The non-template functions of helper virus RNAs create optimal replication conditions to enhance the proliferation of satellite RNAs

As a type of parasitic agent, satellite RNAs (satRNAs) rely on cognate helper viruses to achieve their replication and transmission. During the infection of satRNAs, helper virus RNAs serve as templates for synthesizing viral proteins, including the replication proteins essential for satRNA replication. However, the role of non-template functions of helper virus RNAs in satRNA replication remains unexploited. Here we employed the well-studied model that is composed of cucumber mosaic virus (CMV) and its associated satRNA. In the experiments employing the CMV trans-replication system, we observed an unexpected phenomenon the replication proteins of the mild strain LS-CMV exhibited defective in supporting satRNA replication, unlike those of the severe strain Fny-CMV. Independent of translation products, all CMV genomic RNAs could enhance satRNA replication, when combined with the replication proteins of CMV. This enhancement is contingent upon the recruitment and complete replication of helper virus RNAs. Using the method developed for analyzing the satRNA recruitment, we observed a markedly distinct ability of the replication proteins from both CMV strains to recruit the positive-sense satRNA-harboring RNA3 mutant for replication. This is in agreement with the differential ability of both 1a proteins in binding satRNAs in plants. The discrepancies provide a convincing explanation for the variation of the replication proteins of both CMV strains in replicating satRNAs. Taken together, our work provides compelling evidence that the non-template functions of helper virus RNAs create an optimal replication environment to enhance satRNA proliferation. Satellite RNAs (satRNAs) are a type of subviral pathogens that encode neither replication protein nor coat protein. Consequently, they must compete with helper virus RNAs for viral proteins and host resources to ensure their survival. The competition relationship has led us to conceptualize that helper virus RNAs might exhibit antagonism with satRNAs during replication. Several studies have highlighted the importance of non-template functions of viral RNAs in viral replication, such as viral replicase-mediated RNA recruitment and participating in the assembly of viral replication complex. However, it remains unknown whether this significance extends to satRNA replication. To address this question, we utilized a well-established model involving cucumber mosaic virus and its associated satRNAs. Our data provides compelling evidence to uncover the biological relevance of the non-template functions of helper virus RNAs in enhancing satRNA replication. Thus, our work prompts a reconsideration of the molecular interplay between helper virus RNAs and satRNAs in their replication, previously thought to be mutually competitive.