Involvement of L polymerase and heat shock proteins in the biogenesis of viral circular RNAs derived from respiratory syncytial virus.

Circular RNAs (circRNAs) are new members of noncoding RNAs that have gained increased attention, and reports on viral circRNAs and their functions continue to emerge in recent years. However, the mechanisms underlying the biogenesis of viral circRNAs, especially those produced by RNA viruses, remain largely unexplored. Our recent research systematically identified and characterized viral circRNAs induced by respiratory syncytial virus (RSV), a human RNA virus for which no effective treatments exist. In this study, we further investigated the mechanism underlying the production of RSV circRNAs. We found that the viral L polymerase and the cellular HSP70 and HSP90 proteins associate with viral circRNAs through proteomic analysis of circRNA pulled-down lysates. These interactions were further confirmed using RNA-binding protein immunoprecipitation assays. We also observed that RSV circRNAs colocalize with the L polymerase and HSP70/90 proteins within the synthesis site, inclusion bodies (IBs). Moreover, both the viral L protein and the cellular HSP70 and HSP90 proteins modulate the production of viral circRNAs. Finally, the flanking AT/TA sequences of RSV circRNAs serve as cis-acting elements. Collectively, our study demonstrates that L polymerase and HSP70/90 proteins, identified as novel RSV circRNA-associated proteins, contribute to the biogenesis of viral circRNAs. This study provides new insights into host-RSV interactions and establishes a foundation for the development of innovative anti-RSV strategies.IMPORTANCEAs an RNA virus, respiratory syncytial virus (RSV) imposes a serious global disease burden, and effective treatments for RSV are still lacking. An increasing number of research studies has discovered and validated the presence of viral circular RNAs (circRNAs) and their functions in viral infection. However, elucidating the biogenesis of viral circRNAs, particularly those of RNA viruses, remains an intriguing challenge. In this study, we first explored the production mechanism of RSV circRNAs. We demonstrated that L polymerase and HSP70/90 proteins, identified as novel RSV circRNA-associated proteins, contribute to the biogenesis of viral circRNAs in inclusion bodies (IBs). Our study provides a comprehensive understanding of the crosstalk among RSV circRNA, L polymerase, HSP70/90 proteins, and IBs, which is essential for identifying novel anti-RSV strategies.