FUNCTIONAL ANALYSIS OF DENGUE NONSTRUCTURAL PROTEINS, NS3 AND NS5

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The dengue virus NS3 and NS5 nonstructural proteins are candidates for viral enzymes that are involved in viral RNA transcription and/or replication. In order to examine the gene expression and functional activity of these two proteins, we engineered NS3 and NS5 specific DNA and constructed a recombinant baculovirus which expressed the NS3 or NS5 viral gene. Synthesis of NS3 in recombinant virus-infected cells was detected by indirect immunofluorescence assay. Similarly, the synthesis of NS5 was detected by SDS-polyacrylamide gel analysis of a lysate from recombinant virus infected cells. The protein products were prepared for immunization of rabbits and antisera specific to these proteins were obtained. The availability of NS3 specific sera should facilitate detection of this protein in experiments designed to study the requirement of downstream NS3a and NS4b sequences for expression of NS3. These experiments will be carried out to test the hypothesis that NS1 and NS3 may be analogous since each is followed by two hydrophobic proteins that may serve as proteases that effect specific cleavage of the dengue polyprotein.