Cofolding Organizes Alfalfa Mosaic Virus RNA and Coat Protein for Replication

Guogas, Laura M.; Filman, David J.; Hogle, James M.; Gehrke, Lee

doi:10.1126/science.1103399

Cited by 40 publications

(35 citation statements)

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“…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 (11).…”

supporting

confidence: 49%

“…Evidence for 3Ј organization is provided by the crystal structure of the 3Ј-terminal 39 nucleotides of the AMV RNA in complex with the RNA binding domain of the viral CP (11). These data show that the RNA binding domain of the viral CP forms an ␣-helix containing a crucial arginine (Fig.…”

Section: Discussionmentioning

confidence: 87%

“…We propose that the differences in functional data from the two laboratories may be traced to the use of wild-type cells (described here) versus in vitro analysis and the use of transgenic cells that overexpress replicase proteins (22). The accumulated evidence presented here, along with functional data presented in the accompanying paper (12) and the crystal structure of the AMV RNA-peptide complex (11), suggest that CP binding organizes the 3Ј termini of the viral RNAs for activation of early viral RNA replication.…”

mentioning

confidence: 99%

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

Petrillo

Rocheleau

Kelley-Clarke

et al. 2005

J Virol

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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 ...

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confidence: 49%

Section: Discussionmentioning

confidence: 87%

mentioning

confidence: 99%

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

Petrillo

Rocheleau

Kelley-Clarke

et al. 2005

J Virol

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“…Previously, we described an RNA binding motif (PTxR 17 SxxY) within the N terminus of the viral CP (2). The central role of this arginine for RNA binding has been demonstrated both biochemically (2) and structurally (15). Substituting lysine or alanine for R 17 prevents RNA-peptide binding interactions (2).…”

Section: Resultsmentioning

confidence: 99%

Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent

Guogas

Laforest

Gehrke

2005

J Virol

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Alfalfa mosaic virus (AMV) and ilarvirusRNA-protein interactions play important roles in a variety of biological processes and are of particular importance in the life cycle of positive-strand RNA viruses, where the genomic RNA serves as the template for protein synthesis and replication. Alfalfa mosaic virus (AMV) and ilarviruses provide an interesting model system in which to study RNA-protein interactions because the viral coat protein (CP) has multiple roles in the viral life cycle, not all of which are completely understood. In addition to virion assembly (21, 44) the viral CP has roles in cell-to-cell movement (12, 43), infectivity (6, 23), and translation (28,29).Most bromoviruses have a tRNA-like structure (TLS) on the 3Ј end of the RNA genome. The TLS has been found to be important for recruiting the viral replicase (8, 13). Conversely, AMV and ilarviruses lack a canonical CCA 3Ј end common to the TLS and further require the presence of their own viral CP to initiate replication (6). It has been shown that CP binds specifically to the 3Ј untranslated region found on all three AMV genomic RNAs as well as the subgenomic RNA4 (5, 22, 24). Genomic RNA1 and -2 encode proteins with replicase functions (33). RNA3 is dicistronic, encoding the viral movement protein (MP) and CP. Only the upstream open reading frame, encoding the viral MP, is translated from genomic RNA3. The viral CP is translated from a subgenomic RNA4, which is generated by the internal initiation of transcription from minus-strand RNA3. In vitro-transcribed RNA4 (CP mRNA) can substitute for CP in the activation of replication when inoculated with the genomic RNAs into tobacco protoplasts (46).It was first proposed by Houwing and Jaspars that an RNA conformational change accompanies CP binding to the 3Ј ends of the genomic RNAs and thereby presents a recognition signal for replicase binding (20). Results from circular dichroism analyses and native polyacrylamide gel electrophoresis experiments support the idea that the 3Ј untranslated region (UTR) of AMV RNA3 and -4 undergoes a conformational change upon binding amino-terminal CP peptides (3). Further investigation of this RNA-protein interaction revealed a 26-aminoacid RNA binding motif in the N terminus of the CP that is sufficient to activate replication in mesophyll protoplasts (3,39). A minimal CP binding site was also identified at the terminal 39 nucleotides of the AMV 3Ј UTR (1, 17). While it is known that the genomic RNAs are not infectious in the absence of CP (6, 47), it is unclear how CP initiates infection or how it influences ongoing replication.Several models have been proposed to explain the genome activation phenomenon. The interaction of CP with the genomic RNA 3Ј UTR may serve to protect the RNAs from degradation (30); however, AMV genomic RNAs have been shown to be stable for several hours when inoculated into protoplasts in the absence of CP or polymerase (19). Olsthoorn et al. (31) described the conformational switch model, wherein the protein-free 3Ј UTR assumes a pseu...

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“…TBSV condenses its RNA by virtue of positive charge on the flexible, N-terminal segments, a few of which might fold into a structure that recognizes an RNA packaging signal. The structure determined much later for recognition of alfalfa mosaic virus RNA by its capsid subunit may be a good model (19).…”

Section: Wwwannualreviewsorg • Veritas Per Structurammentioning

confidence: 99%

Veritas per structuram

Harrison

2015

Annu. Rev. Biochem.

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When I entered graduate school in 1963, the golden age of molecular biology had just begun, and myoglobin was the only protein with a known high-resolution structure. The romance of working out the structure of a virus by X-ray crystallography nonetheless captured both my imagination and the ensuing 15 years of my scientific life, during which "protein crystallography" began to morph into "structural biology." The course of the research recounted here follows the broader, 50-year trajectory of structural biology, as I could rarely resist opportunities to capitalize on new technologies when they opened some interesting part of biology to three-dimensional rigor. That fascination shows no sign of subsiding.

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Cofolding Organizes Alfalfa Mosaic Virus RNA and Coat Protein for Replication

Cited by 40 publications

References 21 publications

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

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

Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent

Veritas per structuram

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