[From RNA virus replication to its innovation].
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Gene Expression Regulation, Viral↗
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Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication.
The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.
Antibodies, Monoclonal↗
Replication of hepatitis C virus RNA occurs in a membrane-bound replication complex containing nonstructural viral proteins and RNA.
Biochemical studies revealed that nonstructural proteins of hepatitis C virus (HCV) interacted with each other and were associated with intracellular membranes. The goals of this study were to determine whether nonstructural viral proteins are colocalized at specific intracellular sites where HCV RNA is replicated and to identify the virus components of the HCV replication complex (RC). Immunofluorescence and subcellular fractionation studies were performed to determine the intracellular colocalization of nonstructural HCV proteins and the replicating RNA in a human hepatoma cell line, Huh7, in which a subgenomic HCV RNA was replicated persistently. The replicating HCV RNA was labelled with 5-bromouridine 5'-triphosphate (BrUTP). Results show that each of the nonstructural HCV proteins was colocalized predominantly with the newly synthesized HCV RNA labelled with BrUTP and an endoplasmic reticulum (ER) protein, calnexin. Consistent with these findings, subcellular fractionation and Western blot analyses revealed that the nonstructural HCV proteins were colocalized with HCV RNA mainly in the membrane fractions. Conversely, the viral nonstructural proteins and RNA remained in the soluble fractions upon treatment with detergent, confirming the membrane association of the HCV RC. HCV RNA in the membrane-bound RC was resistant to RNase treatment, whereas it became sensitive to RNases once the membranes were disrupted by treatment with detergent, suggesting that the HCV RC is assembled within membrane structures. Collectively, these findings demonstrate that HCV RNA replication occurs in the perinuclear ER membrane-bound HCV RC, containing nonstructural viral proteins and RNA.
Cell Membrane↗
Replication of RNA viruses: analysis by liquid polymer phase partition of the binding of Q RNA polymerase to nucleic acid.
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Binding Sites↗
mRNA amplification system by viral replicase in transgenic plants.
We have constructed transgenic tobacco plants (M1x2-FCP2IFN plants) expressing viral RNA replication genes of brome mosaic virus (BMV) and BMV RNA3 derivative (FCP2IFN) carrying the human gamma interferon (IFN-gamma) gene. In M1x2-FCP2IFN plants the RNA3 derivative expressed from the integrated cDNA was replicated and subgenomic RNA (i.e. mRNA of IFN-gamma) was produced by BMV replicase. The accumulation level of the mRNA of IFN-gamma was approximately 5-fold higher than that by the cauliflower mosaic virus (CaMV) 35S RNA promoter. In addition IFN-gamma accumulated in M1x2-FCP2IFN plants.
DNA, Complementary↗
Amplification, expression, and packaging of foreign gene by influenza virus.
A system is described that allows use of recombinant DNA technology to modify the genome of influenza virus, a negative-strand RNA virus, and to engineer vectors for the expression of foreign genes. Recombinant RNA is expressed from plasmid DNA in which the coding sequence of the influenza A virus NS gene is replaced with that of the chloramphenicol acetyltransferase gene. When transfected with purified influenza A virus polymerase proteins--in the presence of helper virus--the recombinant RNA is amplified, expressed, and packaged into virus particles, which can be passaged several times. The data indicate that the 22 5' terminal and the 26 3' terminal bases of the influenza A virus RNA are sufficient to provide the signals for RNA transcription, RNA replication, packaging of RNA into influenza virus particles.
Animals↗
Architecture of the poliovirus replicative intermediate RNA.
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Centrifugation, Density Gradient↗
Conservation of a viral RNA genome during replication and translation.
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DNA Replication↗
Effect of interferon on transcription and translation of vesicular stomatitis virus in human and simian cell cultures.
The effect of interferons on vesicular stomatitis virus (VSV) primary transcription, amplified RNA synthesis [i.e. the sum of primary transcription RNA replication (leading to [ - ] RNA) and secondary transcription (leading to [ + ] RNA) and virus protein synthesis were studied. In a human cell line, both human and simian interferons inhibited the initiation of primary transcription and amplified RNA synthesis. In contrast, in a simian cell line tested similarly, the initiation of these activities was not affected, though they decreased as the infection progressed. Nevertheless, virus protein synthesis was completely inhibited. These results demonstrate that the action of interferon on virus transcription and/or translation may depend more on the host cell than on the particular interferon used.
Animals↗
Attachment of avidin-coupled spheres to linear and circular forms of mengovirus double-stranded RNA.
