In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

The 3′ termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3′ UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3′ UTR of AMV RNAs.Alfalfa mosaic virus (AMV) is a plant virus that belongs to one of the five genera in the family Bromoviridae, whose genomes consist of three genomic RNAs (RNAs 1, 2, and 3) and one subgenomic RNA (RNA4) that are capped at the 5′ end and lack polyadenylation at the 3′ terminus (3). RNAs 1 and 2 encode the viral subunits P1 and P2 of the replicase, respectively. RNA3 encodes the movement protein and serves as a template for the synthesis of RNA4, which encodes the coat protein (CP).The role of AMV CP has been the subject of extensive research in the past four decades. Initially, it was found that, in contrast to RNAs of the Bromo-, Cucumo-, and Oleavirus genera, the genomic RNAs of AMV and the closely related genus Ilarvirus were not infectious as such but required the presence of CP in the inoculum (15). This phenomenon was called genome activation and was long considered to compensate for the lack of a tRNA-like structure (TLS) at the 3′ end of their genomic RNAs, a prominent feature of bromo- and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3′ end of AMV RNAs can adopt an alternative conformation that shows many structural similarities to the TLS of other Bromoviridae, although it could not be charged with an amino acid (20). The tRNA-like (TL) conformation (Fig. ​(Fig.1)1) turned out to be the replicative form of the 3′ termini (19, 20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16-18). Although other models have been forwarded (9), we have proposed that switching between these two conformations, mediated by CP binding, plays a fundamental role in the life cycle of AMV and ilarviruses by regulating the competing processes of translation and replication of the viral RNAs.Open in a separate windowFIG. 1.The CPB and the TL conformations of the AMV RNA3 3′ terminus. The two conformers of AMV RNA3 3′ 145 nt are shown. (A) CPB conformer. The two major CP binding sites are indicated by brackets. Base pairing between loop D and stem A promotes TL conformation. (B) Secondary structure of the TL conformer.In the present study, the distribution between CPB and TL conformers was further investigated. We addressed how changes in this distribution would affect recognition of the AMV 3′ untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding ezyme) and the viral polymerase in vitro. We also monitored how the binding affinity to CP is affected by this conformational switch in vivo using the yeast three-hybrid (Y3H) system (2, 11, 24). Together, the in vitro and in vivo data clearly demonstrate the existence and function of the conformational switch in the 3′ UTR of AMV RNAs.