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

Abstract

The flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native state NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine(250) with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12-24 h) in infection. This correlated with our observations that FLSD required approximately 10-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice.

The mechanism by which P250L mutation impairs flavivirus-NS1 dimerization: an investigation based on molecular dynamics simulations

“…The expression of WNV or DENV-1 NS1 β-ladder monomers (C-terminal 172−352 region) alone are enough to produce stable head-to-head dimers, as detected in the crystal lattice and in solution (Edeling et al 2014). Therefore, in agreement with previous experimental reports (Hall et al 1999;Clark et al 2007), the P250L mutation-induced distortions of the β1-strand within the mutated monomer would in fact be a possible explanation for the inhibition of NS1 dimer formation.…”

“…Oligomerization of NS1 is observed in all flaviviruses and the dimeric form of this protein appears to play a role in flavivirus virulence. Studies with Kunjin and Murray Valley encephalitis viruses have demonstrated that a single amino acid substitution at residue 250 from proline to leucine (P250L) is critical for loss of NS1 dimerization (Hall et al 1999;Clark et al 2007). Additionally, according to these reports, the P250L mutation is correlated with retarded virus growth and reduced virulence in mice.…”

DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus

“…(results not shown). The inclusion of a stable proline to leucine mutation at residue 250 in the NS1 protein, shown to attenuate KUN virus in mice (13), into the pKUN1 DNA vaccine backbone should result in further attenuation of the KUN virus-based vaccine, while retaining highly efficient protective immunity…”

“…Inclusion in pKUN1 DNA of a mutation in the KUN NS1 gene, which we have previously shown to reduce KUN virulence in mice (13), ensured significant attenuation of pKUN1-derived virus. Therefore, we predicted that injection of pKUN1 DNA into selected tissues of mice would produce limited virus amplification in the animals and induce protective immunity against KUN virus and the closely related New York strain of WN virus without causing disease.…”

“…KUN-specific antibody responses in unimmunized mice or mice receiving defective DNA could not be detected (ELISA titer Ͻ40). Neutralizing antibody to KUN virus was also detected in sera of mice immunized with pKUN1 DNA (titers of [10][11][12][13][14][15][16][17][18][19][20] or FLSD virus (titers of 40). Slightly higher levels of antibody as measured by ELISA and virus neutralization correlated with a longer period of viremia in FLSD-immunized mice (Table 1).…”