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At least 361 records · Page 20Linked to original sources
PubMed2005

Suppression of HCV RNA replication by baculovirus-mediated shRNA expression.

We describe a HCV core-short-hairpin RNA (shRNA) expressed from a human U6 Pol II promoter, which synergistically enhanced the inhibition of HCV replication in HCV replicon. We also describe the construction of recombinant baculovirus expressing shRNAs of the HCV core gene under the control of the U6 promoter. The anti-HCV shRNAs may provide a practical basis for applying shRNA-based gene therapy to the treatment of HCV.

Baculoviridae↗
PubMed2004

Viral evolution and interferon resistance of hepatitis C virus RNA replication in a cell culture model.

Hepatitis C virus (HCV) replicates through an error-prone process that may support the evolution of genetic variants resistant to the host cell antiviral response and interferon (IFN)-based therapy. We evaluated HCV-IFN interactions within a long-term culture system of Huh7 cell lines harboring different variants of an HCV type 1b subgenomic RNA replicon that differed at only two sites within the NS5A-encoding region. A replicon with a K insertion at HCV codon 2040 replicated efficiently and exhibited sequence stability in the absence of host antiviral pressure. In contrast, a replicon with an L2198S point mutation replicated poorly and triggered a cellular response characterized by IFN-beta production and low-level IFN-stimulated gene (ISG) expression. When maintained in long term-culture, the L2198S RNA evolved into a stable high-passage (HP) variant with six additional point mutations throughout the HCV protein-encoding region that enhanced viral replication. The HP RNA transduced Huh7 cells with more than 1,000-fold greater efficiency than its L2198S progenitor or the K2040 sequence. Replication of the HP RNA resisted suppression by IFN-alpha treatment and was associated with virus-directed reduction in host cell expression of ISG56, an antagonist of HCV RNA translation. Accordingly, the HP RNA was retained within polyribosome complexes in vivo that were refractory to IFN-induced disassembly. These results identify ISG56 as a translational control effector of the host response to HCV and provide direct evidence to link this response to viral sequence evolution, ISG regulation, and selection of the IFN-resistant viral phenotype.

Biological Evolution↗
PubMed1994

Detection and quantification of hepatitis C virus RNA replication in the liver.

To investigate the correlation between the replication of hepatitis C virus in liver and the clinical and histopathological features, we detected and quantified plus and minus strands of HCV-RNA in plasma and in livers of patients with chronic hepatitis C by a quantitative polymerase chain reaction. RNA was extracted from the plasma and liver tissue of ten patients with biopsy-proven chronic hepatitis C. The plus and minus strands of HCV-RNA were detected by a strand-specific reverse transcription with either sense or anti-sense oligonucleotide primers deduced from the hepatitis C virus genome, and a standard HCV-RNA with an enzyme restriction site was used to quantify the amount of HCV-RNA. Both plus and minus strands of HCV-RNA were detected from the liver tissue of all patients included. The amount of plus-stranded HCV-RNA in the liver was 10 times higher than that of minus-stranded HCV-RNA. Plus-stranded HCV-RNA was detected in the plasma in all patients, while the minus strand was not detected in any patient. There was a weak correlation between the amount of both strands of HCV-RNA in the liver and that of the plus strand in plasma. There was no significant correlation between the amount of liver HCV-RNA and serum alanine transaminase and aspartate transaminase levels, or histopathological findings in the liver. The present method of detecting and quantifying liver HCV-RNA is simple and sensitive; it may be used to detect residual hepatitis C virus replication after the disappearance of plasma HCV-RNA in acute hepatitis or in chronic hepatitis after interferon treatment.

Adult↗
PubMed1995

The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid.

The mitochondria of certain natural isolates of Neurospora contain both the Varkud plasmid, which encodes a reverse transcriptase, and a small unrelated RNA (VS RNA) that performs RNA-mediated self-cleavage and ligation reactions. Here, we show that VS RNA is transcribed from a VS plasmid DNA template by the Neurospora mitochondrial RNA polymerase using a promoter located immediately upstream of the RNA self-cleavage site that generates monomeric transcripts. VS RNA is then reverse transcribed by the Varkud plasmid reverse transcriptase to yield a full-length (-) strand cDNA, a predicted replication intermediate. Combined with previous genetic evidence, our results indicate that the VS plasmid replicates by reverse transcription as a satellite of the Varkud plasmid. This mode of replication, unprecedented for a satellite RNA, likely reflects the promiscuity of the Varkud plasmid reverse transcriptase, which does not require a specific primer to initiate cDNA synthesis. Our findings indicate how primitive reverse transcriptases with similar relaxed specificity could have facilitated the evolution of new retroelements.

Base Sequence↗
PubMed1978

Time-course of cowpea chlorotic mottle virus RNA replication.

In a synchronous infection of cowpea chlorotic mottle virus in cowpea leaves, the three largest viral RNA components were synthesized throughout the infection at relatively constant ratios of two molecules of component 3 to one each of components 1 and 2. Component 4 (the coat protein message) was produced minimally during the initial stages of the infection, and as the infection progressed the proportion of component 4 continuously increased. The replicative forms of components 1, 2, and 3 were produced with kinetics similar to those of the corresponding single-stranded molecules.

Kinetics↗
PubMed2004

Dendritic cells transfected with cytopathic self-replicating RNA induce crosspriming of CD8+ T cells and antiviral immunity.

