Naturally occurring mutations in replication proteins of a small RNA virus that alter the number, sizes, and relative abundances of subgenomic RNAs
Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in infected cells. sgRNAs are synthesized by virus-encoded replication proteins (RPs), but whether RPs regulate the number and sizes of sgRNAs remains largely unknown. We report multiple naturally occurring mutations within the RPs of turnip crinkle virus (TCV) that alter the number, sizes, and relative abundances of TCV sgRNAs. TCV is a (+) RNA virus that normally produces two sgRNAs: the 1,724-nucleotide (nt) sgRNA1 expressing movement proteins, and the 1,449-nt sgRNA2 expressing capsid protein. A single amino acid change, A113V, within a region shared by TCV RPs p28 and p88, diminished sgRNA1 levels and delayed viral systemic spread. Interestingly, three second-site RP mutations emerged in infected plants that, alone or in combination with A113V, resulted in over-production of sgRNA1 or accumulation of two alternative sgRNAs of 1,876 and 1,601 nt, and rescued A113V defects. The alternative sgRNAs originated from nearly identical recombination events, their size difference reflecting varying 5 extensions. Structural modeling of the TCV replication complex showed a conical ring architecture containing p28, p88, and a partially double-stranded RNA. While A113 may interact with TCV genomic RNA or host factors to promote (-) sgRNA1 synthesis, the second-site mutations likely influenced binding and entry of RNA template into the p88 active site. They may stall RNA synthesis at specific hotspots and stimulate template switching, thereby generating alternative (-) sgRNAs. Our findings reveal previously unrecognized constraints on viral RPs that ensure production of sgRNAs with precise sizes and abundances. Author summaryMany (+)-strand viruses, such as SARS-CoV-1 and -2, Chikungunya virus, and tomato mosaic virus, synthesize subgenomic RNAs (sgRNAs) during cellular infections. Targeting sgRNA production could prove to be an effective antiviral strategy, as sgRNAs are needed to express diverse proteins critical for viral survival and transmission. Although sgRNA production requires virus-encoded replication proteins (RPs), whether RPs also dictate the number, sizes, and relative abundances of viral sgRNAs remain to be thoroughly investigated. We identified and characterized four naturally occurring mutations in RPs of the plant-infecting turnip crinkle virus that specifically perturbed one of the sgRNAs. Our findings uncover novel constraints on viral RPs that safeguard sgRNA integrity, and avail them as potential targets for controlling pathogenic viruses.