pinecone/core-2020-05-10-deduplication huggingface dataset:
Characterization of the Heparin-Mediated Activation of PKR, the Interferon-Inducible RNA-Dependent Protein Kinase

AbstractHeparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent eIF-2α protein kinase (PKR). We have used heparin oligosaccharides of defined lengths to examine the heparin-mediated activation of human PKR. Heparin oligosaccharide with 8 sugar residues was nearly as efficient as 16-residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas 6-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistically in activating PKR autophosphorylation. The RNA-binding activity of recombinant PKR, measured with adenovirusVARNA, was competed by poly(rI):poly(rC) but not by Hep-16. When the catalytically inactive, histidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a substrate for purified wild-type PKR, the intermolecular phosphorylation of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR but not by heparin-activated PKR. However, eIF-2α phosphorylation was catalyzed by both heparin- and dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosphorylation mediated either by Hep-16 or dsRNA, whereas preincubation with dsRNA either alone or in combination with Hep-16 did not impair subsequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detectable degradation of PKR during preincubation or subsequent autophosphorylation of PKR. These results suggest that, while both dsRNA and heparin are capable of activating PKR autophosphorylation, the structural and functional basis of PKR activation differs for these two classes of polyanionic biomolecules
Variability and Phylogeny of the L1 Capsid Protein Gene of Human Papillomavirus Type 5: Contribution of Clusters of Nonsynonymous Mutations and of a 30-Nucleotide Duplication

AbstractWe analyzed the variability and established the phylogeny of the L1 capsid protein gene of 33 isolates of human papillomavirus type 5 (HPV5) obtained from epidermodysplasia verruciformis patients from different continents. By comparing the sequences of a 419-bp fragment with those published for two Japanese isolates, we found 12.9% variable nucleotide positions, defining 25 variants with mutation rates ranging from 0.2 to 8.8%. Such a high intratypic diversity is unusual among HPVs. Nine of the 139 encoded amino acids were variable and 12 protein variants were identified. Fifteen of the 16 substitutions observed were clustered in two short regions. A 9-amino-acid insert, already reported for the Japanese HPV5b isolate, was found within one of the regions in five isolates. Our data support that the insert arose from the duplication of a 30-nucleotide sequence. Phylogenetic trees distributed the DNA variants into three subtypes (a to c) with a divergence higher than 4.5% and allowed the recognition of European and African lineages. By contrast with the trees based on the HPV5 E6 gene, HPV5a DNA variants and the HPV5b variants lacking the insert constituted a single group in the L1 amino acid tree, probably reflecting different levels of structural constraints for the HPV5 L1 and E6 proteins. In that respect, the short variable L1 sequences should represent less constrained regions
Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses

AbstractThe 3′-terminal half of the beet yellow stunt virus (BYSV) genome, 10,545 nt, has been cloned and sequenced. The sequenced portion of the BYSV genome encompasses 10 open reading frames (ORFs) and 241 nt of the 3′ untranslated region. The sequence spans, in the 5′ to 3′ direction, the C-terminal region of the replication-associated polyprotein gene (ORF 1a) which includes the set of motifs typical of helicases (HEL), the entire 53-kDa polymerase (RdRp) gene (ORF 1b), and genes encoding 30-kDa (ORF 2), 6-kDa (ORF 3), 66-kDa (ORF 4), 61-kDa (ORF 5), 25-kDa (ORF 6), 23.7-kDa (coat protein, CP) (ORF 7), 18-kDa (ORF 8), and 22-kDa (ORF 9) proteins. The double-stranded RNA “replicative form” of the BYSV was demonstrated to have a nontemplate G residue at the 3′ terminus of the (+) strand. The RdRp of BYSV is presumably expressed via a +1 ribosomal frameshift. The five-gene module conserved among closteroviruses was identified in BYSV; it includes a gene array coding for a 6-kDa small hydrophobic protein, a 66-kDa homolog of the cellular HSP70 heat shock proteins, a 61-kDa protein, and a 25-kDa diverged copy of the CP followed by the CP gene itself. Phylogenetic analysis of the replication-associated HEL and RdRp domains as well as proteins from the five-gene module demonstrated the closest relationship between BYSV and two other closteroviruses, beet yellows (BYV) and citrus tristeza (CTV) viruses. Like CTV, the BYSV genome contains a 30-kDa protein gene between the RdRp and the 6-kDa protein genes, and like BYV it has only two genes downstream of the CP gene. The organization of the BYSV genome appears to be intermediate between BYV and CTV, which suggests that these three viruses might represent three distinct but probably close stages in the closterovirus evolution
Nucleotide Sequencing and Generation of an Infectious Clone of Adeno-Associated Virus 3

