In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate

Dorner, Andrew J.; Semler, Bert L.; Jackson, Richard J.; Hanecak, Ronnie; Duprey, E; Wimmer, Eckard

doi:10.1128/jvi.50.2.507-514.1984

Cited by 230 publications

(97 citation statements)

References 35 publications

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“…The hatched rectangle adjacent to p290 Indicates a postulated product of 30-60 kDa, which was not Identified by the antisera In this study (see Dtscusslon). of poliovirus the difference in intracellular and in vitro synthesis is attributable to internal initiation of translation in the in vitro system (Dorner et al, 1984). This does not appear to be the case with MHV, since initiation of ORF 1 a translation appears to be primarily N-terminal in vitro.…”

Section: Synthesis and Processing Of Orf La Products Downstream From P28mentioning

confidence: 95%

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

et al. 1992

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Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

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Section: Synthesis and Processing Of Orf La Products Downstream From P28mentioning

confidence: 95%

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

et al. 1992

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“…Poliovirus mRNA also initiates translation at more than one site, at least in uitro (Celma and Ehrenfeld, 1975), but one cannot attempt an explanation until the sites have been identified. [Dorner et al (1984) claim to have localized an internal initiation site, but they did not prove that the template RNA was intact. The fact that they could demonstrate "internal initiation" in extracts from reticulocytes but not from poliovirusinfected cells hints of an artifact.]…”

Section: Mechanisms That Allow Some Mrnas To Direct the Synthesismentioning

confidence: 98%

Regulation of Protein Synthesis in Virus-Infected Animal Cells

Kozak

1986

Advances in Virus Research Volume 31

178

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“…It has long been known that the majority of cellular mRNAs in eukaryotic organisms initiate translation via a cap-dependent ribosomal scanning mechanism (18,26). However, the initiation of protein translation in picornaviruses occurs by an unusual mechanism involving direct internal binding of the ribosome to a sequence element of the 5Ј UTR of viral RNA (20), termed an internal ribosomal entry site (IRES) (9,21,22). The IRES directs binding of the small ribosomal subunit to viral RNA near the 3Ј border of the IRES, independent of a cap structure at the 5Ј terminus of the RNA.…”

mentioning

confidence: 99%

Specific Inhibition of Coxsackievirus B3 Translation and Replication by Phosphorothioate Antisense Oligodeoxynucleotides

Wang

Cheung

Zhang

et al. 2001

Antimicrob Agents Chemother

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The 5 and 3 untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA form highly ordered secondary structures that have been confirmed to play important regulatory roles in viral cap-independent internal translation initiation and RNA replication. We previously demonstrated that deletions in different regions of the 5 UTR significantly reduced viral RNA translation and infectivity. Such observations suggested strongly that viral RNA translation and replication could be blocked if highly specific antisense oligodeoxynucleotides (AS-ODNs) were applied to target crucial sites within the 5 and 3 UTRs. In this study, seven phosphorothioate AS-ODNs were synthesized, and the antiviral activity was evaluated by Lipofectin transfection of HeLa cells with AS-ODNs followed by infection of CVB3. Analysis by Western blotting, reverse transcription-PCR, and viral plaque assay demonstrated that viral protein synthesis, genome replication, and infectivity of CVB3 were strongly inhibited by the AS-ODNs complementary to different regions of the 5 and 3 UTRs. The most effective sites are located at the proximate terminus of the 5 UTR (AS-1), the proximate terminus of the 3 UTR (AS-7), the core sequence of the internal ribosome entry site (AS-2), and the translation initiation codon region (AS-4). These AS-ODNs showed highly sequence-specific and dose-dependent inhibitory effects on both viral protein synthesis and RNA replication. It is noteworthy that the highest inhibitory activities were obtained with AS-1 and AS-7 targeting the termini of the 5 and 3 UTRs. The percent inhibition values of AS-1 and AS-7 for CVB3 protein VP1 synthesis and RNA replication were 70.6 and 79.6 for AS-1 and 73.7 and 79.7 for AS-7, respectively. These data suggest that CVB3 infectivity can be inhibited effectively by AS-ODNs.

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In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate

Cited by 230 publications

References 35 publications

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

Regulation of Protein Synthesis in Virus-Infected Animal Cells

Specific Inhibition of Coxsackievirus B3 Translation and Replication by Phosphorothioate Antisense Oligodeoxynucleotides

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