Virus replication Proteolytic cleavage Replication complex Running Title: Critical role of NS4A during WNV replication

ow nloaded from The West Nile virus strain Kunjin virus (WNVKUN) NS4A protein is a multifunctional protein involved in membrane proliferation, stimulation of cellular pathways, evasion of host defence and is a major component of the WNVKUN RNA replication complex. We identified a highly conserved region (120P-E-P-E123), upstream of the viral protease dibasic cleavage site and investigated whether this motif was required for WNVKUN replication. Single point mutations to Alanine and a PEPE deletion mutation were created in a full-length infectious WNVKUN molecular clone. All mutations drastically impaired viral replication and virion production, except the P122A mutant which was slightly attenuated. These mutations were subsequently transferred to a WNVKUN replicon to specifically assess effects on RNA replication alone. Again, all mutants, except P122A, showed severely reduced negative sense RNA production as well as decreased viral protein production. Correspondingly, immunofluorescence analyses showed a lack of dsRNA labelling and a dispersed localization of the WNVKUN proteins, suggesting that replication complex formation wa