Functional analysis of the cloverleaf-like structure in the 3' untranslated region of bamboo mosaic potexvirus RNA revealed dual roles in viral RNA replication and long distance movement.

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) RNA was identified to fold into a tertiary structure comprising a cloverleaf-like structure designated ABC domain followed by a major stem-loop D, which in turn is followed by a pseudoknot E and a poly(A) tail. The coat protein accumulation level of the mutant, BaMV40A/DeltaABC, lacking ABC domain was just 15% that of wild-type when inoculated into protoplasts of Nicotiana benthamiana. This suggested that ABC domain might play an important role in BaMV RNA replication. To define the precise role of each of the three stem-loops of ABC domain in RNA replication, three mutants BaMV40A/DeltaA, -/DeltaB, and -/DeltaC each lacking stem-loop A, B, and C, respectively, were created. Our results showed that accumulation of viral products of mutants BaMV40A/DeltaB and -/DeltaC were not as efficient as wild-type. On the contrary, level of accumulation of viral products of BaMV/DeltaA was similar to that of wild-type in protoplasts and inoculated leaves. Interestingly, the accumulation of viral products was not as efficient as that of wild-type in systemic leaves, implying that stem-loop A is dispensable for replication, but signifies a role in systemic accumulation. Using UV cross-linking and competition experiments, it was demonstrated that the E. coli expressed helicase domain of BaMV ORF1 can preferentially interact with the ABC domain.