Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA

Pei Yong Shi, L. I. Wei, Margo A. Brinton

Research output: Contribution to journalArticle

57 Scopus citations

Abstract

The first 96 nucleotides of the 5' noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3'SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3'SL RNA as the probe. Three RNA-protein complexes (complexes 1, 2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross.linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3'SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3'SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.

Original languageEnglish (US)
Pages (from-to)6278-6287
Number of pages10
JournalJournal of virology
Volume70
Issue number9
DOIs
StatePublished - Sep 1996

ASJC Scopus subject areas

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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