Mature rotavirus particles contain equivalent amounts of ^7meGpppGcap and noncapped viral positive-sense RNAs

ABSTRACT Viruses have evolved different strategies to overcome their recognition by the host innate immune system. Addition of cap at their 5’RNA ends is an efficient mechanism to ensure escape from detection by the innate immune system, but also to ensure the efficient synthesis of viral proteins. Rotavirus mRNAs contain a type 1 cap structure at their 5’end that is added by the viral capping enzyme VP3. This is a multifunctional protein with all the enzymatic activities necessary to add the cap, and also functions as an antagonist of the OAS-RNase L pathway. Here, the relative abundance of capped and noncapped viral RNAs during the replication cycle of rotavirus was determined. We found that both classes of rotaviral +RNAs are encapsidated, and they were present in a 1:1 ratio in the mature infectious particles. The capping of viral +RNAs is dynamic since different ratios of capped and noncapped RNAs were detected at different times post infection. Similarly, when the relative amount of capped and uncapped viral +RNAs produced in an in vitro transcription system was determined, we found that the proportion was very similar to that in the mature viral particles and in infected cells, suggesting that the capping efficiency of VP3 both, in vivo and in vitro, might be close to 50%. Unexpectedly, when the effect of simultaneously knocking down the expression of VP3 and RNase L on the cap status of viral +RNAs was evaluated, we found that even though at late times post infection there was an increased proportion of capped viral RNAs in infected cells, the viral particles isolated from this condition contained an equal ratio of capped and noncapped viral RNA, suggesting that there might be a selective packaging of capped-noncapped RNAs. SIGNIFICANCE Rotaviruses have a genome composed of eleven segments of double stranded RNA. Whether all 5’ ends of the positive sense genomic RNA contained in the mature viral particles are modified by a cap structure is unknown. In this work, using a direct quantitative assay we characterized the relative proportion of capped and noncapped viral RNA in rotavirus infected cells and in viral particles. We found that independently of the relative proportions of cap/noncapped RNA present in rotavirus infected cells, there is a similar proportion of these two kinds of 5’-modified positive sense RNAs in the viral particles.