DC-SIGN Binding Contributed by an Extra N-Linked Glycosylation on Japanese Encephalitis Virus Envelope Protein Reduces the Ability of Viral Brain Invasion.

Jian-Jong Liang,Min-Wei Chou,Yi-Ling Lin

doi:10.3389/fcimb.2018.00239

Abstract

The major structural envelope (E) protein of Japanese encephalitis virus (JEV) facilitates cellular binding/entry and is the primary target of neutralizing antibodies. JEV E protein has one N-linked glycosylation site at N154 (G2 site), but the related dengue virus E protein has two glycosylation sites at N67 (G1 site) and N153 (G2 site). We generated three recombinant JEVs with different glycosylation patterns on the E protein. As compared with wild-type (WT) JEV with G2 glycosylation, viral growth in culture cells as well as neurovirulence and neuroinvasiveness in challenged mice were reduced when infected with the G1 mutant (E-D67N/N154A) with glycosylation shifted to G1 site, and the G0 mutant (E-N154A) with non-glycosylation. The G1G2 mutant (E-D67N), with E-glycosylation on both G1 and G2 sites, showed potent in vitro viral replication and in vivo neurovirulence, but reduced neuroinvasiveness. Furthermore, the JEV mutants with G1 glycosylation showed enhanced DC-SIGN binding, which may then lead to reduced brain invasion and explain the reason why WT JEV is devoid of this G1 site of glycosylation. Overall, the patterns of N-linked glycosylation on JEV E proteins may affect viral interaction with cellular lectins and contribute to viral replication and pathogenesis.

Highlights

The flavivirus genus contains many important human pathogens such as Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus, West Nile virus (WNV), and Zika virus (ZIKV)
Mature flaviviral particles have 180 copies of E glycoproteins and M proteins embedded in the viral membrane (Kuhn et al, 2002)
The most notable difference between flaviviruses is in the region near the glycosylation site (Sirohi and Kuhn, 2017)

Summary

Introduction

The flavivirus genus contains many important human pathogens such as Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus, West Nile virus (WNV), and Zika virus (ZIKV). Flaviviral virion is enveloped and contains a positive-sense, single-stranded RNA genome. The viral RNA translates a polyprotein that is proteolytically processed into structural proteins [core, precursor of membrane, and envelope (E)] and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Lindenbach et al, 2007). Embedded in the viral membrane are 180 copies of the E glycoproteins. The E glycoprotein facilitates cellular attachment, binding and entry, and is the primary target of neutralizing antibodies (Mukhopadhyay et al, 2005). The flavivirus receptors included two types of molecules, glycosaminoglycans (GAGs) and cellular proteins. Sulfated GAGs such as heparan sulfate serve as initial attachment factors to gather

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