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.