UMLS:C0019158 - FACTA Search

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Query: UMLS:C0019158 (hepatitis)
30,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Three polymerase proteins of influenza type A virus interact with each other to form the active polymerase complex. Polymerase basic protein 1 (PB1) can interact with PB2 in the presence or absence of polymerase acidic protein. In this study, we investigated the domains of PB1 involved in complex formation with PB2 in vivo, using coexpression and coimmunoprecipitation of the PB1-PB2 complex with monospecific antibodies. Results show that PB1 possesses at least two regions which can interact independently and form stable complexes with PB2. Both of these regions are located at the NH2 terminus of PB1; the COOH-terminal half of PB1 is not involved in interacting with PB2. Deletion analysis further demonstrated that the interacting regions of PB1 encompass amino acids (aa) 48 to 145 and aa 251 to 321. Linker insertions throughout the PB1 sequences did not affect complex formation with PB2. Deletion and linker-insertion mutants of PB1 were tested for polymerase activity in vivo. For this analysis, we developed a simplified assay for viral polymerase activity that uses a reporter chloramphenicol acetyltransferase gene containing the 5' and 3' ends of influenza viral promoter and nontranslating regions (minus sense) of the NS gene joined to a hepatitis delta virus ribozyme at its 3' end. This assay demonstrated that all deletion mutants of PB1 exhibited either background or greatly reduced polymerase activity irrespective of the ability to interact with PB2 and that all linker-insertion mutants except one at the extreme COOH end (L-746) of PB1 were also negative for viral polymerase activity. These results show that compared with complex formation of PB1 with PB2, the polymerase activity of PB1 was extremely sensitive to structural perturbation.
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PMID:Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein 2 at multiple sites. 879 8

The 3'-end of mouse hepatitis virus (MHV) genomic RNA contains a recognition sequence (55 nucleotides [nt]) required for minus-strand RNA synthesis. To determine whether the 3'-end sequence is also involved in subgenomic mRNA transcription, we have constructed MHV defective interfering (DI) RNAs which contain a chloramphenicol acetyltransferase (CAT) gene placed behind an intergenic sequence and a 3'-end sequence with various degrees of internal deletions. The DI RNAs were transfected into MHV-infected cells, and CAT activities, which represent subgenomic mRNA transcription from the intergenic site, were determined. The results demonstrated that the deletions of sequence upstream of the 350 nt at the 3'-end, which include the 3'-untranslated region (3'-UTR), of MHV genomic RNA did not affect subgenomic mRNA transcription. However, deletions that reduced the 3'-end sequences to 270 nt or less completely abolished the mRNA transcription despite the fact that all of these clones synthesized minus-strand RNAs. These results indicated that mRNA transcription from an intergenic site in the MHV DI RNA requires most of the 3'-UTR as a cis-acting signal, which likely exerts its effects during plus-strand RNA synthesis. A substitution of the corresponding bovine coronavirus sequence for the MHV sequence within nt 270 to 305 from the 3'-end abrogated the CAT gene expression, suggesting a very rigid sequence requirement in this region. The deletion of a putative pseudoknot structure within the 3'-UTR also abolished the CAT gene expression. These findings suggest that the 3'-UTR may interact with the other RNA regulatory elements to regulate mRNA transcription.
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PMID:The 3' untranslated region of coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. 879 74

A defective-interfering (DI) RNA of mouse hepatitis virus (MHV) was developed as a vector for expressing MHV hemagglutinin/esterase (HE) protein. The virus containing an expressed HE protein (A59-DE-HE) was generated by infecting cells with MHV-A59, which does not express HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. A similar virus (A59-DE-CAT) expressing the chloramphenicol acetyltransferase (CAT) was used as a control. These viruses were inoculated intracerebrally into mice, and the role of the HE protein in viral pathogenesis was evaluated. Results showed that all mice infected with parental A59 or A59-DE-CAT succumbed to infection by 9 days postinfection (p.i.), demonstrating that inclusion of the DI did not by itself alter pathogenesis. In contrast, 60% of mice infected with A59-DE-HE survived infection. HE- or CAT-specific subgenomic mRNAs were detected in the brains at days 1 and 2 p.i. but not later, indicating that the genes in the DI vector were expressed only in the early stage of viral infection. No significant difference in virus titer or viral antigen expression in brains was observed between A59-DE-HE- and A59-DE-CAT-infected mice, suggesting that virus replication in brain was not affected by the expression of HE. However, at day 3 p.i. there was a slight increase in the extent of inflammatory cell infiltration in the brains of the A59-DE-HE-infected mice. Surprisingly, virus titers in the livers of A59-DE-HE-infected mice were 3 log10 lower than that of the A59-DE-CAT-infected mice at day 6 p.i. Also, substantially less necrosis and viral antigen were detected in the livers of the A59-DE-HE-infected mice. This may account for the reduced mortality of these mice. The possible contribution of the host immune system to this difference in pathogenesis was analyzed by comparing the expression of four cytokines. Results showed that both tumor necrosis factor-alpha and interleukin-6 mRNAs increased in the brains of the A59-DE-HE-infected mice at day 2 p.i., whereas interferon-gamma and interleukin-1 alpha mRNAs were similar between A59-DE-HE- and A59-DE-CAT-infected mice. These data suggest that the transient expression of HE protein enhances an early innate immune response, possibly contributing to the eventual clearance of virus from the liver. This study indicates the feasibility of the DI expression system for studying roles of viral proteins during MHV infection.
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PMID:Expression of hemagglutinin/esterase by a mouse hepatitis virus coronavirus defective-interfering RNA alters viral pathogenesis. 950 Oct 44

