pinecone/core-2020-05-10-deduplication huggingface dataset:
Hepatitis C Virus Core Protein Is Necessary for the Maintenance of Immortalized Human Hepatocytes

AbstractHepatitis C virus (HCV) core protein has many intriguing properties and plays an important role in cell growth regulation. We have recently shown that the HCV core protein from genotype 1a promotes primary human hepatocytes to an immortalized phenotype. Here, we investigated whether the presence of core protein is necessary for maintenance of the immortalized hepatocytes and investigated its consequences on cellular gene expression. The introduction of an antisense orientation of the core gene into immortalized hepatocytes led to the onset of cell death. Further analysis suggested that cell death occurred through apoptosis associated with the activation of tumor suppressor pathways. Antisense core gene expression in immortalized hepatocytes increased p53 expression at both the mRNA and the protein levels. A decreased telomere length and reduced c-myc protein expression were also observed in hepatocytes when the antisense core gene was introduced. Results from these studies suggested that modulation of cell cycle regulatory genes by repression of core protein expression is responsible for reversion of the immortalized phenotype of the hepatocytes. Thus, targeted inhibition may contribute to the development of new therapeutic modalities for prevention of HCV core protein function
Mutations in NS5B Polymerase of Hepatitis C Virus: Impacts on in Vitro Enzymatic Activity and Viral RNA Replication in the Subgenomic Replicon Cell Culture

AbstractHepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique β-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and β-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the β-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR345), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416–8428)
Viremia and Immunogenicity in Nonhuman Primates of a Tetravalent Yellow Fever–Dengue Chimeric Vaccine: Genetic Reconstructions, Dose Adjustment, and Antibody Responses against Wild-type Dengue Virus Isolates

AbstractChimeric yellow fever (YF)–dengue (DEN) viruses (ChimeriVax–DEN) were reconstructed to correct amino acid substitutions within the envelope genes of original constructs described by Guirakhoo et al. (2001, J. Virol. 75, 7290–7304). Viruses were analyzed and compared to the previous constructs containing mutations in terms of their growth kinetics in Vero cells, neurovirulence in mice, and immunogenicity in monkeys as monovalent or tetravalent formulations. All chimeras grew to high titers [∼7 to 8 log10, plaque-forming units (PFU)/ml] in Vero cells and were less neurovirulent than YF 17D vaccine in mice. For monkey experiments, the dose of DEN2 chimera was lowered to 3 log10 PFU in the tetravalent mixture in an effort to reduce its dominant immunogenicity. The magnitude of viremia in ChimeriVax–DEN immunized monkeys was similar to that of YF-VAX, but significantly lower than those induced by wild-type DEN viruses. All monkeys developed high levels of neutralizing antibodies against homologous (chimeras) or heterologous (wild-type DEN viruses isolated from different geographical regions) viruses after a single dose of monovalent or tetravalent vaccine. Administration of a second dose of tetravalent vaccine 2 months later increased titers to both homologous and heterologous viruses. A dose adjustment for dengue 2 chimera resulted in a more balanced response against dengue 1, 2, and 3 viruses, but a somewhat higher response against chimeric dengue 4 virus. This indicates that further formulations for dose adjustments need to be tested in monkeys to identify an optimal formulation for humans
Structural Features of Envelope Proteins on Hepatitis C Virus-like Particles as Determined by Anti-envelope Monoclonal Antibodies and CD81 Binding

