Synthesis of dengue virus RNA in vitro: initiation and the involvement of proteins NS3 and NS5.

An assay for flavivirus RNA-dependent RNA polymerase activity in vitro was established using extracts of Vero cells infected with dengue virus type 2 (DEN-2) or Kunjin virus (KUN). RNA synthesis was initiated on a template of viral replicative form (RF) and RF was converted to the replicative intermediate (RI). The RNA-dependent RNA polymerase complex of DEN-2 utilised either DEN-2 or KUN RF as template, and similarly the KUN polymerase complex utilised either DEN-2 or KUN RF template. In addition, antibodies against the nonstructural proteins NS3 and NS5 inhibited the conversion of RF to RI, indicating that NS3 and NS5 are involved in viral RNA replication.