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

Abstract

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 5Ј end and lack polyadenylation at the 3Ј 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 3Ј end of their genomic RNAs, a prominent feature of bromo-and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3Ј 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. 1) turned out to be the replicative form of the 3Ј termini (19,20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16)(17)(18). Although other models have been forwarded (9), we have proposed that ...

Non-canonical Translation in Plant RNA Viruses

“…In addition to the CPB form, AMV RNAs 3 ′ termini also fold into a pseudoknot structure that resembles a TLS conformation. The 3 ′ -UTR can be recognized by a tRNA-specific enzyme and by the viral replicase and this recognition is inhibited by the addition of CP (Olsthoorn et al, 1999;Chen and Olsthoorn, 2010). Thus, it suggests that TLS conformation acts as a minus-strand promoter and the CP interaction and pseudoknot stability may regulate a conformational switch between translation and replication (Chen and Olsthoorn, 2010).…”

Non-encapsidation activities of the capsid proteins of positive-strand RNA viruses

“…One model suggests that the interaction stimulates RNA synthesis by reorganizing the conformation of 3′ UTR to facilitate the recognition of the promoter for minus-strand synthesis by the viral RdRp (Guogas et al, 2004;Reichert et al, 2007). An alternative model proposes that the change in 3′ UTR conformation upon CP binding blocks RNA synthesis (Chen and Olsthoorn, 2010;Olsthoorn et al, 1999). A key to reconciling these discrepant observations may be that the CP strongly stimulated viral RNA replication at low concentrations, but inhibits replication at higher concentrations (Guogas et al, 2005).…”

A conserved RNA pseudoknot in a putative molecular switch domain of the 3′-untranslated region of coronaviruses is only marginally stable

“…Riboswitches are a particularly dramatic example of conformational switching, where the direct binding of a small molecule metabolite can drive a global conformational change that results in the formation of one RNA conformation to the exclusion of others; when found in noncoding regions, riboswitches play a role in the regulation of the expression of downstream genes (Wakeman et al 2007;Baird et al 2010). Other RNA motifs often found in the 59-or 39-untranslated regions (UTRs) of viral RNAs are postulated to undergo conformational switching (Chen and Olsthoorn 2010;Hammond et al 2010); however, how the conformational landscape is changed to accommodate a pair of mutually exclusive structures is often not well understood. RNA pseudoknotting, which defines a specific RNA topology, is often intimately associated with RNA conformational switching (Hammond et al 2009;Kang et al 2009;Klein et al 2009;Rieder et al 2009;Chen and Olsthoorn 2010).…”

“…Other RNA motifs often found in the 59-or 39-untranslated regions (UTRs) of viral RNAs are postulated to undergo conformational switching (Chen and Olsthoorn 2010;Hammond et al 2010); however, how the conformational landscape is changed to accommodate a pair of mutually exclusive structures is often not well understood. RNA pseudoknotting, which defines a specific RNA topology, is often intimately associated with RNA conformational switching (Hammond et al 2009;Kang et al 2009;Klein et al 2009;Rieder et al 2009;Chen and Olsthoorn 2010). Here, we investigate the energetics that characterize a putative molecular switch in the 39 UTR of the model animal coronavirus, mouse hepatitis virus (MHV) (Goebel et al 2004a;Zust et al 2008).…”