Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1

The flavivirus nonstructural protein NS1 has been implicated in viral RNA r eplication, although its precise role has not been identified. In its nativ e slate NS1 exists as a heat labile homodimer that is thought to be require d for NS1 function and secretion. However, we have recently identified a cD NA clone of KUN virus (FLSD) that replicates efficiently in cell culture bu t produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to le ucine) when compared with the parental KUN virus. When site-directed mutage nesis was used to substitute leucine(250) with proline in FLSD to produce t he clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly fas ter than FLSD and produced 100-fold more infectious virus early (12-24 h) i n infection. This correlated with our observations that FLSD required appro ximately IO-fold more infectious virus than 250pro to produce disease in we anling mice after intraperitoneal inoculation. Taken together our results i ndicate that mutation from proline to leucine at residue 250 in KUN NS1 abl ates dimer formation, slows virus replication, and reduces virulence in mic e. (C) 1999 Academic Press.