RNA-dependent RNA polymerase

About: RNA-dependent RNA polymerase is a research topic. Over the lifetime, 13904 publications have been published within this topic receiving 767954 citations. The topic is also known as: RdRp & RNA replicase.

RNA is a structural element in retrovirus particles

Abstract: A single retroviral protein, Gag, is sufficient for virus particle assembly While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as “scaffolding” in the particle To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA We found that these particles contain cellular mRNA in place of genomic RNA This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles Furthermore, we found that the retroviral cores were disrupted by treatment with RNase Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles

Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

Abstract: A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ∼31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes. RNA transcripts derived from the full-length MHV-A59 construct were infectious, although transfection frequencies were enhanced 10- to 15-fold in the presence of transcripts encoding the nucleocapsid protein N. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar growth kinetics, plaque morphology, and cytopathology in murine cells as did wild-type MHV-A59. Molecularly cloned viruses recognized the MHV receptor (MHVR) for docking and entry, and pretreatment of cells with monoclonal antibodies against MHVR blocked virus entry and replication. Cells infected with molecularly cloned MHV-A59 virus expressed replicase (gene 1) proteins identical to those of laboratory MHV-A59. Importantly, the molecularly cloned viruses contained three marker mutations that had been derived from the engineered component clones. Full-length infectious constructs of MHV-A59 will permit genetic modifications of the entire coronavirus genome, particularly in the replicase gene. The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.

SARS Coronavirus nsp1 Protein Induces Template-Dependent Endonucleolytic Cleavage of mRNAs: Viral mRNAs Are Resistant to nsp1-Induced RNA Cleavage

Abstract: SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5′-end of a reporter mRNA having a short 5′ untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5′ untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5′ cap structure and 3′ poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5′-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.

RNA Methodologies: A Laboratory Guide for Isolation and Characterization

Abstract: RNA and the cellular biochemistry revisited - a rationale transcription and the organization of eukaryotic gene sequences messenger RNA resilient ribonucleases RNA isolation strategies isolation of polyadenylated RNA electrohoresis - principles and parameters electrophoresis of RNA the Northern blot nucleic acid probe technology practical nucleic acid hybridization principles of detection quantititation of specific mRNAs by the S1 nuclease assay analysis of nuclear RNA transcription rate assays extraction of nuclear RNA for steady state analysis an RNA paradigm.

Structure and replication of the genome of the hepatitis delta virus

Abstract: The hepatitis delta virus can be found in the serum and liver of some hepatitis B virus patients. We now report that the RNA genome of serum-derived delta virus is single-stranded and circular. Livers of infected chimpanzees or woodchucks contained as many as 300,000 copies of genomic strand RNA per average cell, and at least some of this RNA had a circular conformation. Also present in the livers were RNA species complementary to the virion RNA. The genomic RNA was 5-22 times more abundant than this antigenomic strand. Some of the antigenomic RNA was complexed with genomic RNA, as evidenced by the fact that at least 34% of the antigenomic RNA was resistant to digestion with either RNase A in 0.3 M NaCl or S1 nuclease. Some of the antigenomic RNA was in a circular conformation. These and other findings showed that the structure and replication of hepatitis delta virus are in many ways similar to those of the previously described plant viroids, virusoids, and satellite RNAs.