AbstractWe have determined the complete nucleotide sequences of adeno-associated virus 3 (AAV-3) and generated an infectious clone. The single-stranded DNA genome of AAV-3 is 4726 nucleotides in length. The positive strand contains two large open reading frames; the left open reading frame encodes the nonstructural proteins and the right open reading frame encodes the structural proteins. The coding regions are flanked by identical inverted terminal repeat sequences containing palindromes. AAV-3 has little homology with the autonomous parvoviruses or erythroviruses but has 82% overall sequence homology with AAV-2. At the amino acid level there was 88% homology with AAV-2 nonstructural (Rep) proteins and 87% homology with AAV-2 capsid proteins. In addition, AAV-3 differed importantly from AAV-2 in the lack of a typical promoter sequence (TATA box) at p40 and the presence of the consensus sequence for adenovirus-related transcription factor E4F binding within the upstream region of the p5 promoter. These results suggest that AAV-3 not only consists of serologically distinct structural proteins but that viral propagation also may be controlled by different gene regulatory elements at the transcription level. The infectious clone confirmed the sequence and may be useful for developing new vectors for gene therapy
Evidence That the Soluble Factors Secreted by Activated Immune Cells Suppress Replication of Human Neurotropic JC Virus DNA in Glial Cells

AbstractCoordination of the immune response to viral infection and disease in the brain is believed to involve bidirectional interaction between the immune system and the central nervous system (CNS). Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the CNS that generally affects patients exhibiting an immunocompromised condition due to various illnesses. The human polyomavirus, JCV, which infects greater than 70% of the adult population is the etiological agent of this disease. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical signals. However, under immunocompromised conditions, the virus enters the lytic cycle, and upon cytolytic destruction of glial cells, causes PML. To understand the molecular mechanism underlying immune regulation of JCV replication, we have developed a cell culture system and have investigated the effect of soluble factors from T-cell cultures on replication of JCV DNA in glial cells. Our data demonstrate that replication of JCV DNA in the presence of PMA-stimulated T-cell supernatant is substantially decreased in transfected glial cells. Heat-inactivation and size-fractionation studies revealed participation of a heat labile factor(s) which loses its maximum activity at 60° and ranges between 30 and 100 kDa in size. The unfractionated T-cell supernatant and the fraction enriched in 30- to 100-kDa proteins reduced the level of viral DNA replication during the early phase of the lytic cycle. These observations suggest that regulatory factors which are secreted by immune cells may modulate the level of JCV DNA replication in glial cells. The importance of these observations in reactivation of JCV in immunocompromised individuals and development of PML is discussed
Both Codon Context and Leader Length Contribute to Efficient Expression of Two Overlapping Open Reading Frames of a Cucumber Necrosis Virus Bifunctional Subgenomic mRNA

