The novel trans-complementation system for chikungunya virus

Abstract

Alphaviruses (Togaviridae) are mosquito transmitted positive-sense RNA viruses that cause severe and sometimes even lethal diseases in their vertebrate hosts. Numerous outbreaks have been brought on by alphaviruses worldwide in the last few decades. Chikungunya virus (CHIKV) stands out as a major global health concern due to recurrent outbreaks and debilitating chronic arthralgia caused by CHIKV infection. The arthralgia might be due to the persistent replication of CHIKV. CHIKV non-structural protein 4 (nsP4) is the RNA-dependent RNA polymerase (RdRp), which enables the replication of the viral genome and therefore plays a critical role in virus infection. In this work, we established the inducible stable cell lines for CHIKV nsP4 expression. NsP4 expressed in these cells can functionally trans-complement CHIKV mutant genomes harboring lethal mutations in nsP4 or lacking the nsP4 region entirely. This system enables the production of CHIKV virions that are infectious for nsP4 expressing cell lines but limited to early stages of infection (attachment, internalization, and replicase protein expression) in other cells. Moreover, our system was found to be suitable for the analysis of compounds inhibiting CHIKV replication; for example, RdRp inhibitor 4’-Fluorouridine significantly inhibited viral replication in nsP4 expressing cells. Additionally, our study revealed that CHIKV nsP4 can form functional replicases with P123 polyproteins of heterologous alphaviruses using trans-replication assay. This finding expands the versatility of the CHIKV nsP4 stable cell lines. We extended these findings and constructed inducible cell lines that express both nsP1 and nsP4 components of replicase and demonstrated that these cells can complement for lethal defects introduced into both nsP1 and nsP4 of the CHIKV genome. Taken together, CHIKV nsP4 inducible stable cell lines are valuable tools for alphavirus research, can be used to produce virions that are infections only in specific cell lines and therefore be handled without biosafety risk, such tools open new possibilities for studies of alphavirus replication as well as for screening and analysis of inhibitors of alphavirus replication.