Woodchuck hepatitis virus (WHV) enhancer II (EnII) is located upstream of the major pregenomic RNA promoter and is thought to play an important role in the insertional activation of the N-myc2 gene during WHV hepatocarcinogenesis. WHV EnII is recognized by at least three host transcription factors: HNF-1, HNF-4, and Oct-1. Here, the roles of these EnII-binding factors in viral transcription and replication have been further examined. In HepG2 cells transiently transfected with a chloramphenicol acetyltransferase (CAT) gene whose expression is dependent upon EnII, mutations in either the HNF-1 or the HNF-4 site strongly reduced CAT activity, while ablation of the Oct-1 site decreased CAT expression only twofold. Mutations in more than one site completely abolished reporter expression. These same mutations were also tested in an overlength WHV genome for their impact on viral replication and gene expression. In transfected HepG2 cells, lesions in the HNF-1 site inactivated pregenomic RNA expression and viral reverse transcription, with only minimal effects on the expression of other viral mRNAs. By contrast, Oct-1 site lesions had no effect on either viral RNA synthesis or DNA replication, and HNF-4 site lesions produced a modest reduction of pregenomic RNA but had no impact on viral DNA synthesis. Testing of the mutants in susceptible woodchucks revealed that, as expected, viruses with lesions in the HNF-1 site were nearly noninfectious, while mutants with lesions at the Oct-1 site were fully replication competent. HNF-4 site mutants were replication competent but may display reduced levels of replication in the intact animal host. We conclude that (i) EnII is primarily devoted to the regulation of pregenomic RNA in WHV, (ii) HNF-1 is essential for EnII function in vivo, and (iii) HNF-4 plays a demonstrable but adjunctive role in EnII function.
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PMID:In vivo effects of mutations in woodchuck hepatitis virus enhancer II. 965 6

The mouse hepatitis virus (MHV) nucleocapsid protein stimulated translation of a chimeric reporter mRNA containing an intact MHV 5'-untranslated region and the chloramphenicol acetyltransferase (CAT) coding region. The nucleocapsid protein binds specifically the tandemly repeated-UCYAA- of the MHV leader. This RNA sequence is the same as the intergenic motif found in the genome RNA. Preferential translation of viral mRNA in MHV infected cells is stimulated in part by this interaction and represents a specific, positive translational control mechanism employed by coronaviruses.
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PMID:Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs. 978 98

We have developed a defective-interfering (DI) RNA of mouse hepatitis virus (MHV) as a vector for expressing a variety of cellular and viral genes including the chloramphenicol acetyltransferase (CAT), hemagglutinin' esterase (HE), and gamma interferon. Here, we used the HE-expressing DI RNA for examining the role of HE protein in viral pathogenesis. The pseudorecombinant virus containing an expressed HE protein was generated by infecting cells with MHV-A59, which does not express, HE, and transfecting the in vitro-transcribed DI RNA containing the HE gene. These pseudorecombinant viruses (DE-HE A59) were then inoculated intracerebrally into mice. Viruses recovered from cells infected with A59 and transfected with DI RNA expressing the CAT gene (DE-CAT A59) were used as a control. At various time points after inoculation, mice were observed for clinical symptoms. Tissues (brains and livers) were obtained for determining the replication of DI RNA by RT-PCR, virus replication by plaque assay, antigen expression by immunohistochemistry, and pathological changes. Results showed that all mice infected with DE-CAT A59 succumbed to infection by 9 days postinfection (d p.i). These data are identical to the pathogenesis of the parental A59 virus, demonstrating that inclusion of the DI RNA did not by itself alter pathogenesis. In contrast, only 40% of mice infected with DE-HE A59 succumbed to infection. The subgenomic mRNAs transcribed from the DI vector were detected at 1 and 2 d p.i. but not at subsequent time points, indicating that the genes in the DI vector were expressed only at an early stage of viral infection. No significant difference in virus replication in the brains was detected between these two groups of mice, suggesting that virus replication in brains was not affected by the expression of the HE. Histopathological examination showed only a small increase in the extent of inflammatory cell infiltration and reduced viral antigen in the mice infected with DE-HE A59. There was no difference in virus replication in the livers at 2 and 4 d p.i., but a 3 log10 reduction was detected in the livers of mice infected with DE-HE A59 at 6 d p.i. Histological examination showed a significant reduction in viral antigen, inflammation and necrosis in mice infected with DE-HE A59. These results indicate that the expression of HE from the DI vector altered the viral pathogenesis. This study thus demonstrates the usefulness of this system in studying the role of viral or cellular genes expressed locally at the sites of viral infection in viral pathogenesis.
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PMID:Using a defective-interfering RNA system to express the HE protein of mouse hepatitis virus for studying viral pathogenesis. 978 24

