Proteomic Discovery of VEEV E2-Host Partner Interactions Identifies GRP78 Inhibitor HA15 as a Potential Therapeutic for Alphavirus Infections.

Michael D Barrera,Aarthi Narayanan,Victoria Callahan,Kylene Kehn-Hall,Ivan Akhrymuk,Weidong Zhou,Catherine Campbell,Nishank Bhalla

doi:10.3390/pathogens10030283

Michael D Barrera, Aarthi Narayanan + Show 6 more

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https://doi.org/10.3390/pathogens10030283

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Abstract

Alphaviruses are a genus of the Togaviridae family and are widely distributed across the globe. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV), cause encephalitis and neurological sequelae while chikungunya virus (CHIKV) and Sindbis virus (SINV) cause arthralgia. There are currently no approved therapeutics or vaccines available for alphaviruses. In order to identify novel therapeutics, a V5 epitope tag was inserted into the N-terminus of the VEEV E2 glycoprotein and used to identify host-viral protein interactions. Host proteins involved in protein folding, metabolism/ATP production, translation, cytoskeleton, complement, vesicle transport and ubiquitination were identified as VEEV E2 interactors. Multiple inhibitors targeting these host proteins were tested to determine their effect on VEEV replication. The compound HA15, a GRP78 inhibitor, was found to be an effective inhibitor of VEEV, EEEV, CHIKV, and SINV. VEEV E2 interaction with GRP78 was confirmed through coimmunoprecipitation and colocalization experiments. Mechanism of action studies found that HA15 does not affect viral RNA replication but instead affects late stages of the viral life cycle, which is consistent with GRP78 promoting viral assembly or viral protein trafficking.

Highlights

Alphaviruses are a genus of the Togaviridae family that are significant human and veterinary pathogens
To facilitate protein interaction analysis, a V5 epitope tag was added to the N terminus of the Venezuelan equine encephalitis virus (VEEV) E2 glycoprotein in the context of the VEEV TC-83 genome, referred to as VEEV TC-83 V5-E2 (Figure 1A)
To determine the effects of the inserted tag on viral replication kinetics and E2 expression, the virus was grown in HEK 293T cells

Summary

Introduction

Alphaviruses are a genus of the Togaviridae family that are significant human and veterinary pathogens. Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are endemic to the Western Hemisphere. VEEV is endemic to the United States, Central and South America [1], while EEEV is endemic to. Sindbis virus (SINV) is widely distributed and found in Africa, Asia, Europe, and Australia while chikungunya virus (CHIKV) is endemic to Africa, South American and Central America [3,4]. Alphaviruses cause general symptoms of malaise, fever, and headaches, but alphaviruses, such as VEEV and EEEV are more neurovirulent and can cause encephalitis and neurological sequelae whereas SINV and CHIKV cause arthralgia [3,5,6]. As recently as 2019 an outbreak of EEEV occurred in the United States where

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