in the LTR of the vector; both β-galactosidase and green fluorescent protein reporter gene activities were strongly down-regulated in both murine and human CD8+ cells, but not in CD4+ lymphoid cell lines and freshly isolated lymphocytes transduced with this vector, compared with the findings using a control vector carrying wild-type LTRs. Titration experiments on NIH-3T3 cells revealed that inclusion of the CD4 silencer in the LTRs did not reduce the titer of the vectors. These findings indicate that a cellular silencer can be successfully included in retroviral vectors, where it maintains its transcription-regulatory function, thus suggesting a novel approach to transcriptional targeting
Distribution and Quantification of Human Immunodeficiency Virus Type 1, Strain JC499, Proviral DNA in Tissues from an Infected Chimpanzee

AbstractData are accumulating to show that the natural history of human immunodeficiency virus type 1 (HIV-1) in chimpanzees closely reproduces that in humans and is influenced by biologic properties of the infecting HIV-1 strain. To determine the distribution and relative amounts of HIV-1, proviral DNA in multiple tissues from a chimpanzee euthanized because of an abdominal tumor and kidney failure was quantified by nested PCR limiting-dilution assays. At death, 21 months after infection with HIV-1JC499, this animal had a CD4+ T-cell count of 268 and 1.7 × 105 copies of virion RNA/ml of plasma. The highest proviral burdens were in peripheral lymph nodes and blood, followed by lung, colon, and spleen; values ranged from 130 to 3350 proviral copies/μg DNA (equivalent to DNA in 150,000 cells). The lowest levels of virus were in the spinal cord, brain, and cecum (0.3 to 2.5 copies/μg DNA), with all other tissues harboring intermediate levels (6.8 to 114 copies/μg DNA). Viral burdens in all tissues were comparable to or greater than those reported for HIV-1-infected humans in all stages of disease. Immunohistochemistry for HIV-1 p24 Gag antigen revealed (i) trapping of HIV-1 on follicular dendritic cells in lymph node germinal centers and (ii) virus in the brain, where it was localized primarily to capillary endothelial cells in the cerebral cortex. Analysis of the genetic diversity of the Env V3 loop in tissues indicated that there was no apparent compartmentalization of HIV-1 variants. Of interest, in 83 of 94 (88.3%) clones sequenced, the unique GYGR motif at the tip of the V3 loop of HIV-1JC499 had reverted to the more common GPGR. The results support the conclusion that HIV-1 has the potential to maintain high viral burdens in chimpanzees and to disseminate to most organs, including the central nervous system. The use of the chimpanzee model with HIV-1JC499 (or related strains) in vaccine efficacy studies should prove valuable, especially when assessing protection against disease. Furthermore, comparison of both replicative properties of HIV-1JC499 with SIVcpz strains and immune responses of chimpanzees infected with these viruses might provide new information about HIV pathogenesis
In Vitro Assembly of Bacteriophage P4 Procapsids from Purified Capsid and Scaffolding Proteins

AbstractBacteriophage P4 is a satellite virus of bacteriophage P2, which has acquired the ability to utilize the structural gene products of P2 to assemble its own capsid. The normal P2 capsid has a T = 7 icosahedral structure comprised of the gpN-derived capsid protein, whereas the capsid produced under the control of P4 has a smaller, T = 4 structure. The protein responsible for this size determination is the P4-coded gene product Sid, which forms an external scaffold on the P4 procapsid. Using an in vitro assembly system, we show that gpN and Sid can coassemble into procapsid-like particles, indistinguishable from those produced in vivo, in the absence of any other gene products. The fidelity of the assembly reaction is enhanced by the inclusion of PEG and has a pH optimum between 8.0 and 8.5. Analysis of the assembly properties of truncated versions of Sid and gpN suggests that the amino-terminal part of Sid is involved in gpN binding, while the carboxyl-terminal part forms trimeric Sid–Sid interactions, and that the first 31 amino acids of gpN are required for binding to Sid as well as for size determination
Identification and Analysis of Vaccinia Virus Palmitylproteins

