Essential role of cyclization sequences in Flavivirus RNA replication

A possible role in RNA replication for interactions between conserved compl ementary (cyclization) sequences in the 5'- and 3'-terminal regions of Flav ivirus RNA was previously suggested but never tested in vivo. Using the M-f old program for RNA secondary-structure predictions, we examined for the fi rst time the base-pairing interactions between the covalently linked 5' gen omic region (first similar to 160 nucleotides) and the 3' untranslated regi on (last similar to 115 nucleotides) for a range of mosquito borne Flavivir us species. Base-pairing occurred as predicted for the previously proposed conserved cyclization sequences. In order to obtain experimental evidence o f 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 com plementarity. None of the RNAs with separate mutations in only the 5' or on ly the 3' cyclization sequences was able to replicate after transfection in to BHK cells, while replicon RNA with simultaneous compensatory mutations i n both cyclization sequences was replication competent. This was detected b y immunofluorescence for expression of the major nonstructural protein NS3 and by Northern blot analysis for amplification and accumulation of replico n RNA. We then used the M-fold program to analyze RNA secondary structure o f the covalently linked 5'- and 3'-terminal regions of three tick borne vir us species and identified a previously undescribed additional pair of conse rved complementary sequences in locations similar to those of the mosquito borne species. They base-paired with DeltaG values of approximately -20 kca l, equivalent or greater in stability than those calculated for the origina lly proposed cyclization sequences. The results show that the base-pairing between 5' and 3' complementary sequences, rather than the nucleotide seque nce 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.