Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus.

Daniel Westlake,Natalie A Prow,Roy A Hall,Helle Bielefeldt-Ohmann

doi:10.3390/v13020147

Daniel Westlake, Natalie A Prow + Show 2 more

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https://doi.org/10.3390/v13020147

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Journal: Viruses	Publication Date: Jan 20, 2021
Citations: 3	License type: CC BY 4.0

Affiliation: University of Queensland, University of South Australia

Abstract

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV3929), including the hinge region (E273–277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV3929 binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV3929 exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV3929 as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV3929 in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273–277 and E154–156 of ALFV3929 were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV3929, with the E327/E273–277 combination most dominant. These data demonstrate that attenuation of ALFV3929 is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Highlights

Alfuy virus (ALFV) is a member of the Japanese encephalitis virus (JEV) sero-complex of flaviviruses and is closely related genetically and antigenically to the pathogenic MurrayValley encephalitis virus (MVEV), which is associated with encephalitis in humans and equines [1]
In this study we identified a novel flavivirus attenuation marker in the EDIII of ALFV
The substitution of glutamine with a large positively charged residue at E327, substantially enhanced GAG binding and reduced plaque size in ALFV. This substitution reduced neuro-invasiveness of infectious clones containing the ALFV prM-E genes in an Murray Valley encephalitis virus (MVEV) genome backbone. This extends our understanding of the mechanisms of attenuation in ALFV and the JEV serogroup of flaviviruses

Summary

Introduction

Valley encephalitis virus (MVEV), which is associated with encephalitis in humans and equines [1]. While it shares 84% deduced amino acid sequence identity with. MVEV (89% in the viral envelope (E) protein), ALFV is neither pathogenic in humans nor neuro-invasive in mice [1,2]. ALFV is a positive sense RNA virus with a genome of 11.4kb, encoding a single polyprotein co- and post-translationally processed into three structural proteins, capsid (C), pre-membrane/membrane (prM/M) and envelope (E), in addition to seven non-structural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Inside the outer layer is the nucleocapsid consisting of the C protein and viral RNA [3].

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