Mechanistic insights into Sindbis virus infection: noncapped genomic RNAs enhance the translation of capped genomic RNAs to promote viral infectivity

Alphaviruses are globally distributed, vector-borne RNA viruses with high outbreak potential and no clinical interventions, posing a significant global health threat. Previously, the production and packaging of both viral capped and noncapped genomic RNAs (cgRNA and ncgRNA) during infection was reported. Studies have linked ncgRNA production to viral infectivity and pathogenesis, but its precise role remains unclear. To define the benefits of ncgRNAs, pure populations of capped and noncapped Sindbis virus (SINV) gRNAs were synthesized and transfected into host cells. The data showed that mixtures of cgRNAs and ncgRNAs had higher infectivity compared to pure cgRNAs, with mixtures containing low cgRNA proportions exceeding linear infectivity expectations. This enhancement depended on co-delivery of cgRNAs and ncgRNAs to the same cell and required the noncapped RNAs to be viral in origin. Contrary to the initial hypothesis that the ncgRNAs serve as replication templates, the cgRNAs were preferentially replicated. Further analysis revealed that viral gene expression, viral RNA (vRNA) synthesis and particle production were enhanced in the presence of ncgRNAs, which function to promote cgRNA translation early in infection. Our findings highlight the importance of ncgRNAs in alphaviral infection, showing they enhance cgRNA functions and significantly contribute to viral infectivity.