Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs

McClure, Marcella A.; Perrault, Jacques

doi:10.1093/nar/13.19.6797

Cited by 20 publications

(19 citation statements)

References 40 publications

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“…A mild cross-reaction between the RNA probes and cellular rRNA was observed when nucleic acid from uninfected monkey kidney cells was tested by using the cRNA probes. Similar observations have been reported in other viral systems (14,15), and this was believed to be caused by small homologous regions between viral and cellular RNAs. Our results suggest that this potential problem can be solved by performing simultaneous hybridizations by using cRNA and vRNA HAV ssRNA probes separately with duplicates of dotted samples.…”

Section: Discussionsupporting

confidence: 87%

Detection of hepatitis A virus by hybridization with single-stranded RNA probes

Jiang

Estes

Metcalf

1987

Appl Environ Microbiol

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An improved method of dot-blot hybridization to detect hepatitis A virus (HAV) was developed with single-stranded RNA (ssRNA) probes. Radioactive and nonradioactive ssRNA probes were generated by in vitro transcription of HAV templates inserted into the plasmid pGEM-1. 32P-labeled ssRNA probes were at least eightfold more sensitive than the 32P-labeled double-stranded cDNA counterparts, whereas biotin-labeled ssRNA probes showed a sensitivity comparable with that of the 32P-labeled double-stranded cDNA counterparts. Hybridization of HAV with the ssRNA probes at high stringency revealed specific reactions with a high signal-to-noise ratio. The differential hybridization reactions seen with probes of positive and negative sense (compared with HAV genomic RNA) were used to detect HAV in clinical and field samples. A positive/negative ratio was introduced as an indicator that permitted a semiquantitative expression of a positive HAV reaction. Good agreement of this indicator was observed with normal stool samples and with HAV-seeded samples. By using this system, HAV was detected in estuarine and freshwater samples collected from a sewage-polluted bayou in Houston and a saltwater tributary of Galveston Bay.

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Section: Discussionsupporting

confidence: 87%

Detection of hepatitis A virus by hybridization with single-stranded RNA probes

Jiang

Estes

Metcalf

1987

Appl Environ Microbiol

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“…Minor background hybridization was detected from RNA isolated from the mock-transfected cells corresponding to 28S RNA (Fig. 2A, lane C); previous studies have also noted background hybridization between 28S RNA and poliovirus RNA (16). The hybridization signal for RNA derived from in vitro transcription of pT7IC at a position consistent with the known size for full-length poliovirus RNA (7.5 kb) was evident at increasing times posttransfection ( Fig.…”

Section: Resultsmentioning

confidence: 53%

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons

Choi

Pal-Ghosh

Morrow

1991

J Virol

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Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-Pl fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

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“…However, also for this group of viruses, insertions of host cell sequences occur. The integration of 28S ribosomal RNA fragments was documented for influenza virus (Khatchikian, Orlich, & Rott, 1989) and poliovirus (Charini, Todd, Gutman, & Semler, 1994;McClure & Perrault, 1985), while in the genomes of Sindbis virus mutants, tRNA sequences were detected (Monroe & Schlesinger, 1983;Tsiang, Monroe, & Schlesinger, 1985). The integration of cellular protein-coding sequences is to the best of our knowledge for RNA viruses unique to pestiviruses.…”

Section: Mechanisms Of Rna Recombination and Biological Relevance Of mentioning

confidence: 99%