While the 5' cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA transcription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize the cis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of the cis-acting signal for replication in the 5' untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5'-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA transcription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5' untranslated region, suggesting that the secondary structure formed by this 35-nt sequence may facilitate the downstream consensus sequence accessible for the discontinuous RNA transcription. This may provide a mechanism by which the 5' cis-acting sequence regulates subgenomic RNA transcription. The 5'-most 24 nt are not essential for transcription, while the 9 nt immediately downstream of the leader enhances RNA transcription. The sequence between nt 86 and 135 had little effect on transcription. This study thus defines the cis-acting transcription signal at the 5' end of the DI genome.
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PMID:The leader RNA of coronavirus mouse hepatitis virus contains an enhancer-like element for subgenomic mRNA transcription. 1104 1

Infectious hematopoietic necrosis virus (IHNV) is a Novirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14 degrees C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.
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PMID:Recovery of NV knockout infectious hematopoietic necrosis virus expressing foreign genes. 1107 23

Subgenomic RNA transcription of coronaviruses involves the interaction between the leader (or antileader) and the intergenic (IG) sequences. However, it is not clear how these two sequences interact with each other. In this report, a previously unrecognized minor species of subgenomic mRNA, termed mRNA5-1, was identified in cells infected with mouse hepatitis virus (MHV) strains JHM2c, JHM(2), JHM(3), A59, and MHV-1. Sequence analysis revealed that the leader-body fusion site of the mRNA is located at approximately 150 nucleotides (nt) downstream of the consensus IG sequence for mRNA 5 and did not have sequence homology with any known IG consensus sequences. To determine whether this sequence functions independently as a promoter, we cloned a 140-nt sequence (from approximately 70 nt upstream to approximately 70 nt downstream of the fusion site) from viral genomic RNA and placed it in front of a reporter gene in the defective-interfering (DI) RNA-chloramphenicol acetyltransferase (CAT) reporter vector. Transfection of the reporter RNA into MHV-infected cells resulted in synthesis of a CAT-specific subgenomic mRNA detected by reverse transcription-polymerase chain reaction (RT-PCR). The strength of this promoter was similar to that of the IG7 (for mRNA 7) as measured by the CAT activity. Deletion analysis showed that the sequence as few as 13 nt was sufficient to initiate mRNA transcription, while mutations within the 13-nt abolished mRNA transcription. In vitro translation study confirmed that the envelope (E) protein was translated from mRNA5-1, which encodes the open reading frame (ORF) 5b at its 5'-end, indicating that mRNA5-1 is a functional message. Furthermore, when the ORF5b was replaced with the CAT gene and placed in the DI in the context of viral mini-genome, CAT was expressed not only from the first ORF of mRNA5-1 but also from the second and third ORF of mRNA5 and genomic DI RNA, respectively, suggesting that more than one mechanism is involved in regulation of ORF5b expression. Our findings thus support the notion that base-pairing between the leader (or antileader) and the IG is not the sole mechanism in subgenomic RNA transcription.
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PMID:Identification of a noncanonical signal for transcription of a novel subgenomic mRNA of mouse hepatitis virus: implication for the mechanism of coronavirus RNA transcription. 1111 83

A recombinant mesogenic NDV strain, Beaudette C, and an engineered recombinant NDV expressing an additional gene were generated entirely from cloned cDNAs. For this purpose, a full-length cDNA clone of the virus genome, represented in eight different subgenomic fragments, was assembled in a transcription plasmid between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme sequence. Infectious NDV could be generated in the cells infected with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinant virus was then amplified and recovered, either after inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that of the parental wild-type virus. By using this system, a recombinant NDV containing a foreign gene encoding chloramphenicol acetyltransferase (CAT) was generated. To do this, the CAT transcription cassette containing the CAT open reading frame, flanked by NDV gene start and gene end sequence motifs, was inserted into the region between the HN and L genes of the full-length cDNA. This construct was then used in the generation of a recombinant NDV expressing CAT protein. The CAT gene was maintained stably for at least eight passages without any detectable loss of the gene from the recombinant. Generation of the recombinant virus, however, was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease in yield. In addition, the virus showed an increase in mean death time for eggs and a lower intracerebral pathogenicity index in day-old chicks, implicating attenuation of the recombinant virus. Thus, introduction of an additional gene into the NDV genome represents a method to achieve growth retardation and attenuation. These results also indicate that NDV can be engineered to express foreign protein stably and can be manipulated in the future for use as a vaccine vector.
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PMID:Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation. 1111 92

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