Picornavirus-infected cells contain a double-stranded RNA (replicative form [RF] RNA), composed of viral genomic RNA hydrogen bonded to cRNA of similar nucleotide length. Mengovirus RF RNA reacted with a succinimide ester of biotin was shown by electron microscopy to bind avidin-coupled polymethacrylate spheres. These binding sites are taken to indicate the presence of VPg protein molecular by methods previously applied to poliovirus RF RNA (Richards et al., Proc. Natl. Acad. Sci. U.S.A. 76:676-680, 1979). One sphere was bound at or very near one terminus of linear RF molecules while a second sphere was bound at a site which was 1 to 4% of the genome length from the other terminus. Assignment of VPg positions was limited by the physical dimension (ca. 60-nm diameter) of the heavy metal-contrasted sphere observed by electron microscopy. A third site of sphere binding was detected at a lower frequency of occurrence at a site which was 10 to 20% of the genome length from one or the other terminus. Circular RF RNA molecules were also detected with two spheres attached at juxtaposed sites. The termini of the linear RF RNA were located within the circular structures by sphere attachment at sites which were very close to and obscured a nucleic acid projection which we have described to occur on circular mengovirus RF RNA (D. L. Robberson, M. V. Marshall, G. B. Thornton, and R. B. Arlinghaus, manuscript submitted for publication). CsCl gradient fractions of RNA reacted with avidin-coupled spheres were highly enriched in circular structures.
Avidin↗
Hepatitis delta antigens enhance the ribozyme activities of hepatitis delta virus RNA in vivo.
The mechanism of regulation for the ribozyme activity of hepatitis delta virus (HDV) RNA in infected cells is unknown. Previously, we developed a direct assay capable of detecting the ribozyme activity of HDV dimer or trimer RNAs in vivo (K.-S. Jeng, A. Daniel, and M. M. C. Lai, J. Virol, 70:2403-2410, 1996). In this study, we used this method to examine the effects of hepatitis delta antigen (HDAg) on the ribozyme activities of HDV RNA in vivo. The HDV multimer cDNAs were cotransfected with plasmids encoding either HDV small delta antigen (SHDAg) or large delta antigen (LHDAg), and the self-cleavage of the primary transcripts from the HDV cDNA was analyzed at day 2 postransfection. The results were as follows. (i) Both HDAgs, particularly LHDAg, enhanced the self-cleavage activity of HDV RNA; however, HDAgs are not required for HDV RNA cleavage. (ii) HDAg could not restore the ribozyme activity of mutant HDV RNAs which have lost the ribozyme function. (iii) The enhancement of ribozyme activity by HDAg does not require HDV RNA replication. (iv) RNA-binding activity of HDAg is required for the enhancement of RNA cleavage. (v) The self-ligation activities of HDV ribozyme also were enhanced by HDAg. These results suggest that HDAg can regulate the cleavage and ligation of HDV RNA during the HDV life cycle.
Cell Line↗
FUNCTIONS OF THE 3'-UNTRANSLATED REGIONS OF POSITIVE STRAND RNA VIRAL GENOMES.
Positive strand RNA viral genomes are unique in the viral world in serving a dual role as mRNA and replicon. Since the origin of the minus-strand RNA replication intermediate is at the 3'-end of the genome, the 3'-untranslated region (UTR) clearly plays a role in viral RNA replication. The messenger role of this same RNA likely places functional demands on the 3'-UTR to serve roles typical of cellular mRNAs, including the regulation of RNA stability and translation. Current understanding indicates varied roles for positive strand RNA viral 3'-UTRs, with the dominant roles differing between viruses. Three case studies are discussed: turnip yellow mosaic virus RNA, whose 3' tRNA mimicry is thought to negatively regulate minus strand synthesis; brome mosaic virus, whose 3'-UTR contains a unique promoter element directing minus strand synthesis; and tobacco mosaic virus, whose 3'-UTR contains an enhancer of translational expression.
Journal Article↗
[Study of fusion of bacteriophage f2 double-stranded RNA, poly(A).poly(U), and poly(G).poly(C) in the presence of tetraethylammonium bromide].
The data on the dependence of the melting curve parameters of double-stranded RNA (replicative form of RNA of f2 bacteriophage) poly(A) times poly(U) and poly(G) times poly(C) on the concentration of (C2H5)4NBr were obtained. The RNA melting range width is shown to pass through the minimum value T =2.1+/-0.1degrees at the point of inversion of relative stability of GC and AU pairs that corresponds to 4.0+/-0.1 M concentration of (C2H5)4NBr. Using the melting temperatures of poly(A) times poly(U) and poly(G) times poly(C) the rependence of Tgc-Tau parameter on (C2H5)4NBr concentration was shown. It was concluded from these data that the effect of the double-stranded RNA stacking heterogeneity was negligible in the 0-3 M range of (C2H5)4NBr concentration. Melting curves of RNA were obtained at various values of Tgc-Tau parameter. It was shown that the profile of fine structure of melting curves depends on the value of Tgc-Tau parameter.
Bacteriophages↗
Cross-catalytic replication of an RNA ligase ribozyme.
A self-replicating RNA ligase ribozyme was converted to a cross-catalytic format whereby two ribozymes direct each other's synthesis from a total of four component substrates. Each ribozyme binds two RNA substrates and catalyzes their ligation to form the opposing ribozyme. The two ribozymes are not perfectly complementary, as is the case for replicating nucleic acid genomes in biology. Rather, the ribozymes contain both template elements, which are complementary, and catalytic elements, which are identical. The specificity of the template interactions allows the cross-catalytic pathway to dominate over all other reaction pathways. As the concentration of the two ribozymes increases, the rate of formation of additional ribozyme molecules increases, consistent with the overall autocatalytic behavior of the reaction system.
