Decreased Virulence of Ross River Virus Harboring a Mutation in the First Cleavage Site of Nonstructural Polyprotein Is Caused by a Novel Mechanism Leading to Increased Production of Interferon-Inducing RNAs.

Xiang Liu,Jayaram Bettadapura,Andres Merits,Age Utt,Lara J Herrero,Suresh Mahalingam,Anni Lepland,Penny A Rudd,Margit Mutso,Adam Taylor

doi:10.1128/mbio.00044-18

Abstract

ABSTRACTInfection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo. Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease.

Highlights

IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV)
Reports have shown that a mutation in the region corresponding to the junction of nonstructural protein 1 and nsP2 regions in nonstructural polyproteins P123/P1234 of SINV and RRV has a substantial effect on type I IFN induction independent of virusmediated host shutoff [16]
Infectious-center assays (ICA) performed using BHK-21 cells revealed that both viruses were efficiently rescued

Summary

Introduction

IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). Reports have shown that a mutation in the region corresponding to the junction of nonstructural protein 1 (nsP1) and nsP2 regions (referred to hereafter as 1/2 site) in nonstructural polyproteins P123/P1234 of SINV and RRV has a substantial effect on type I IFN induction independent of virusmediated host shutoff [16]. Mutations in nsP2 of SFV affect type I IFN induction independently of host shutoff [13] This region has been identified as an important virulence factor for SFV [17] and SINV [18,19,20,21,22]; the molecular mechanism(s) behind these effects has not been revealed. The data suggest that for RRV, the A532V mutation has an effect on the inductive phase of the type I IFN response [16]

Objectives

Methods

Results

Conclusion