Evaluation of the Conformational Switch Model for Alfalfa Mosaic Virus RNA Replication

Abstract

Key elements of the conformational switch model describing regulation of alfalfa mosaic virus (AMV) replication (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999) have been tested using biochemical assays and functional studies in nontransgenic protoplasts. Although comparative sequence analysis suggests that the 3 untranslated regions of AMV and ilarvirus RNAs have the potential to fold into pseudoknots, we were unable to confirm that a proposed pseudoknot forms or has a functional role in regulating coat protein-RNA binding or viral RNA replication. Published work has suggested that the pseudoknot is part of a tRNA-like structure (TLS); however, we argue that the canonical sequence and functional features that define the TLS are absent. We suggest here that the absence of the TLS correlates directly with the distinctive requirement for coat protein to activate replication in these viruses. Experimental data are evidence that elevated magnesium concentrations proposed to stabilize the pseudoknot structure do not block coat protein binding. Additionally, covarying nucleotide changes proposed to reestablish pseudoknot pairings do not rescue replication. Furthermore, as described in the accompanying paper (L. M. Guogas, S. M. Laforest, and L. Gehrke, J. Virol. 79:5752-5761, 2005), coat protein is not, by definition, inhibitory to minus-strand RNA synthesis. Rather, the activation of viral RNA replication by coat protein is shown to be concentration dependent. We describe the 3 organization model as an alternate model of AMV replication that offers an improved fit to the available data.Regulation of the switch between translation and replication is a fundamental problem in understanding the biology of positive-stranded RNA viruses. Olsthoorn et al. (22) published the conformational switch model to propose a mechanism for regulating the switch in alfalfa mosaic virus (AMV). The conformational switch model asserts that the 3Ј termini of the viral RNAs fold into two mutually exclusive conformations that have distinct functions, that is, pseudoknotted (coat protein [CP]-free) and extended (CP-bound) forms. In the absence of viral CP, the RNA 3Ј terminus is said to adopt a pseudoknotted tRNA-like structure (TLS) that would make it structurally homologous to many other bromovirus RNAs. Regions 1 and 2 of the viral RNA (see Fig. 1B) have the potential to form a pseudoknot, and nucleotide variations across the ilarviruses suggest covarying substitutions that would maintain the longrange pseudoknot pairing (22). Equally interesting, however, is the fact that the nucleotides in region 3 of the viral RNAs also covary with changes in region 2, thereby also maintaining the potential to form the downstream hairpin by short-range folding (hairpin from nucleotides 869 through 877) (Fig. 1B). Recent data confirm that hairpin from nucleotides 869 through 877 is present in the crystal structure of the 39-nucleotide 3Ј-terminal RNA fragment in complex with the RNA binding domain of the viral CP ...

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

“…Finally, Petrillo et al (21) observed that RNA3 containing the pseudoknot compensatory mutant GC3 does not replicate in nontransgenic cells, in contrast to our previous data using transgenic cells expressing the viral replicase proteins (20). Although in transgenic cells the accumulation of GC3 was only ϳ20% of wild type, it was still much higher than that of any of the single mutants, G3 and C3, whose accumulation was almost undetectable.…”

“…These data are in contrast to our earlier findings with wild-type AMV CP (20) and to those of Aparicio et al (1) with a related ilarvirus for which we also proposed the existence of a TLS (4). This discrepancy may have been caused by the lack of specificity of CP26; the bandshifts observed by Petrillo et al (21) are independent of the integrity of hpA since mutant C3 in which all base-pairs of hpA are disrupted binds this peptide as well as the wild type or any other RNA with an intact hpA. This means that CP26 is apparently binding elsewhere in the 3Ј UTR and therefore may not be a good representative of the wild-type CP.…”

“…Although in transgenic cells the accumulation of GC3 was only ϳ20% of wild type, it was still much higher than that of any of the single mutants, G3 and C3, whose accumulation was almost undetectable. It is possible that the assay of Petrillo et al (21) was not sufficiently sensitive to detect a fivefold reduction in RNA accumulation. We think that the lower accumulation of GC3 is a reflection of the strong conservation of 5Ј pyrimidine (Py)-3Ј purine (Pu) base pairs in the 3Ј stem of the pseudoknot of AMV and related ilarvirus RNAs (4).…”

“…Recently, the existence of the conformational switch has been disputed by Gehrke and coworkers (21). By performing band-shift assays using a synthetic 26-amino-acid N-terminal peptide (CP26) representing the CP RNA binding domain and mutants of the 3Ј terminal 170-nt AMV RNA, it was shown that the presence of the pseudoknot does not interfere with the binding of CP26.…”

Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent

“…This conformational change converts the RNA to a more compact form in a manner similar to that described for folding active catalytic RNAs (7,45) or rRNA-protein complexes (4). It has been proposed that CP-free AMV RNAs fold into a TLS (31); however, AMV and ilarvirus RNAs lack key structural and functional determinants of TLS, including the canonical CCA 3Ј terminus, the ability to be charged by an aminoacyl synthetase, and activity as a substrate for nucleotidyltransferase (discussed in the accompanying paper [32] and refer- VOL. 79, 2005 CP REQUIREMENT FOR AMV REPLICATION 5759 ences 14, 16, and 31).…”