A Chikungunya Virus <i>trans</i> -Replicase System Reveals the Importance of Delayed Nonstructural Polyprotein Processing for Efficient Replication Complex Formation in Mosquito Cells

Abstract

Chikungunya virus (CHIKV) is a medically important alphavirus that is transmitted by <i>Aedes aegypti</i> and <i>Aedes albopictus</i> mosquitoes. The viral replicase complex consists of four nonstructural proteins (nsPs) expressed as a polyprotein precursor and encompasses all enzymatic activities required for viral RNA replication. nsPs interact with host components of which most are still poorly understood, especially in mosquitos. A CHIKV <i>trans</i>-replicase system that allows the uncoupling of RNA replication and nsP expression was adapted to mosquito cells and subsequently used for analysis of universal and host-specific effects of 17 different nonstructural polyprotein (ns-polyprotein) mutations. It was found that mutations blocking nsP enzymatic activities as well as insertions of enhanced green fluorescent protein (EGFP) into different nsPs had similar effects on <i>trans</i>-replicase activity regardless of the host (i.e., mammalian or mosquito). Mutations that slow down or accelerate ns-polyprotein processing generally had no effect or reduced <i>trans</i>-replicase activity in mammalian cells, while in mosquito cells most of them increased <i>trans</i>-replicase activity prominently. Increased RNA replication in mosquito cells was counteracted by an antiviral RNA interference (RNAi) response. Substitution of the W258 residue in the membrane binding peptide of nsP1 resulted in a temperature-sensitive defect, in the context of both the <i>trans</i>-replicase and infectious CHIKV. The defect was compensated for by secondary mutations selected during passaging of mutant CHIKV. These findings demonstrate the value of alphavirus <i>trans</i>-replicase systems for studies of viral RNA replication and virus-host interactions.<b>IMPORTANCE</b> Chikungunya virus is an important mosquito-transmitted human pathogen. This virus actively replicates in mosquitoes, but the underlying molecular mechanisms and interactions of viral and host components are poorly understood. This is partly due to the lack of reliable systems for functional analysis of viral nonstructural polyproteins (ns-polyproteins) and nonstructural proteins (nsPs) in mosquito cells. Adaption of a CHIKV <i>trans</i>-replicase system allowed study of the effects of mutations in the ns-polyprotein on RNA replication in cells derived from mammalian and mosquito hosts. We found that a slowdown of ns-polyprotein processing facilitates replication complex formation and/or functioning in mosquito cells and that this process is antagonized by the natural RNAi defense system present in mosquito cells. The mosquito-adapted CHIKV <i>trans</i>-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.