UCLA

A virus has one main goal: to replicate itself. To achieve this goal, many kinds of viruses must encode their own replication machinery to amplify their genomes in the host cell. This study focuses on a simple, positive-sense, single-stranded RNA virus, Nodamura, which has a genome consisting of two mRNA molecules. The translation product of RNA1 is an RNA-dependent RNA polymerase (RdRp), while its RNA2 codes for the capsid protein (CP); the RdRp binds to and replicates each of these molecules, and the CP packages the two of them into a single capsid. The focus of the current study is to assess the relative levels at which these two viral RNA molecules compete to be bound and replicated by the RdRp. Understanding replication phenomena such as this has important implications in numerous other many-molecule-mRNA-genome virus infection scenarios, in addition to potential translational medicine applications where it is useful to amplify therapeutic mRNAs using an RdRp. In vitro transcribed Nodamura RNA constructs were created in which a fluorescent reporter gene is added to RNA1, and the CP gene in RNA2 is replaced entirely by a different fluorescent reporter. These two constructs were then transfected into mammalian (BHK-21) cells. Fluorescence intensity assays and quantitative PCR experiments were performed at various time points post-transfection to study the replication competition between Nodamura’s RNA1 and RNA2 in the absence and presence of one another. These assays showed that Nodamura RNA1 replication decreases by as much as a factor of two when in the presence of RNA2, while RNA2 is not amplified at all in the presence of the RdRp encoded by RNA1. These results indicate how in cis and in trans replication dynamics determine the differential expression of the viral genomic RNAs.