AbstractThe importance of codon context and leader length in the translational regulation of p20 and p21 from the bifunctional 0.9-kb subgenomic mRNA of cucumber necrosis virus was investigated. Nucleotide substitutions introduced into the −3 and +4 positions of the p21 AUG codon (where the A of the AUG is +1) verified that purines in these positions are favored and demonstrated the similar contribution of the −3 and +4 positions to the efficiency of initiation codon selection in plants. The effect of nucleotide substitutions in the +5 position, most clearly demonstrated when pyrimidines occupy the −3 and +4 positions, also provided direct insight into the influence of the +5 position in plants. The codon context of the upstream p21 initiation codon affected expression from the downstream p20 AUG codon. In addition, an increase in the length of the subgenomic mRNA leader decreased expression from the downstream p20 initiation site. These latter observations are in accordance with the “Kozak rules” for accession of internal AUG codons by leaky ribosomal scanning and provide the first example of an effect of leader length on the efficiency of translation initiation in a plant (viral) mRNA
Lymphocytes Are the Major Reservoir for Foamy Viruses in Peripheral Blood

AbstractSimian and human foamy virus (FV) DNA can be readily detected in peripheral blood leukocytes. However, it is unknown which leukocyte populations harbor the virusin vivo.We, therefore, analyzed blood samples from nine African green monkeys, four chimpanzees, and two humans for the presence of foamy virus proviral DNA in different FACS-purified leukocyte populations, using a highly sensitive nested polymerase chain reaction (PCR). The CD8+lymphocytes were PCR positive in all 15 samples and the average viral burden was highest in this population. FV DNA was detected in 10 of 15 cell samples enriched for B lymphocytes, and 4 of 9 CD4+lymphocyte, 3 of 13 CD14+monocyte, and 4 of 13 polymorphonuclear leukocyte samples. A highly sensitive reverse transcriptase PCR was performed to detect viral transcripts in peripheral blood leukocytes. All samples were negative. In conclusion, lymphocytes, and especially CD8+T lymphocytes, were found to be a major target for foamy virus in the peripheral blood, but viral gene expression was not detected
Characterization of NIa Protease from Turnip Mosaic Potyvirus Exhibiting a Low-Temperature Optimum Catalytic Activity

AbstractNuclear inclusion protein a (NIa) protease of turnip mosaic potyvirus is responsible for the processing of the viral polyprotein into functional proteins. The NIa protease was found to exhibit its optimum catalytic activity at approximately 15° and a bell-shaped pH-dependent activity profile with a maximum at approximately pH 8.5. Kinetic studies showed that bothKmandVmaxvalues were lower at 12 than at 25° in all three different pH conditions, pH 7.0, 7.4, and 8.3, indicating that the higher activity at 12° is due to the lower value ofKm. Interestingly, the self-cleavage of the 27-kDa protease to generate the 25-kDa protease occurred more rapidly at 25 than at 12°, implying that the C-terminal self-cleavage site may interfere with the binding of the peptide substrate to the active site of the protease. Mutations and deletions at the C-terminal cleavage site had no effect on the temperature dependence of the proteolytic activity, demonstrating that the C-terminal self-cleavage is not related to the low-temperature optimum catalytic activity. The fluorescence measurement of the NIa protease upon temperature variation revealed that the protease undergoes a large conformational change between 2 and 42° and a drastic transition near 45°, suggesting that the low-temperature optimum catalytic activity is due to the highly flexible structure of the NIa protease
Rotavirus Stimulates IL-8 Secretion from Cultured Epithelial Cells

AbstractRotavirus is the most important cause of severe gastroenteritis in children worldwide. We have investigated cytokine responses to rotavirus infection of cultured intestinal epithelial cells. Interleukin 8 (IL-8) is a chemotactic and cell-activating cytokine that is synthesized by epithelial cells and induced in response to bacterial enteric pathogens. Rotavirus inoculation increased IL-8 mRNA levels in cultured intestinal epithelial cells within 2 hr of infection. IL-8 secretion increased 102- to 103-fold by 8 hr postinfection. Secretion of TNFα or IL-1β, cytokines which themselves increase IL-8 secretion, was not induced by rotavirus, nor was that of TNFα, IFNα, IFNγ, or IL-6. Neutralizing antibodies to TNFα or IL-1α/β did not affect the IL-8 response. Secretion of IL-8 was dependent on an intact viral capsid, as single-shell particles were inert. Neutralizing monoclonal antibodies (vp7-specific) that do not block cell attachment did block rotavirus stimulation of IL-8 secretion, indicating that attachment to the cell surface is not a sufficient stimulus to induce IL-8. Genetically inactivated rotavirus was also effective for IL-8 induction, indicating that viral replication was not required. These data suggest that epithelial cytokine IL-8 may be an important mediator of the host response to viral gastroenteritis pathogens such as rotavirus
Superinfection of HIV-2-Preinfected Macaques after Rectal Exposure to a Primary Isolate of SIVmac251

