Role of Glycosylation of Envelope (E) and Non-structural Protein 1 (NS1) in Zika Virus Replication and Pathogenesis

Zika virus (ZIKV), a mosquito-transmitted flavivirus, re-emerged in the last decade causing serious human diseases including congenital microcephaly and Guillain-Barré syndrome. To address the changes in the viral genome that may have led to increased virulence of the virus, I constructed an infectious cDNA clone for the historical ZIKV isolate MR766. Recombinant MR766 (rMR) virus recovered from the full-length cDNA clone mimicked growth and pathogenic properties of the parental virus both in vitro and in vivo. Analyses of full-length genomes of over 300 ZIKV isolates revealed that one sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic, being absent in many of the African isolates while present in all isolates from the recent outbreaks. Here, I interrogated the role of this sequence motif and glycosylation of the E protein in pathogenicity of ZIKV. The rMR virus, which contains the VNDT motif, was highly pathogenic and caused lethality in a mouse model. In contrast, recombinant viruses with deletion of VNDT motif (m1MR) or mutation of N-linked glycosylation site (m2MR), were highly attenuated and non-lethal. The mutant viruses replicated poorly in the brain of infected mice when inoculated subcutaneously but replicated well following intracranial inoculation, suggesting that the N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion. Since the nonstructural protein 1 (NS1) is also glycosylated and known to play a role in transmission and pathogenicity, I mutated the glycosylation sites in NS1 (N130 and N207) individually or in combination in the background of m2MR virus. All mutant viruses grew to titers similar to the rMR virus in cell culture except the m5MR virus (triple glycosylation defective), which grew to lower titers. Mice inoculated with m5MR virus did not exhibit morbidity and survived. Interestingly, these mice developed high titers of neutralizing antibodies and cellular immune responses that are protective upon lethal challenge with the rMR virus. Our results suggest that glycosylation of both E and NS1 proteins plays an important role in virus pathogenicity, and m5MR virus could be developed as a live attenuated viral vaccine for ZIKV