AbstractThe envelope glycoprotein E2 of hepatitis C virus (HCV) is a major component of the viral envelope. Knowledge of its topologic features and antigenic determinants in virions is crucial in understanding the viral binding sites to cellular receptor(s) and the induction of neutralizing antibodies. The lack of a robust cell culture system for virus propagation has hampered the characterization of E2 presented on the virion. Here we report the structural features of hepatitis C virus-like particles (HCV-LPs) of the 1a and 1b genotypes as determined by various mouse and human monoclonal anti-envelope antibodies. Our results show that the E2 protein of HCV-LPs reacts with human monoclonal antibodies recognizing conformational determinants. Monoclonal antibodies (mAbs) specific for the hypervariable region 1 (HVR-1) sequence reacted strongly with HCV-LPs, suggesting that the HVR-1 is exposed on the viral surface. Several mAbs recognized both HCV-LPs with equally high affinity, indicating that the corresponding epitopes [amino acids (aa) 192–217 of E1 and aa 412–423, aa 522–531, and aa 640–653 of E2] are conserved in both genotypes and exposed on the surface of the HCV-LP. The E2 and E1/E2 dimers of 1a bound strongly to the recombinant large extracellular loop (LEL) of CD81 (CD81–LEL) of human and African green monkey, while the HCV-LP of 1a bound weakly to human CD81–LEL. E1/E2 dimers and the HCV-LPs of 1b did not bind CD81–LEL, consistent with the notion that CD81 recognition by E2 is strain-specific and does not correlate with permissiveness of infection. A model of the topology and exposed antigenic determinants of the envelope proteins of HCV is proposed
Human Cytomegalovirus-Mediated Induction of MIF in Fibroblasts

AbstractHuman cytomegalovirus (HCMV) infection of fibroblasts induces the proinflammatory mediator macrophage migration inhibitory factor (MIF). Our in vitro experiments show that active HCMV infection alone is required to induce an early and sustained induction of MIF mRNA and protein production. Unlike in other infection models, in which MIF has been described to be released from preformed stores, our data conclusively show that HCMV infection triggers de novo synthesis and subsequent secretion of MIF. The kinetics of MIF protein production during HCMV infection points to an efficacious immune modulation, in which lymphocytes and monocytes are initially recruited by the early release of chemokines to sites of infection and locally activated by increasing concentrations of MIF
Mapping of B-Cell Epitopes of Hemagglutinin Protein of Rinderpest Virus

AbstractMonoclonal antibodies (mAbs) against secreted hemagglutinin (H) protein of rinderpest virus (RPV) expressed by a recombinant baculovirus were generated to characterize the antigenic sites on H protein and regions of functional significance. Three of the mAbs displayed hemagglutination inhibition activity and these mAbs were unable to neutralize virus infectivity. Western immunoblot analysis of overlapping deletion mutants indicated that three mAbs recognize antigenic regions at the extreme carboxy terminus (between amino acids 569 and 609) and the fourth mAb between amino acids 512 and 568. Using synthetic peptides, aa 569–577 and 575–583 were identified as the epitopes for E2G4 and D2F4, respectively. The epitopic domains of A12A9 and E2B6 mAbs were mapped to regions encompassing aa 527–554 and 588–609. Two epitopes spanning the extreme carboxy terminal region of aa 573 to 587 and 588 to 609 were shown to be immunodominant employing a competitive ELISA with polyclonal sera form vaccinated cattle. The D2F4 mAb which recognizes a unique epitope on RPV-H is not present on the closely related peste des petits ruminant virus HN protein and this mAb could serve as a tool in the seromonitoring program after rinderpest vaccination
Growth Characteristics of a Highly Virulent, a Moderately Virulent, and an Avirulent Strain of Equine Arteritis Virus in Primary Equine Endothelial Cells Are Predictive of Their Virulence to Horses

AbstractEquine viral arteritis (EVA) is an endotheliotropic viral disease of horses caused by equine arteritis virus (EAV). Although there is only one serotype of EAV, there is marked variation in the virulence of different strains of the virus. The replication and cytopathogenicity of three well-characterized strains of EAV of different virulence to horses were compared in rabbit kidney (RK-13) and primary equine pulmonary artery endothelial cells (ECs). Viral protein expression, plaque size, and cytopathogenicity of all three viruses were similar in RK-13 cells, whereas two virulent strains of EAV were readily distinguished from an avirulent strain by their plaque morphology and cytopathogenicity in primary equine ECs. Furthermore, EAV nucleocapsid protein was detected by flow cytometric analysis significantly later in ECs infected with the avirulent than those infected with the virulent strains of EAV. Primary equine ECs provide a convenient and relevant model for in vitro characterization of the pathogenesis of EVA and the virulence determinants of EAV
Role of G Protein β3 Subunit C825T and HLA Class II Polymorphisms in the Immune Response after HBV Vaccination

