CELL-PROTEINS BIND SPECIFICALLY TO WEST NILE VIRUS MINUS-STRAND 3' STEM-LOOP RNA

The first 96 nucleotides of the 5' noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynami cally predicted stem-loop (SL) structures that are conserved among fla viviruses. 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' m inus-strand stem-loop RNA [WNV (-)3'SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were perf ormed 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 specifici ty of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell protei ns with molecular masses of 108, 60, 50, and 42 kDa were detected by U V-induced cross-linking to the WNV (-)3'SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotid es of the WNV 3' minus-strand RNA, the region that contains the termin al SL. A flavivirus resistance phenotype was previously shown to be in herited in mice as a single, autosomal dominant allele. The efficienci es of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, s usceptible cells (C3H/He). Three RNA-protein complexes and four W-indu ced cross-linked cell proteins with mobilities identical to those dete cted 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 resista nt 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.