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Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

ABSTRACT: We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severely defective as templates for either polymerase. We also evaluated the infectivity of substitution mutant WNV genome-length RNAs. All WNV RNAs containing the DV2 3'SL were unable to replicate. However, WNV RNAs containing substitutions of the 5'NCR, the capsid gene, and/or 3'NCR nt sequences upstream from the WNV 3'SL, by the analogous DV2 nt sequences, were infectious. Combined results suggested that replication was not dependent upon species homology between the 3'SL and NS5.

SUBMITTER: Yu L

PROVIDER: S-EPMC3368002 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

Yu Li L Nomaguchi Masako M Padmanabhan R R Markoff Lewis L

Virology 20080129 1

We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severel ...[more]

PMID: 18234265

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Project description:A possible role in RNA replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flavivirus RNA was previously suggested but never tested in vivo. Using the M-fold program for RNA secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne Flavivirus species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence of the predicted interactions, the putative cyclization sequences (5' or 3') in the replicon RNA of the mosquito-borne Kunjin virus were mutated either separately, to destroy base-pairing, or simultaneously, to restore the complementarity. None of the RNAs with separate mutations in only the 5' or only the 3' cyclization sequences was able to replicate after transfection into BHK cells, while replicon RNA with simultaneous compensatory mutations in both cyclization sequences was replication competent. This was detected by immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replicon RNA. We then used the M-fold program to analyze RNA secondary structure of the covalently linked 5'- and 3'-terminal regions of three tick-borne virus species and identified a previously undescribed additional pair of conserved complementary sequences in locations similar to those of the mosquito-borne species. They base-paired with DeltaG values of approximately -20 kcal, equivalent or greater in stability than those calculated for the originally proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide sequence per se, is essential for the replication of mosquito-borne Kunjin virus RNA and that more than one pair of cyclization sequences might be involved in the replication of the tick-borne Flavivirus species.

Dataset Information

Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

Publications

Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication.

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