RNA determinants of a specific RNA-Coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs

Ansel-McKinney, Patricia; Gehrke, Lee

doi:10.1006/jmbi.1998.1656

Cited by 41 publications

(23 citation statements)

References 88 publications

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“…These data are consistent with prior evidence showing that mutations in the loop nucleotide sequences did not have a significant effect on viral RNA-coat protein interactions (1,26). The lack of involvement by the loop nucleotides distinguishes the AMV RNA-coat protein interaction from a number of other RNA-protein complexes, wherein specific binding is dependent on interactions with bulge or loop nucleotides (16,24,29).…”

Section: Discussionsupporting

confidence: 90%

“…The apex of hairpin 847 to 864, including paired nucleotides 851 and 860 and 852 and 859, represented a G-C rich environment in the selection, while the base of the proposed 3Ј hairpin 869 to 877 was similarly G-C rich. Highly represented nucleotides U844 (29 of 30 clones), C846 (28 of 30 clones), and A878 (29 of 30 clones) were also identified as being important for coat protein binding in previous chemical modification interference and hydroxyl radical footprinting experiments (1, 2), providing additional evidence in support of a two-site binding model (1,18). Conversely, two 3Ј-terminal nucleotides were found to be highly variable in the selection (Fig.…”

Section: Resultsmentioning

confidence: 66%

“…The stoichiometry of peptide binding to the RNA is 2:1 (1), and the boxes enclose nucleotides that are protected from hydroxyl radical cleavage by coat protein or the coat protein peptides (2). The red and blue letters represent nucleotides that were identified as important for coat protein binding by chemical modification interference analysis (1). The localization of nucleotides in the chemical modification interference study suggested that there are two repeated, partially overlapping peptide binding sites on the RNA.…”

Section: Resultsmentioning

confidence: 99%

“…2 and 3A), a finding that is consistent with the results of a higher complexity selection (14). A second internal control relates to the finding that nucleotides U844, C846, and A877 were identified as important for coat protein binding both by chemical modification interference analysis (1) and by the selection in this study. The convergence of the selection and chemical modification interference data is consistent with a two-site binding model (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Previous biochemical characterization has defined specific binding determinants in both the RNA and coat protein (1,3,14,15,18,(26)(27)(28). An amino acid mutation that disrupts coat protein binding to the viral RNA (R17A) also blocks RNA replication (37), suggesting that coat protein binding is directly linked to replication functions.…”

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confidence: 99%

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Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Rocheleau

Petrillo

Guogas

et al. 2004

J Virol

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The alfalfa mosaic virus (AMV) RNAs are infectious only in the presence of the viral coat protein; however, the mechanisms describing coat protein's role during replication are disputed. We reasoned that mechanistic details might be revealed by identifying RNA mutations in the 3-terminal coat protein binding domain that increased or decreased RNA replication without affecting coat protein binding. Degenerate (doped) in vitro genetic selection, based on a pool of randomized 39-mers, was used to select 30 variant RNAs that bound coat protein with high affinity. AUGC sequences that are conserved among AMV and ilarvirus RNAs were among the invariant nucleotides in the selected RNAs. Five representative clones were analyzed in functional assays, revealing diminished viral RNA expression resulting from apparent defects in replication and/or translation. These data identify a set of mutations, including G-U wobble pairs and nucleotide mismatches in the 5 hairpin, which affect viral RNA functions without significant impact on coat protein binding. Because the mutations associated with diminished function were scattered over the 3-terminal nucleotides, we considered the possibility that RNA conformational changes rather than disruption of a precise motif might limit activity. Native polyacrylamide gel electrophoresis experiments showed that the 3 RNA conformation was indeed altered by nucleotide substitutions. One interpretation of the data is that coat protein binding to the AUGC sequences determines the orientation of the 3 hairpins relative to one another, while local structural features within these hairpins are also critical determinants of functional activity.Alfalfa mosaic virus (AMV) has a number of exceptional features that make it a useful system for studying RNA-protein interactions (reviewed in references 6 and 17). The three positive-sense genomic AMV RNAs (RNAs 1 to 3) and the subgenomic coat protein mRNA (RNA 4) are packaged individually into four bacilliform, aphid-transmitted particles. The viral genomic RNAs are infectious only in the presence of the viral coat protein (7), which binds specifically to the RNA 3Ј termini. Unlike the arginine-rich RNA binding domains of the human immunodeficiency virus Tat and Rev proteins, the AMV coat protein RNA binding domain is lysine rich (3); however, it includes an arginine residue whose position and side chain identity are crucial for specific, high-affinity RNA binding (2). The AMV RNAs lack a poly(A) tail and are further distinguished from closely related plant viruses (e.g., brome mosaic virus) by the absence of a canonical CCA 3Ј terminus and the inability to be charged by aminoacyl synthetases, both of which are defining features of tRNA-like ends (12).The relationship between the formation of the AMV coat protein-RNA complex and functional activity in viral RNA translation, replication, and assembly is poorly understood. Previous biochemical characterization has defined specific binding determinants in both the RNA and coat protein (1,3,14,15,18,(26)(27)(28...

