Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Abstract

Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

“…A stable full-length cDNA clone of Zika virus was recently assembled into the linear vector pJAZZ (Annamalai et al, 2017;Godiska et al, 2010). Although not reported to date, linear plasmids may also be used to generate stable infectious clones of plant viruses.…”

A single nonsynonymous mutation on gene encoding E protein of Zika virus leads to increased neurovirulencein vivo

“…4B ). Of note, N154 is a unique glycosylation site on Zika virus E protein, and it has been shown to support Zika virus infection in adult immune-deficient mice by either enhancing virus neuroinvasion or facilitating DC-SIGN binding (38, 39). However, when Zika virus was inoculated intracranially in neonatal mice, the deletion of N154 glycosylation had no impact on virus virulence (40).…”

“…However, similar to West Nile virus (WNV) and Japanes encephalitis virus (JEV), Zika virus has only one N154 glycosylation site on E protein (40, 43). Indeed, glycosylation on N153 or N154 of E protein helps WNV or JEV to invade CNS in mammals (38,39,44,45). Surprisingly, an additional use of N67 glycosylation on JEV attenuates virus pathogenesis and reduces viral neuro-invasion in vivo (45), despite the N67 glycosylation mediates enhanced infectivity of WNV or DENV by facilitating interaction with DC-SIGN molecule on cell surface in cell culture models (45–47).…”

Pathogenic viruses: Molecular detection and characterization

“…The virus entry and replication stimulates the host antiviral and proinflammatory responses, apoptosis, and disrupts regulation of glial cell proliferation (Wang et al, 2017). The N-linked glycosylation of the envelope (E) protein and the precursor membrane (prM) protein (Nambala and Su, 2018) have been indicated to be important determinants of the Zika virus virulence and neuroinvasion (Annamalai et al, 2017). Although ZIKV has been known as an arthropod-borne virus with Aedes species mosquitoes as the primary vector, multiple routes of ZIKV transmission have been identified.…”