Maintaining the structural integrity of thebamboo mosaic virus 3′ untranslated region isnecessary for retaining the catalytic constant forminus-strand RNA synthesis

Abstract

Background: Bamboo mosaic virus (BaMV) and the Potato virus X (PVX) are members of the genus Potexvirus andhave a single-stranded positive-sense RNA genome. The 3′-untranslated region (UTR) of the BaMV RNA genomewas mapped structurally into ABC (a cloverleaf-like), D (a stem-loop), and E (pseudoknot) domains. The BaMVreplicase complex that was isolated from the infected plants was able to recognize the 3′ UTR of PVX RNA toinitiate minus-strand RNA synthesis in vitro.Results: To investigate whether the 3′ UTR of PVX RNA is also compatible with BaMV replicase in vivo, weconstructed chimera mutants using a BaMV backbone containing the PVX 3′ UTR, which was inserted in or used toreplace the various domains in the 3′ UTR of BaMV. None of the mutants, except for the mutant with the PVX3′ UTR inserted upstream of the BaMV 3′ UTR, exhibited a detectable accumulation of viral RNA in Nicotianabenthamiana plants. The in vitro BaMV RdRp replication assay demonstrated that the RNA products were generatedby the short RNA transcripts, which were derived from the chimera mutants to various extents. Furthermore, theVmax/KM of the BaMV 3′ UTR (rABCDE) was approximately three fold higher than rABCP, rP, and rDE in minus-strandRNA synthesis. These mutants failed to accumulate viral products in protoplasts and plants, but were adequatelyreplicated in vitro.Conclusions: Among the various studied BaMV/PVX chimera mutants, the BaMV-S/PABCDE that containednon-interrupted BaMV 3′ UTR was the only mutant that exhibited a wild-type level of viral product accumulation inprotoplasts and plants. These results indicate that the continuity of the domains in the 3′ UTR of BaMV RNA wasnot interrupted and the domains were not replaced with the 3′ UTR of PVX RNA in vivo