Maintaining the structural integrity of the bamboo mosaic virus 3′ untranslated region is necessary for retaining the catalytic constant for minus-strand RNA synthesis

BACKGROUND: Bamboo mosaic virus (BaMV) and the Potato virus X (PVX) are members of the genus Potexvirus and have a single-stranded positive-sense RNA genome. The 3′-untranslated region (UTR) of the BaMV RNA genome was mapped structurally into ABC (a cloverleaf-like), D (a stem-loop), and E (pseudoknot) domains. The BaMV replicase complex that was isolated from the infected plants was able to recognize the 3′ UTR of PVX RNA to initiate minus-strand RNA synthesis in vitro. RESULTS: To investigate whether the 3′ UTR of PVX RNA is also compatible with BaMV replicase in vivo, we constructed chimera mutants using a BaMV backbone containing the PVX 3′ UTR, which was inserted in or used to replace the various domains in the 3′ UTR of BaMV. None of the mutants, except for the mutant with the PVX 3′ UTR inserted upstream of the BaMV 3′ UTR, exhibited a detectable accumulation of viral RNA in Nicotiana benthamiana plants. The in vitro BaMV RdRp replication assay demonstrated that the RNA products were generated by the short RNA transcripts, which were derived from the chimera mutants to various extents. Furthermore, the V(max)/K(M) of the BaMV 3′ UTR (rABCDE) was approximately three fold higher than rABCP, rP, and rDE in minus-strand RNA synthesis. These mutants failed to accumulate viral products in protoplasts and plants, but were adequately replicated in vitro. CONCLUSIONS: Among the various studied BaMV/PVX chimera mutants, the BaMV-S/PABCDE that contained non-interrupted BaMV 3′ UTR was the only mutant that exhibited a wild-type level of viral product accumulation in protoplasts and plants. These results indicate that the continuity of the domains in the 3′ UTR of BaMV RNA was not interrupted and the domains were not replaced with the 3′ UTR of PVX RNA in vivo.