A Single Amino Acid Substitution in the West Nile Virus Nonstructural Protein NS2A Disables Its Ability To Inhibit Alpha/Beta Interferon Induction and Attenuates Virus Virulence in Mice

Abstract

Alpha/beta interferons (IFN-α/β) are key mediators of the innate immune response against viral infection. The ability of viruses to circumvent IFN-α/β responses plays a crucial role in determining the outcome of infection. In a previous study using subgenomic replicons of the Kunjin subtype of West Nile virus (WNV_KUN), we demonstrated that the nonstructural protein NS2A is a major inhibitor of IFN-β promoter-driven transcription and that a single amino acid substitution in NS2A (Ala30 to Pro [A30P]) dramatically reduced its inhibitory effect (W. J. Liu, H. B. Chen, X. J. Wang, H. Huang, and A. A. Khromykh, J. Virol. 78:12225-12235). Here we show that incorporation of the A30P mutation into the WNV_KUN genome results in a mutant virus which elicits more rapid induction and higher levels of synthesis of IFN-α/β in infected human A549 cells than that detected following wild-type WNV_KUN infection. Consequently, replication of the WNV_KUNNS2A/A30P mutant virus in these cells known to be high producers of IFN-α/β was abortive. In contrast, both the mutant and the wild-type WNV_KUN produced similar-size plaques and replicated with similar efficiency in BHK cells which are known to be deficient in IFN-α/β production. The mutant virus was highly attenuated in neuroinvasiveness and also attenuated in neurovirulence in 3-week-old mice. Surprisingly, the mutant virus was also partially attenuated in IFN-α/βγ receptor knockout mice, suggesting that the A30P mutation may also play a role in more efficient activation of other antiviral pathways in addition to the IFN response. Immunization of wild-type mice with the mutant virus resulted in induction of an antibody response of similar magnitude to that observed in mice immunized with wild-type WNV_KUN and gave complete protection against challenge with a lethal dose of the highly virulent New York 99 strain of WNV. The results confirm and extend our previous original findings on the role of the flavivirus NS2A protein in inhibition of a host antiviral response and demonstrate that the targeted disabling of a viral mechanism for evading the IFN response can be applied to the development of live attenuated flavivirus vaccine candidates.