Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity.

Courtney N Dial,Thomas M Kicmal,Bryan C Mounce,Patrick M Tate

doi:10.3390/v11050403

Abstract

Polyamines are small positively-charged molecules abundant in eukaryotic cells that are crucial to RNA virus replication. In eukaryotic cells, polyamines facilitate processes such as transcription, translation, and DNA replication, and viruses similarly rely on polyamines to facilitate transcription and translation. Whether polyamines function at additional stages in viral replication remains poorly understood. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to polyamine depletion both in vitro and in vivo; however, precisely how polyamine function in picornavirus infection has not been described. Here, we describe CVB3 mutants that arise with passage in polyamine-depleted conditions. We observe mutations in the 2A and 3C proteases, and we find that these mutant proteases confer resistance to polyamine depletion. Using a split luciferase reporter system to measure protease activity, we determined that polyamines facilitate viral protease activity. We further observe that the 2A and 3C protease mutations enhance reporter protease activity in polyamine-depleted conditions. Finally, we find that these mutations promote cleavage of cellular eIF4G during infection of polyamine-depleted cells. In sum, our results suggest that polyamines are crucial to protease function during picornavirus infection. Further, these data highlight viral proteases as potential antiviral targets and highlight how CVB3 may overcome polyamine-depleting antiviral therapies.

Highlights

Coxsackievirus B3 (CVB3) is a positive-sense RNA virus, belonging to the Picornaviridae family.CVB3 commonly infects children, resulting in a self-limiting illness that can lead to the development of muscle, lung, and heart maladies
Our previous work demonstrating that CVB3 was sensitive to polyamine depletion via DFMO
To ascertain how CVB3 evolves in response to repeated replication cycles in the absence of polyamine depletion, we passaged the virus in the presence of DFMO

Summary

Introduction

Coxsackievirus B3 (CVB3) is a positive-sense RNA virus, belonging to the Picornaviridae family.CVB3 commonly infects children, resulting in a self-limiting illness that can lead to the development of muscle, lung, and heart maladies. Viral proteases, including enteroviral proteases, play crucial roles in the replication of RNA viruses [3]. Picornavirus RNA is directly translated via host ribosomes into a polyprotein, containing both structural and nonstructural proteins. This large protein is subsequently cleaved to generate functionally distinct viral proteins. The ability of viral proteases to cleave the viral polyprotein is essential for productive infection, as limiting viral protease activity significantly hinders virus infection. Viral proteins cleave several host proteins, including translation factors, immune effectors, and signaling molecules [5,6,7,8,9,10], to establish a proviral environment

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