AbstractThe G protein β3 (GNB3) subunit and HLA are candidate genes predictive of immune response capacity. We therefore studied the influence of both gene systems on cellular and humoral immunity against hepatitis B virus (HBV) in 79 HBV booster-vaccinated healthy volunteers and an independent group of 77 probands after HBV basic immunization. Following booster vaccination, lymphocyte in vitro proliferation after stimulation with HBV surface antigen was 2.5-fold increased in GNB3 825T (TC + TT) vs CC allele carriers (P = 0.01) and was not influenced by HLA-DRB1 or DQB1 alleles. In addition, anti-HBs antibody titers in both groups were 2-fold increased in TC vs CC and decreased in TT vs CC allele carriers. However, antibody titers after HBV booster immunization were elevated in HLA-DQB1*0301 carriers (P corrected = 0.027). In summary, the GNB3 825T allele appears as a marker particularly predictive of cellular and HLA-DQB1*0301 of humoral immune responses following HBV vaccination
Interferon Regulatory Factor-1, Interferon-β, and Reovirus-Induced Myocarditis

AbstractViral myocarditis is an important human disease, and reovirus-induced myocarditis in mice provides an excellent model to study direct viral damage to the heart. Previously, we showed that reovirus induction of and sensitivity to interferon-β (IFN-β) is an important determinant of viral pathogenicity in the heart and that the transcription factor interferon regulatory factor-3 (IRF-3) is required for reovirus induction of IFN-β in primary cardiac myocyte cultures. Given several lines of evidence suggesting a possible distinctive environment for IRFs in the heart, we have now focused on IRF-1. Previous studies demonstrated that viruses, double-stranded-RNA (dsRNA), and IFN-α/β can each induce IRF-1 and that IRF-1 plays a role in dsRNA, but perhaps not viral, induction of IFN-α/β. Importantly, none of these studies used a virus with a dsRNA genome (such as reovirus), none of them used a highly differentiated nonlymphoid cell type, and none of them addressed whether viral induction of IRF-1 is direct or is mediated through viral induction of IFN-β. Indeed, as recently as this year it has been assumed that viral induction of IRF-1 is direct. Here, we found that reovirus induced IRF-1 in primary cardiac myocyte cultures, but that IRF-1 was not required for reovirus induction of IFN-β. Surprisingly, we found that reovirus failed to induce IRF-1 in the absence of the IFN-α/β response. This provides the first evidence that viruses may not induce IRF-1 directly. Finally, nonmyocarditic reovirus strains induced more cardiac lesions in mice deficient for IRF-1 than they did in wildtype mice, directly demonstrating a protective role for IRF-1. Together, the results indicate that while IRF-1 is downstream of the IFN-β response, it plays an important protective role against viral myocarditis
A Novel Class of Large and Infectious Defective RNAs of Citrus Tristeza Virus

AbstractCitrus tristeza virus (CTV)-infected plants contain one or more populations of defective RNAs (dRNAs), mostly with a size range of ca. 2.0 to 5.0 kb. Several CTV dRNAs have been characterized and found to consist mainly of the two termini of the genomic RNA, with extensive internal deletions. The present paper describes a new class of large (∼12.0 kb) dRNAs from three different CTV isolates with two unusual features. First is their composition with intact replicase genes. These dRNAs contained a large 5′ portion of the genomic RNA terminus, which apparently corresponded to the recently described 5′ large single-stranded subgenomic RNA (sgRNA) of ORF1a+1b (Che et al., 2001). The 3′ portion of the large dRNAs varied among the 10 different cDNA clones examined in this work. In 2 dRNAs this portion consisted of truncated ORF10 (p20), and in 5 dRNAs it contained truncated ORF11 (p23). Two dRNA molecules were found with a 3′ portion that started in the exact 5′ position of the intergenic region between the p20 and p23 ORFs. In one dRNA, this portion coincided with the full-length sgRNA corresponding to ORF10. The second unusual feature was their ability to be readily transmitted mechanically to citrus plants by stem slashing and also to Nicotiana benthamiana protoplasts. The possibility that these dRNAs may be encapsidated and be capable of self-replication is discussed
Molecular Interactions and Viral Stability Revealed by Structural Analyses of Chemically Treated Cypovirus Capsids