AbstractTo test the protection afforded by a weakly pathogenic HIV-2 isolate against the superinfection or development of SIV-induced disease, we intrarectally challenged six HIV-2-preinfected rhesus monkeys with a pathogenic isolate of SIVmac251. At the time of SIV challenge, none of these HIV-2-infected animals was positive for virus isolation, p27-Gag antigenemia, or HIV-2 provirus detection in PBMCs or peripheral lymph nodes. However, all monkeys exhibited anti-HIV-2 antibody titers ranging from 102to 103. Neutralizing antibodies against the challenge SIV strain were also detected in two animals. After rectal exposure to SIVmac251, five of the six HIV-2-preinfected macaques were superinfected. SIVmac251 DNA sequences were detected repeatedly in the PBMCs of the five superinfected animals and the two controls, whereas no HIV-2 provirus was detected for 14 months postchallenge. The one monkey that resisted superinfection was negative for all SIV infection criteria. This monkey exhibited the highest anti-SIV ELISA and cross-neutralizing antibody titers on the day of SIV challenge. Preinfection with a weakly pathogenic HIV-2 ROD isolate protected one of six macaques from infection with the closely related pathogenic SIVmac251 isolate, but no protection from the progression of disease was evidenced in the other five
The 5′ Terminal Sequence of Alfalfa Mosaic Virus RNA 3 Is Dispensable for Replication and Contains a Determinant for Symptom Formation

AbstractTransgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AlMV), can be infected with RNA 3 of the tripartitite AlMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter andnosterminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5′ terminal 79 nucleotides (nt). When the sequence corresponding to the 5′ terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5′ terminal deletion of 79 nt was produced. The 5′ truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3′-extended or 3′-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants
A Dominant Mutant Form of the Herpes Simplex Virus ICP8 Protein Decreases Viral Late Gene Transcription

AbstractThe herpes simplex virus (HSV) infected cell protein 8 (ICP8) is required for viral DNA replication and normal viral gene expression. Previous work in our laboratory has shown that ICP8 may play a role in stimulating late gene expression. In V2.6 cells which express thed105 mutant form of ICP8, synthesis of late proteins and accumulation of late gC mRNA are reduced during HSV infection (Gao, M., and Knipe, D. M.,J. Virol.65, 2666–2675, 1991). To determine if the negative effect ofd105 ICP8 on late gene expression was exerted at the transcriptional level, we measured the levels of mRNAs and transcription from three late genes, gC, UL47, and gD, in V2.6 cells and Vero cells infected with the HSV-1 wild-type virus. In infected V2.6 cells, the levels of late gC and UL47 mRNA were 7- to 12-fold lower than those of infected Vero cells under conditions where the levels of viral DNA replication in these two cell types were similar. The transcription levels of these two late genes in infected V2.6 cells were reduced to similar extents (9- to 14-fold). The levels of accumulated mRNA and transcription of the early–late gD gene also showed parallel reductions in infected V2.6 cells (about 6-fold). In contrast, transcription of the β pol gene was reduced only slightly (about 2-fold) byd105 ICP8. These results demonstrate that thed105 ICP8 inhibits expression of three viral late genes at the transcriptional level, and in general, the effect ofd105 ICP8 on viral gene expression appears to correlate with the extent to which expression of the gene is stimulated by viral DNA synthesis
Terminal Region Sequence Variations in Variola Virus DNA

