Functional analysis of glycosylation of Zika virus envelope and NS1 proteins

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. ZIKV has two important glycoproteins, both the envelope (E) protein, and nonstructural protein 1 (NS1). The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. NS1 forms a homodimer necessary for viral replication and it is also secreted as a hexamer and is involved in immune system evasion and pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154 and the NS1 is glycosylated at amino acids N130 and N207. To study the function of ZIKV protein glycosylation, recombinant ZIKV viruses were generated that lack the glycosylation sites, and the mutant viruses were analyzed in mammalian and mosquito hosts. In mouse models, the mutants were attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; in contrast to the NS1 mutant, the knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the E mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that the E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Additionally, a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation (ZIKV-NS1-LAV) protected mice against transmission to the fetus. Vaccinated dams challenged subcutaneously with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their continued development in humans to prevent congenital ZIKV syndrome seems warranted.