Base Sequence↗
[The role of polymerase protein genes of influenza A virus in the transition from the early to late stage of replication].
Regulation of influenza virus RNA replication was studied with the use of A/FPV/Rostock/34 strain ts-mutants. Mutants C44, C15, C45 possessing the ts-defects in the PB2, PB1 and PA genes respectively were used for the infection of chick embryo cultured cells and H-uridine-labelled nucleocapsid-associated RNA was analysed in polyacrylamide gel electrophoresis to assess the kinetics of vRNA synthesis. A typical early-late transition of the pattern of vRNA synthesis was observed in the cells infected with C44, whereas the other two mutants exhibited a slightly changed (C15) or strongly distorted (C45) pattern. In shift up experiments after the transfer to non-permissive temperature all the mutants exhibited partial reversion to an early pattern of vRNA synthesis. The results are discussed in connection with the mechanism of the early-late transition of influenza virus-specific RNA synthesis.
Autoradiography↗
The bunyavirus nucleocapsid protein is an RNA chaperone: possible roles in viral RNA panhandle formation and genome replication.
Cellular RNA chaperones are crucial for the genesis of correctly folded functional RNAs. Using several complementary in vitro assays we find that the bunyavirus nucleocapsid protein (N) is an RNA chaperone. In the Bunyaviridae genomic RNA is in stable "panhandle" formation that arises through the hydrogen bonding of the terminal nucleotides of the RNA. The RNA chaperone function of N facilitates panhandle formation even though the termini are separated by >2 kb. RNA panhandle formation is likely driven by the exceptionally high base-pairing specificity of the terminal nucleotides as evidenced by P-num analysis. N protein can nonspecifically dissociate RNA duplexes. In addition, following panhandle formation, the RNA chaperone activity of N also appears to be involved in dissociation of the RNA panhandle and remains in association with the 5' terminus of the viral RNA following dissociation. Thus, N likely functions in the initiation of genome replication to allow efficient initiation of RNA synthesis by the viral polymerase. The RNA chaperone activity of N may be facilitated by an intrinsically disordered domain that catalyzes RNA unfolding driven by reciprocal entropy transfer. These observations highlight the essential features that are probably common to all RNA chaperones in which the role of the chaperone is to nonspecifically dissociate higher order structure and formation of functional higher order structure may often be predicted by RNA P-num value. The data also highlight features of N that are probably specifically important during replication of bunyavirus RNA.
Amino Acid Sequence↗
Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene.
A picornavirus hybrid genome was constructed in which the internal ribosomal entry site (IRES) of encephalomyocarditis virus was inserted between the 5' non-translated region and the open reading frame of poliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybrid RNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could be removed from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) in which the translation of the PV open reading frame normally promoted by the type 1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. This result indicates that these elements are not likely to contain cis-acting elements necessary for PV replication or encapsidation. A foreign gene (bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPO cDNA between the PV and encephalomyocarditis virus IRES elements. The dicistronic RNA replicated in HeLa cells and yielded a derivative of PV (W1-DICAT) with a genome 17% longer than that of wild-type PV. CAT assays and immunoblot analyses showed that the viral RNA efficiently expressed the foreign gene in cell culture. The CAT activity diminished somewhat with each passage of the dicistronic virus, an observation which suggested that the inserted gene had a deleterious effect on viral replication. However, even after five virus passages, a significant quantity of the foreign gene was still expressed. Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNA species that replicated and expressed luciferase for up to 20 hr after transfection. However, this elongated RNA was not encapsidated.
Base Sequence↗
Screening for hepatitis C virus antiviral activity with a cell-based secreted alkaline phosphatase reporter replicon system.
We describe a phased screening system for discovery of compounds with antiviral activity against hepatitis C virus (HCV). The primary assay utilizes dicistronic subgenomic HCV replicons in which the upstream cistron was modified to express the human immunodeficiency virus (HIV) tat protein. When these replicons are stably transfected into Huh-7-derived cells that express secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV long terminal repeat promoter, there is a strong correlation between intracellular HCV RNA abundance and the activity of SEAP secreted into the culture medium. Thus, active compounds are easily identified by direct enzymatic quantification of SEAP in the medium without post-assay processing. Compounds that reduce SEAP activity without causing cellular toxicity are next evaluated in a second Huh-7-derived cell line constitutively expressing SEAP under control of the tat-HIV promoter axis, independent of HCV RNA replication. This specificity control identifies compounds that cause reductions in SEAP that are unrelated to suppression of HCV RNA replication. Compounds showing HCV-specific activity in primary assays are next evaluated by real-time RT-PCR to directly quantify reductions in HCV RNA. We have found excellent agreement between the SEAP and RT-PCR assays. This phased system provides an efficient and cost-effective screen for compounds with antiviral activity against HCV.
Alkaline Phosphatase↗