A Chikungunya Virus trans -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 and 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 -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-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 -replicase activity in mammalian cells, while in mosquito cells most of them increased-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 -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-replicase systems for studies of viral RNA replication and virus-host interactions. 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 -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-replicase system represents a valuable tool to study alphavirus-mosquito interactions at the molecular level and to develop advanced antiviral strategies.

Design and Use of Chikungunya Virus Replication Templates Utilizing Mammalian and Mosquito RNA Polymerase I-Mediated Transcription

“…Incorporation of one or more reporter-coding sequences into template RNA constructs allows the use of the much more convenient measurement of reporter activities as a proxy for the synthesis of corresponding positive-strand RNAs and is also applicable in a high-throughput format. Therefore, in the current study, as previously (8,36,39), firefly luciferase (Fluc) was used as a substitute for most of the ns ORF and Gaussia luciferase (Gluc) was used as a substitute for the structural ORF in template constructs ( Fig. 1B and 2B).…”

“…The effect is due to the high background activity of the reporter resulting from its efficient translation using RNA polymerase II-generated capped transcripts. This activity is often comparable to or even higher than the reporter activity produced from much more abundant viral replicase-generated full-length positive-strand RNAs (36,39).…”

“…In the case of alphaviruses, decoupled replication systems are generally designed to amplify truncated RNA templates where the ns and structural ORFs of alphavirus genome are replaced by sequences encoding different reporter proteins; such systems are often referred to as trans-replication systems. trans-Replication systems have been developed for SINV, SFV, and CHIKV and used to study alphavirus replication complex biogenesis (31)(32)(33) and RNA template sequence requirements (34), for analysis of the impact of template length on the size of replication complexes (35), for tagging of different replicase proteins (36), and for analysis of the impact of different mutations introduced into replicase proteins on viral RNA replication in mammalian (8,37,38) and mosquito (39) cells. While these studies have universally found the alphavirus transreplication systems to be efficient and robust tools, they have also revealed certain technical limitations.…”

“…While these studies have universally found the alphavirus transreplication systems to be efficient and robust tools, they have also revealed certain technical limitations. Namely, to produce replication-competent template RNA and mRNA for expression of replicase polyproteins, alphavirus trans-replication systems have traditionally used either bacteriophage T7 RNA polymerase and the corresponding promoters (33) or cellular RNA polymerase II promoters, such as the immediate early promoter of human cytomegalovirus (CMV) or the Aedes aegypti polyubiquitin promoter (Ubi) (36,39). The major drawback of the use of bacteriophage T7 RNA polymerase promoters is that the use of such trans-replication systems is restricted to cell lines expressing T7 RNA polymerase, which are generally not available for cell types relevant for in vivo alphavirus infections.…”

ADP-ribosyl–binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication

“…For this system, one plasmid encodes the CHIKV P1234 polyprotein behind a human cytomegalovirus (hCMV)-driven polymerase II (pol II) promoter, and a second plasmid encodes a pol I promoter-driven viral template RNA for synthesis of luciferase reporters from both the genomic 5′ UTR (firefly luciferase, Fluc) and subgenomic 5′ UTR (Gaussia luciferase, Gluc) by the viral proteins encoded by the P1234 plasmid. The pol II promoter/terminator was substituted for the previously described hCMV-Fluc-Gluc reporter to reduce the background reporter expression (69,71) and improve assay sensitivity. All previously analyzed nsP3 mutations (27) were introduced into viral polymerasecompetent hCMV-P1234 and polymerase-mutated control CMV-P1234 GAA plasmids by site-directed mutagenesis and were transfected into NSC34 cells along with the pol I-Fluc-Gluc template plasmid (Fig.…”