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At least 397 records · Page 22Linked to original sources
PubMed2003

The optimal temperature for RNA replication in cells infected by Soil-borne wheat mosaic virus is 17 degrees C.

Systemic infection of wheat plants with Soil-borne wheat mosaic virus (SBWMV) requires temperatures below 20 degrees C. Here we examine the cause of the temperature sensitivity by inoculating infectious in vitro transcripts of SBWMV RNA1 and RNA2 to barley mesophyll protoplasts. After RNA inoculation, protoplasts were incubated at temperatures between 15 and 25 degrees C for up to 48 h. Western blot analysis showed that the capsid protein accumulated most abundantly at 17 degrees C but was not detectable at 25 degrees C. Northern blot analysis showed that the wild-type RNA1 and RNA2 and their subgenomic RNAs accumulated most abundantly at 17 degrees C but were barely detectable at 25 degrees C. An RNA1 mutant in which the p152 and p211 replicase genes were placed between the 5'- and 3'-untranslated regions also replicated most efficiently at 17 degrees C but not at 25 degrees C. Thus, the requirement for temperatures lower than 20 degrees C for SBWMV infection is primarily determined by replication of RNA1, which encodes the viral RNA replicase.

Cells, Cultured↗
PubMed1991

The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation.

Infection of cells with poliovirus results in a rapid inhibition of host RNA and protein synthesis. Concordant with this shutoff, the p220 subunit of the cap-binding protein complex is cleaved, probably indirectly, by the poliovirus proteinase p2A (2Apro). To elucidate the mechanism of action of 2Apro in inhibiting protein synthesis in vivo, we studied the effect of transient expression of 2Apro in COS-1 monkey kidney cells. In cells transfected with a 2Apro expression plasmid, p220 was cleaved and the 2Apro mRNA was reduced 30-fold compared to an identical plasmid containing a translation termination codon within the 2Apro coding region. The reduced expression from the 2Apro vector results from a 4-fold reduction in DNA replication and 22-fold reduction in transcription by RNA polymerase II from the adenovirus major late promoter/SV40 enhancer utilized in this vector. In contrast, no decrease in transcription of the adenovirus virus-associated I RNA gene by RNA polymerase III was observed. The effect of 2Apro expression on cap-dependent mRNA translation was studied by producing a dicistronic beta-globin mRNA harboring the encephalomyocarditis virus leader and 2Apro coding region within the 3' end of the mRNA to mediate cap-independent translation of 2Apro. Expression of this mRNA was also reduced 25-fold compared to an identical plasmid harboring a termination codon within the 2Apro coding region. Translation of the beta-globin marker gene from this mRNA was reduced 3-fold when corrected for mRNA level. These results suggest that p220 cleavage itself is not sufficient for complete inhibition of host translation and that an important effect of 2Apro expression on host protein synthesis is a reduction in RNA polymerase II transcription and to a lesser extent, DNA replication. This reduction could be a primary effect of 2Apro, or a secondary effect caused by the inhibition of translation.

Animals↗
PubMed1989

Inhibition of hepatitis delta virus RNA replication in primary woodchuck hepatocytes.

Cell cultures of primary woodchuck hepatocytes can be infected with hepatitis delta virus (HDV) as demonstrated by the appearance of genomic HDV RNA 7 days after inoculation. This tissue culture system was used to study the effect of antiviral substances. Ribavirin inhibited HDV replication at a concentration of 10 micrograms/ml, if added up to three days post infection. Suramin had an inhibitory effect only when added simultaneously with the virus, at a concentration of 200 micrograms/ml. This concentration had no toxic effect on primary woodchuck hepatocytes. alpha-Amanitin showed a weak inhibitory effect only at the highest nontoxic concentration of 0.1 microgram/ml. Acyclovir had no effect.

Amanitins↗
PubMed1993

The secondary structure of the 5'-noncoding region of beet necrotic yellow vein virus RNA 3: evidence for a role in viral RNA replication.

Secondary structure-sensitive chemical and enzymatic probes have been used to produce a model for the folding of the first 312 residues of the long 5'-noncoding region of beet necrotic yellow vein virus RNA 3. The structure consists of two major domains, one of which includes long distance base-pairing interactions between two short sequence elements (Box I and Box II) situated between positions 237 and 292 and complementary elements (Box I' and II') near the 5'-terminus. Previous studies have shown that base pairing between these sequence elements (in either the plus-strand or minus-strand RNA) is important for RNA 3 accumulation during infection. RNA 3 transcripts were produced containing mutations which preferentially disrupted Box II-II' base pairing in either the plus- or minus-strand. In infection experiments, transcripts with mutations which disrupted the Box II-II' interaction in the plus-strand structure replicated less efficiently than mutants in which the Box II-II' interaction was disrupted in the minus-strand. These findings indicate that the complex 5'-proximal plus-strand structure to which the Box II-II' interaction contributes comprises at least part of the promoter for plus-strand RNA synthesis.

Base Sequence↗
PubMed1990

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.

Antibodies, Monoclonal↗
PubMed2003

Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication.

The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by approximately 37 and approximately 60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes.

Base Sequence↗
PubMed2000

Genetic dissection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement.

Coat protein (CP) of alfalfa mosaic virus (AMV) binds as a dimer to the 3' termini of the three genomic RNAs and is required for initiation of infection, asymmetric plus-strand RNA accumulation, virion formation, and spread of the virus in plants. A mutational analysis of the multiple functions of AMV CP was made. Mutations that interfered with CP dimer formation in the two-hybrid system had little effect on the initiation of infection or plus-strand RNA accumulation but interfered with virion formation and reduced or abolished cell-to-cell movement of the virus in plants. Six of the 7 basic amino acids in the N-terminal arm of CP (positions 5, 6, 10, 13, 16, and 25) could be deleted or mutated into alanine without affecting any step of the replication cycle except systemic movement in plants. Mutation of Arg-17 interfered with initiation of infection (as previously shown by others) and cell-to-cell movement of the virus but not with plus-strand RNA accumulation or virion formation. The results indicate that in addition to the RNA-binding domain, different domains of AMV CP are involved in initiation of infection, plus-strand RNA accumulation, virion formation, cell-to-cell movement, and systemic spread of the virus.

Alfalfa mosaic virus↗
PubMed2006

Chemical synthesis of picornaviral protein primers of RNA replication.

Naturally occurring nucleopeptidic replication primers (VPg-pUpU) of poliovirus and coxsackie virus were chemically synthesized. The synthesis was accomplished via block-coupling of two minimally protected fragments of the target structures: a short RNA-nucleopeptide and a longer peptide segment containing diverse side-chain functionalities. The synthetic VPg-pUpU of coxsackie virus was characterized by NMR spectroscopy.

Base Sequence↗