AbstractGenome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of ≥98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of ≥65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted
Host Cell Effect upon Glycosylation and Antigenicity of Human Respiratory Syncytial Virus G Glycoprotein

AbstractInfection of different human epithelial cell lines with human respiratory syncytial virus (HRSV) revealed significant differences in the electrophoretic mobility of the viral attachment glycoprotein (G). Cell-type specific differences in G protein glycosylation were observed with certain lectins and sugar-specific reagents. Furthermore, substantial changes in the reactivity of the G glycoprotein with anti-G monoclonal antibodies were associated to the infected cell type. Strain-specific epitopes—present in a limited number of HRSV isolates of the same antigenic group—were particularly susceptible to cell-type-specific modifications of the mature G protein. Some of these epitopes, which were either exposed in the unglycosylated precursor or reproduced with synthetic peptides, were nonetheless masked in the mature G protein expressed in certain cell lines. Antigenic and electrophoretic mobility changes of the G glycoprotein were reverted in extracts of HEp-2 cells infected with HRSV grown in other cell types, indicating that phenotypic traits rather than selection of variants were associated to the above stated changes. These results highlight the importance of cell-type-specific modifications for HRSV G glycoprotein antigenicity and raise questions about the actual antigenic structure of this molecule when HRSV replicates in the respiratory tract
Genetic Characterization and Phylogeny of Sabiá Virus, an Emergent Pathogen in Brazil

AbstractSabiá virus, one of five arenaviruses from South America known to cause hemorrhagic fever in humans, emerged in 1990 when it was isolated from a fatal case in Sao Paulo, Brazil. Subsequently, it has caused two laboratory-acquired infections. Its natural distribution and host are still unknown. Using viral RNA and multiple polymerase chain reaction products as templates, the nucleotide sequence of the small (S) RNA segment of Sabiá virus, which codes for the nucleocapsid (N) and glycoprotein precursor, was determined. This virus shares an ambisense genome in common with other arenaviruses, although it has a unique predicted three stem–loop structure in the S RNA intergenic region. Phylogenetic analysis of a portion of the N gene sequence confirmed that Sabiá virus is distinct from all other members of the Arenaviridae and shares a progenitor with Junin, Machupo, Tacaribe, and Guanarito viruses
Syncytia Formation Induced by Coronavirus Infection Is Associated with Fragmentation and Rearrangement of the Golgi Apparatus

AbstractCoronavirus mouse hepatitis virus (MHV) possesses a membrane glycoprotein (M) which is targeted to the Golgi apparatus (GA). We used immunocytochemistry with an organelle-specific antiserum to investigate the morphologic changes of the GA during infection of L2 murine fibroblasts with MHV-A59. Twenty-four hours after infection the GA was fragmented and translocated in the center of syncytia, while the microtubular network was also rearranged displaying radiating elements toward the center of syncytia. Two fusion-defective mutants, which contain an identical amino acid substitution in the cleavage signal sequence of the spike glycoprotein (S), induced fragmentation of the GA. However, the GA migrated only partially to the centers of syncytia during infection with these mutants. Revertant viruses, in which the above mutation was corrected, had fusion properties and GA staining similar to wtMHV-A59. Experiments with brefeldin A (BFA), which induces redistribution of the GA into the rough endoplasmic reticulum (RER), revealed that an intact GA for a period of 4–16 hr postinfection, is required for coronavirus replication and syncytia formation. Thus, during MHV infection, syncytia formation is associated with fragmentation of the GA, followed by a previously undescribed phenomenon of migration of the organelle into the centers of syncytia. The fragmentation of the GA, however, may occur without the formation of syncytia. Therefore, two distinct mechanisms may be responsible for the fragmentation of the GA and its subsequent migration to the center of syncytia