Construction and functional analysis of stable cell lines expressing nsP1 of alphaviruses

Abstract

Alphaviruses are positive-strand RNA viruses that are transmitted by mosquitoes and cause various (often serious) diseases in their vertebrate hosts, including humans. Similar to other positive-strand RNA viruses, alphaviruses encode for RNA replicase. RNA replicase of alphaviruses consists of four virus-encoded subunits, called non-structural proteins 1-4 (nsP1-4). nsP1 is a membrane anchor or the replicase complex. It forms a dodecameric ring that holds other components in their place, its guanine-7-methyltransferase (MTase) and guanylyltransferase (GTase) activities are essential for the capping of viral RNA genomes. In this study, tetracycline-inducible stable cell lines expressing nsP1 or its mutants of different alphaviruses were generated. Trans- complementation assay was performed by supplementing homologous or heterologous wild-type nsP1 or its mutant version to functionally defective replicase of Chikungunya virus (CHIKV) in which the defect was caused by mutations of nsP1. We found that expression of wild-type nsP1 of matching or closely related alphaviruses can significantly compensate for capping defects of CHIKV replicase. The compensation was less effective, or not observed at all, for replicase mutants where the defect was due to its compromised membrane attachments. Adding the defective nsP1 to the CHIKV replicase which harbours the same mutation worsens the activity of replicase; however, activities of mutated replicase were restored when it was supplemented with nsP1 harbouring different functional defects. Furthermore, expression of these findings expands our knowledge about alphavirus RNA replication and trans-complementation of replicase-associated defects. Besides, tetracycline-inducible stable cell lines can be used as tools to trans-complement alphaviruses with lethal defects in nsP1 allowing obtaining conditionally infectious alphaviruses virions which can be used at low biosafety level laboratories for high-throughput neutralization and antiviral testing.