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Section: Discussionsupporting

confidence: 90%

Section: Resultsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Rocheleau

Petrillo

Guogas

et al. 2004

J Virol

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Base‐pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA‐coat protein complex

Boyce

Scott

Guogas

et al. 2005

J of Molecular Recognition

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The three-dimensional structure of the 3' terminus of alfalfa mosaic virus RNA in complex with an amino-terminal coat protein peptide revealed an unusual RNA fold with inter-AUGC basepairing stabilized by key arginine residues (Guogas, et al., 2004). To probe viral RNA interactions with the full-length coat protein, we have used in vitro genetic selection to characterize potential folding patterns among RNAs isolated from a complex randomized pool. Nitrocellulose filter retention, electrophoretic mobility bandshift analysis, and hydroxyl radical footprinting techniques were used to define binding affinities and to localize the potential RNA-protein interaction sites. Minimized binding sites were identified that included both the randomized domain and a portion of the constant regions of the selected RNAs. The selected RNAs, identified by their ability to bind full-length coat protein, have the potential to form the same unusual inter-AUGC Watson-Crick base pairs observed in the crystal structure, although the primary sequences diverge from the wild-type RNA. A constant feature of both the wild-type RNA and the selected RNAs is a G ribonucleotide in the third position of an AUGC-like repeat. Competitive binding assays showed that substituting adenosine for the constant guanosine in either the wild-type or selected RNAs impaired coat protein binding. These data suggest that the interactions observed in the RNA-peptide structure are likely recapitulated when the full-length protein binds. Further, the results underscore the power of in vitro genetic selection for probing RNA-protein structure and function.

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Genetic Dissection of the Multiple Functions of Alfalfa Mosaic Virus Coat Protein in Viral RNA Replication, Encapsidation, and Movement

Tenllado

Bol

2000

Virology

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Coat protein (CP) of alfalfa mosaic virus (AMV) binds as a dimer to the 3' termini of the three genomic RNAs and is required for initiation of infection, asymmetric plus-strand RNA accumulation, virion formation, and spread of the virus in plants. A mutational analysis of the multiple functions of AMV CP was made. Mutations that interfered with CP dimer formation in the two-hybrid system had little effect on the initiation of infection or plus-strand RNA accumulation but interfered with virion formation and reduced or abolished cell-to-cell movement of the virus in plants. Six of the 7 basic amino acids in the N-terminal arm of CP (positions 5, 6, 10, 13, 16, and 25) could be deleted or mutated into alanine without affecting any step of the replication cycle except systemic movement in plants. Mutation of Arg-17 interfered with initiation of infection (as previously shown by others) and cell-to-cell movement of the virus but not with plus-strand RNA accumulation or virion formation. The results indicate that in addition to the RNA-binding domain, different domains of AMV CP are involved in initiation of infection, plus-strand RNA accumulation, virion formation, cell-to-cell movement, and systemic spread of the virus.

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RNA determinants of a specific RNA-Coat protein peptide interaction in alfalfa mosaic virus: conservation of homologous features in ilarvirus RNAs

Cited by 41 publications

References 88 publications

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Degenerate In Vitro Genetic Selection Reveals Mutations That Diminish Alfalfa Mosaic Virus RNA Replication without Affecting Coat Protein Binding

Base‐pairing potential identified by in vitro selection predicts the kinked RNA backbone observed in the crystal structure of the alfalfa mosaic virus RNA‐coat protein complex

Genetic Dissection of the Multiple Functions of Alfalfa Mosaic Virus Coat Protein in Viral RNA Replication, Encapsidation, and Movement

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