A potential shortcoming of nonlive vaccines is their relative inefficiency in generating T cell responses, thus limiting their application in infections requiring cellular immunity. Here, we present a system to induce cellular immunity and to study the immunological implications of time-delayed dendritic cell (DC) apoptosis and antigen reprocessing in vivo. We generated a self-replicating cytopathic pestivirus RNA to enhance production and presentation of hepatitis C virus (HCV) antigens and to induce apoptosis in DC 24-48 hr after transfection. Replicon-transfected H-2(b) DCs used to immunize HLA-A2 transgenic mice induced protection upon challenge with a vaccinia virus expressing HCV antigens. Induction of cell death enhanced the immunogenicity of DC-associated antigen. Transfer of cellular material from vaccine DCs to endogenous antigen presenting cells was visualized in lymph nodes and spleen, and crossprimed CD8(+) T cells were characterized. The findings are relevant for the rational design of vaccines against noncytopathic pathogens like HCV.

Animals↗
PubMed1996

Chaotic interactions of self-replicating RNA.

A general system of high-order differential equations describing complex dynamics of replicating biomolecules is given. Symmetry relations and coordinate transformations of general replication systems leading to topologically equivalent systems are derived. Three chaotic attractors observed in Lotka-Volterra equations of dimension n = 3 are shown to represent three cross-sections of one and the same chaotic regime. Also a fractal torus in a generalized three-dimensional Lotka-Volterra Model has been linked to one of the chaotic attractors. The strange attractors are studied in the equivalent four-dimensional catalytic replicator network. The fractal torus has been examined in adapted Lotka-Volterra equations. Analytic expressions are derived for the Lyapunov exponents of the flow in the replicator system. Lyapunov spectra for different pathways into chaos has been calculated. In the generalized Lotka-Volterra system a second inner rest point--coexisting with (quasi)-periodic orbits--can be observed; with an abundance of different bifurcations. Pathways from chaotic tori, via quasi-periodic tori, via limit cycles, via multi-periodic orbits--emerging out of periodic doubling bifurcations--to "simple" chaotic attractors can be found.

Models, Theoretical↗
PubMed2003

Analysis of hepatitis C virus/classical swine fever virus chimeric 5'NTRs: sequences within the hepatitis C virus IRES are required for viral RNA replication.

Hepatitis C virus (HCV) is classified in the genus Hepacivirus of the family Flaviviridae, whose members have a single-stranded RNA genome of positive polarity, which encodes a single polyprotein. Within this family, HCV is closely related to viruses of the genus Pestivirus, which includes classical swine fever virus (CSFV). Translation of the hepaci- and pestiviral polyprotein is initiated by internal entry of ribosomes, promoted by the 5'NTR. The secondary and tertiary RNA structures of the HCV and pestivirus 5'NTRs are well conserved, despite the fact that their sequences differ significantly from one another. By analogy with other positive-stranded RNA viruses, the 5'NTR of HCV is likely to contain cis-acting determinants for replication as well as the determinants for translation. Studies on both signals could be complicated, as these signals might overlap. In this study, this problem was addressed by constructing chimeric HCV/CSFV 5'NTRs. A two-step analysis of these 5'NTRs was performed: (a) in a translation assay, which provided the possibility to study translation independently of the possible effects on replication; and (b) in a replication assay, in which were studied only the chimeric 5'NTRs for which IRES-dependent translation was demonstrated. An overlap was observed between HCV RNA elements involved in these processes. Exchange of domain II had a minor effect on the translation efficiency of the chimeric 5'NTRs, while replication of subgenomic replicons with these chimeric 5'NTRs was abolished. Exchange of domain III subdomains severely decreased translation activity, while replication was maintained.

5' Untranslated Regions↗
PubMed1980

Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.

Animals↗
PubMed2005

The topology of bulges in the long stem of the flavivirus 3' stem-loop is a major determinant of RNA replication competence.

All flavivirus genomes contain a 3'terminal stem-loop secondary structure (3'SL) formed by the most downstream approximately 100 nucleotides (nt) of the viral RNA. The 3'SL is required for virus replication and has been shown to bind both virus-coded and cellular proteins. Results of the present study using an infectious DNA for WN virus strain 956 initially demonstrated that the dengue virus serotype 2 (DEN2) 3'SL nucleotide sequence could not substitute for that of the WN 3'SL to support WN genome replication. To determine what WN virus-specific 3'SL nucleotide sequences were required for WN virus replication, WN virus 3'SL nucleotide sequences were selectively deleted and replaced by analogous segments of the DEN2 3'SL nucleotide sequence such that the overall 3'SL secondary structure was not disrupted. Top and bottom portions of the WN virus 3'SL were defined according to previous studies (J. L. Blackwell and M. A. Brinton, J. Virol. 71:6433-6444, 1997; L. Zeng, L., B. Falgout, and L. Markoff, J. Virol. 72:7510-7522, 1998). A bulge in the top portion of the long stem of the WN 3'SL was essential for replication of mutant WN RNAs, and replication-defective RNAs failed to produce negative strands in transfected cells. Introduction of a second bulge into the bottom portion of the long stem of the wild-type WN 3'SL markedly enhanced the replication competence of WN virus in mosquito cells but had no effect on replication in mammalian cells. This second bulge was identified as a host cell-specific enhancer of flavivirus replication. Results suggested that bulges and their topological location within the long stem of the 3'SL are primary determinants of replication competence for flavivirus genomes.

3' Untranslated Regions↗