Simple Summary Based on analysis of the tRNA-like structure (TLS) mutation in cucumber mosaic virus (CMV), mutation repair is correlated with several levels of triggered factors, including the dose of inoculation of virus mutants, the quantity effect on corresponding viral RNA, and the quality effect on corresponding viral RNA. All types of TLS mutation in different RNAs of CMV can be repaired at a low dose around the dilution end-point. At a high dose of inoculation, TLS mutations in RNA2 and RNA3, but not RNA1, can be repaired, which correlates with the relative quantity defect of RNA2 or the genome size defect of RNA3. In addition, all the above types of mutation repair necessarily require cell-to-cell movement, which presents the obligated effect of cell-to-cell movement on mutation repair. Some debilitating mutations in RNA viruses are repairable; however, the triggering factors of mutation repair remain largely unknown. In this study, multiple triggering factors of mutation repair are identified based on genetic damage to the TLS in CMV. TLS mutations in different RNAs distinctively impact viral pathogenicity and present different types of mutation repair. RNA2 relative reduction level or RNA3 sequence change resulting from TLS mutation is correlated with a high rate of mutation repair, and the TLS mutation of RNA1 fails to be repaired at the high inoculum dose. However, the TLS mutation of RNA1 can be repaired at a low dose of inoculation, particularly around the dilution end-point or in the mixed inoculation with RNA2 having a pre-termination mutation of the 2b gene, an RNAi suppressor. Taken together, TLS mutations resulting in quality or quantity defects of the viral genome or TLS mutations at low doses around the dilution end-point are likely to be repaired. Different levels of TLS mutation repair necessarily require cell-to-cell movement, therefore implying its obligated effect on the evolution of low-fitness viruses and providing a new insight into Muller's ratchet. This study provides important information on virus evolution and the application of mild viral vaccines.