AbstractCytoplasmic polyhedrosis virus (CPV, genus Cypovirus) is a unique member of the family Reoviridae which lacks the outer protective shells that exist in all other members, yet exhibits unusual stability. We have analyzed the effects of different acidic, basic, detergent, and urea treatments on CPV capsids. The integrity of the CPV capsids was unaffected under high-pH conditions that disrupted the orthoreovirus inner core, consistent with its ability to maintain structural integrity in extremely alkaline environments during infection. However, it was sensitive to low pH, detergents, and urea, similarly to other viruses in this family. The three-dimensional structure comparisons by electron cryomicroscopy of the intact empty CPV capsid with the “spikeless” capsid whose turrets were removed by chemical treatments revealed the interaction footprint of the turret on the capsid shell. The observed structural changes associated with the removal of the turret suggest critical structural roles of the turret in maintaining capsid integrity in addition to its enzymatic activities
Characterization of Neutralization Specificities of Outer Capsid Spike Protein VP4 of Selected Murine, Lapine, and Human Rotavirus Strains

AbstractNeutralization specificities of outer capsid spike protein VP4 of murine rotavirus strains EW (P?[16],G3) and EHP (P?[20],G3) and lapine rotavirus strains Ala (P?[14],G3), C11 (P?[14],G3), and R2 (P?[14],G3) as well as human rotavirus strains PA169 (P?[14],G6) and HAL1166 (P?[14],G8) were determined by two-way cross-neutralization. This was done by generating and characterizing (i) three murine × human, three lapine × human, and two human × human single gene substitution reassortant rotaviruses, each of which bore identical human rotavirus DS-1 strain VP7 (G2), and (ii) guinea pig hyperimmune antiserum raised against each reassortant. Reference rotavirus strains employed in the study represented 10 established VP4 (P) serotypes, including 1A[8], 1B[4], 2A[6], 3[9], 4[10], 5A[2], 5B[2], 5B[3], 6[1], 7[5], 8[11], 9[7], and 10[16] as well as a P serotype unknown P[18]. Murine rotavirus strains EW and EB were demonstrated to share the same P serotype (P10[16]) distinct from (i) 9 established P serotypes, (ii) lapine and human rotavirus strains bearing the P[14] genotype, and (iii) an equine rotavirus strain bearing the P[18] genotype. Both lapine (Ala, C11, and R2) and human (PA169 and HAL1166) rotaviruses were shown to belong to the same VP4 serotype, which represented a distinct new P serotype (P11[14]). P serotype 13[20] was assigned to murine rotavirus EHP strain VP4, which was shown to be distinct from all the P serotypes/genotypes examined in the present study
Interleukin-8 Gene Regulation in Intestinal Epithelial Cells Infected with Rotavirus: Role of Viral-Induced IκB Kinase Activation

AbstractRotavirus is the major etiologic agent of diarrhea in children and the most common cause of severe pediatric gastroenteritis. Rotavirus infection is limited to mature enterocytes that line the villi of the small intestine. Gut epithelial cells, upon infection and cytokine stimulation, are able to produce chemokines, a family of small chemotactic cytokines that regulate the migration and activation of leukocytes. We have previously shown that rotavirus infection of the intestinal epithelial cell line HT-29 induces increased expression of the CXC chemokine interleukin- (IL) 8. Mechanisms responsible for the transcriptional regulation of the IL-8 gene in intestinal epithelial cells during viral infections have not been fully elucidated. Therefore, the purpose of this study was to define the molecular mechanisms of IL-8 gene expression in HT-29 cells infected with rotavirus. Transient transfection analysis of 5′ deletions and mutations of the IL-8 promoter driving expression of luciferase reporter gene indicates that the activating protein- (AP) 1 and nuclear factor- (NF) κB elements are necessary for IL-8 promoter activation during rotavirus infection. The importance of NF-κB activation for IL-8 gene expression was further demonstrated by the inhibition of rotavirus-induced IL-8 gene transcription and protein synthesis following blockade of degradation of the NF-κB cytoplasmic inhibitor IκB-α. Rotavirus infection of HT-29-induced IκB kinase (IKK) activation and overexpression of a dominant negative mutant of IKK-β greatly reduced rotavirus-induced IL-8 promoter activation and NF-κB-driven transcription, indicating that IKK is involved in rotavirus-induced IL-8 gene expression and NF-κB activation
The Role of Endothelial Cell-Derived Inflammatory and Vasoactive Mediators in the Pathogenesis of Bluetongue

