Xiaoyu Sun, University of Helsinki

Xiaoyu Sun (1), Serban L. Ilca (2), Katri Eskelin (1), Juha T. Huiskonen (2,3), Minna M. Poranen (1) (1) Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland (2) Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom (3) Helsinki Institute of Life Science, University of Helsinki, Finland

Virus Replication: Entry, Exit and Everything in Between

The viral-encoded RNA-dependent RNA polymerase (RdRp) is a key factor in the replication and transcription of dsRNA viruses. Typically, the RdRp is sequestered into a protein capsid, the polymerase complex, for intra-capsid genome replication and transcription. However, the free RdRp subunits of several dsRNA viruses have been reported to be functional also in the absence of other viral proteins, and able to replicate heterologous templates. Thus, free RdRps synthetized in infected cells could potentially convert cellular mRNA into dsRNA, leading to toxic effects on the host. How to ensure that the RdRp is successfully assembles into viral particles without fatally interrupting the host mRNA metabolism is an intriguing question. Here we used the in vitro assembly and replication system of cystovirus phi6 together with biochemical and structural methods (e.g., microscale thermophoresis, asymmetric flow field flow fractionation, cryo-EM) to study interactions between the phi6 RdRp P2 and assembly cofactor P7, and the potential impact of these interactions on the viral RNA synthesis. We show that P2 and P7 interact prior to their incorporation into the polymerase complex, mainly through electrostatic forces. This interaction reduces P2 RdRp affinity to ssRNA, which downregulates RNA replication. The P2-P7 interaction also facilitates the incorporation of P7 into the empty polymerase complex during the self-assembly of the capsid. However, at high ssRNA concentration P7 is displaced enabling P2 binding to RNA, and RNA polymerization. Furthermore, we identified P7-P2 complexes and describe their interaction with viral dsRNA in transcribing viral core particles.