AbstractVaccinia virus encodes at least eight proteins that incorporate label from tritiated palmitic acid when it is added to infected cell cultures. Three of these palmitylproteins are encoded by the A33R, B5R, and F13L open reading frames and migrate by gel electrophoresis with relative molecular masses of 23–28, 42, and 37 kDa, respectively. In this report we provide evidence that the A22R and A36R open reading frames also encode palmitylproteins with apparent molecular masses of 22 and 50–55 kDa, respectively. Furthermore, the hemagglutinin protein (A56R) from the Copenhagen strain is shown to be palmitylated while the hemagglutinin protein from the WR and IHD-J strains is not. A 94-kDa VV palmitylprotein appears to be a multimeric complex composed of the B5R protein and possibly others. All vaccinia-encoded palmitylproteins are present in the membranous fraction of cells and are specific for the trans-Golgi network membrane-enveloped forms of the virus, suggesting that these proteins play a role in the envelopment and egress of virions or the infectivity of released virus
Single Amino Acid Differences Are Sufficient for CD4+ T-Cell Recognition of a Heterologous Virus by Cattle Persistently Infected with Bovine Viral Diarrhea Virus

AbstractCattle that are persistently infected (PI) with one strain of bovine viral diarrhea virus (BVDV) can resolve infection with a second, antigenically heterologous strain but not the homologous strain. Since CD4+ T cells are thought to be critical for the resolution of acute BVDV infection (Howard et al., 1992, Vet. Immunol. Immunopathol. 32, 303–314), we have examined the recognition of a heterologous virus (NADL) by CD4+ T cells from Pe515-PI animals. The immune response of non-PI control cattle challenged with NADL or Pe515ncp was strain cross-reactive, whereas Pe515-PI animals responded to NADL only. The immune repertoire of both groups included NS3, which differs by approximately 1% (9/683) amino acids between these two viruses. Lymphoproliferative responses to proteins and synthetic peptides corresponding to three nonconservative differences in NS3 demonstrated that CD4+ T cells from non-PI control animals responded well to proteins but poorly to the peptides from both viruses. In contrast, PI animals were responsive to heterologous proteins and peptides but nonresponsive to the homologous equivalents. A single amino acid difference between the two sequences was sufficient to allow responsiveness
Involvement of Oxidative Stress, NF-IL-6, and RANTES Expression in Dengue-2-Virus-Infected Human Liver Cells

AbstractThe liver has been suspected to be one of the major targets of dengue virus infection. Here, we detected increasing secretion of the chemokine RANTES (regulated upon activation, normal T cell expressed and secreted), which functions to recruit the immune cells, in dengue-virus-infected liver cells and patients. Three luciferase reporter genes with various deletions at the 5′-end of the RANTES promoter were constructed to explore the RANTES activation mechanism in human liver cells. The reporter gene was optimally activated by dengue-2 virus when the RANTES promoter contains the region from the transcription starting site (+1) to the nucleotide at the −181 position. NF-IL-6 and an undefined factor forming DNA–protein complexes in the RANTES promoter E and A/B regions in the infected cells were demonstrated by electrophoretic mobility shift assay. Further analysis showed that oxidative stress was an upstream inducer of NF-IL-6 and RANTES signaling in dengue-virus-infected liver cells. This finding was demonstrated by three antioxidants (N-acetyl-l-cysteine, nitro-l-arginine methyl ester, and pyrrolidine dithiocarbamate) used to suppress the activation. In contrast, the DNA binding activity of the undefined factor was not affected by the antioxidant treatment, indicating the existence of an oxidant-independent pathway. We hypothesize that dengue virus infection of the liver cells may trigger both an oxidant-dependent and an oxidant-independent pathway to up-regulate RANTES mRNA expression through activating NF-IL-6 and an undefined factor, respectively. In conclusion, the present study suggests a new direction for the study of liver pathogenesis involving RANTES in host immune responses during dengue virus infection