AbstractBluetongue is an insect-transmitted disease of sheep and wild ruminants that is caused by bluetongue virus (BTV). Cattle are asymptomatic reservoir hosts of BTV. Infection of lung microvascular endothelial cells (ECs) is central to the pathogenesis of BTV infection of both sheep and cattle, but it is uncertain as to why sheep are highly susceptible to BTV-induced microvascular injury, whereas cattle are not. Thus, to better characterize the pathogenesis of bluetongue, the transcription of genes encoding a variety of vasoactive and inflammatory mediators was quantitated in primary ovine lung microvascular ECs (OLmVECs) exposed to BTV and/or inflammatory mediators. BTV infection of OLmVECs increased the transcription of genes encoding interleukin- (IL) 1 and IL-8, but less so IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. In contrast, we previously have shown that transcription of genes encoding all of these same mediators is markedly increased in BTV-infected bovine lung microvascular ECs and that BTV-infected bovine ECs produce substantially greater quantities of prostacyclin than do sheep ECs. Thus, sheep and cattle were experimentally infected with BTV to further investigate the role of EC-derived vasoactive mediators in the pathogenesis of bluetongue. The ratio of thromboxane to prostacyclin increased during BTV infection of both sheep and cattle, but was significantly greater in sheep (P = 0.001). Increases in the ratio of thromboxane to prostacyclin, indicative of enhanced coagulation, coincided with the occurrence of clinical manifestations of bluetongue in BTV-infected sheep. The data suggest that inherent species-specific differences in the production and activities of EC-derived mediators contribute to the sensitivity of sheep to BTV-induced microvascular injury
Rotavirus Protein NSP3 Shuts Off Host Cell Protein Synthesis

AbstractA recombinant vaccinia virus encoding rotavirus protein NSP3 driven by an internal ribosome entry site (IRES) from the encephalomyocarditis (EMC) virus was able to abate protein synthesis in BSC1 cells by 25-fold, with as much as 30% of the remaining protein synthesis being NSP3. Hence NSP3 shuts off host cell protein synthesis down to the level seen during rotavirus infection but is unable to prevent translation from EMC IRES-driven genes. This effect was abolished by deletions in the eIF4G-binding (aa 274–313) and the dimerization (aa 150–206) but not the viral mRNA-binding (aa 83–149) domains, supporting that NSP3 functions in vivo as a dimer. Binding of eIF4G by NSP3 has been implicated in interfering with mRNA 5′-3′ circularization, hence such circularization is essential for translation in mammalian cells
Hormone Response Element in SV40 Late Promoter Directly Affects Synthesis of Early as Well as Late Viral RNAs

AbstractWe previously demonstrated that the presence of a hormone response element surrounding the transcription initiation site of the SV40 major late promoter (+1 HRE) confers a replication advantage to the virus in a cell-type-specific manner. We determine here the mechanism by which the +1 HRE confers this advantage by analyzing in detail the various stages of the viral life cycle of wild-type versus a +1 HRE mutant in MA-134 cells. We show that the mutant overexpresses late genes at the expense of early genes at early times after infection. This initial underproduction of early RNA leads, subsequently, to an underproduction of large T-antigen, viral DNA, and infectious virions. We conclude that the +1 HRE is necessary for the proper initial regulation of transcription from the early as well as late promoter so the cascade of subsequent events can be executed for the optimal production of virions