Replication of dengue virus requires first making a minus-strand copy of the plus-strand genome, followed by making plus-strand genomes from the minus strand. Approximately the first 100 and the last 300 nucleotides of the dengue genome are not part of the open reading frame, yet are conserved. We presume that the 3' untranslated region (UTR) and the complement of the 5' UTR contain cis-acting sequences essential for the initiation of replication of the minus-strand and the plus-strand, respectively. We previously constructed a clone containing the CAT gene surrounded by the 5' and 3' UTRs of dengue virus type 4. An SP6 polymerase promoter was used to make a capped 1.5 kb transcript which resembles a miniature version of the dengue genome, except it encodes CAT instead of the dengue polyprotein. This RNA was transfected into cells alone or along with dengue virus RNA to provide trans-acting functions, and CAT activity was monitored 5 days later. Unfortunately, the CAT activity was higher in the cells transfected without helper. We were surprised at the longevity of the CAT activity in the absence of helper. The reason for the diminution upon co-transfection with dengue RNA is not certain, but it could be due to an effect on transfection efficiency, or perhaps to an effect of dengue replication on the health of the cells. We are in the process of deriving a clone which will express the lacZ gene from the SP6 promoter, with no dengue sequences, for use as an internal standard in transfection experiments. Also, we are deriving another version of the CAT clone, where the SP6 promoter will transcribe the minus-strand. Transfection of such a minus-strand RNA into cells should not result in CAT activity unless plus-strand copies are made. Ultimately, we hope to engineer mutations into the dengue UTRs to better define critical sequences.
