scholarly journals Multifunctional Roles for the N-Terminal Basic Motif of Alfalfa mosaic virus Coat Protein: Nucleolar/Cytoplasmic Shuttling, Modulation of RNA-Binding Activity, and Virion Formation

2012 ◽  
Vol 25 (8) ◽  
pp. 1093-1103 ◽  
Author(s):  
Mari Carmen Herranz ◽  
Vicente Pallas ◽  
Frederic Aparicio
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Alfalfa Mosaic Virus ◽  
Binding Activity ◽  
Systemic Movement ◽  
Virion Formation

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.

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

2005 ◽  
Vol 79 (9) ◽  
pp. 5743-5751 ◽  
Author(s):  
Jessica E. Petrillo ◽  
Gail Rocheleau ◽  
Brenna Kelley-Clarke ◽  
Lee Gehrke
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Rna Binding ◽  
Alfalfa Mosaic Virus ◽  
Rna Replication ◽  
Comparative Sequence Analysis ◽  
Viral Rna ◽  
Conformational Switch ◽  
Functional Studies ◽  
Functional Features

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.

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

2004 ◽  
Vol 78 (15) ◽  
pp. 8036-8046 ◽  
Author(s):  
Gail Rocheleau ◽  
Jessica Petrillo ◽  
Laura Guogas ◽  
Lee Gehrke
Keyword(s):  
Coat Protein ◽  
Protein Binding ◽  
Mosaic Virus ◽  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Genetic Selection ◽  
Alfalfa Mosaic Virus ◽  
Rna Replication ◽  
Viral Rna

ABSTRACT 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.

scholarly journals The Photosystem II Oxygen-Evolving Complex Protein PsbP Interacts With the Coat Protein of Alfalfa mosaic virus and Inhibits Virus Replication

2014 ◽  
Vol 27 (10) ◽  
pp. 1107-1118 ◽  
Author(s):  
Muthukumar Balasubramaniam ◽  
Bong-Suk Kim ◽  
Heather M. Hutchens-Williams ◽  
L. Sue Loesch-Fries
Keyword(s):  
Coat Protein ◽  
Photosystem Ii ◽  
Mosaic Virus ◽  
Virus Replication ◽  
Alfalfa Mosaic Virus ◽  
Bimolecular Fluorescence Complementation ◽  
Oxygen Evolving Complex ◽  
In Planta ◽  
Oxygen Evolving

Alfalfa mosaic virus (AMV) coat protein (CP) is essential for many steps in virus replication from early infection to encapsidation. However, the identity and functional relevance of cellular factors that interact with CP remain unknown. In an unbiased yeast two-hybrid screen for CP-interacting Arabidopsis proteins, we identified several novel protein interactions that could potentially modulate AMV replication. In this report, we focus on one of the novel CP-binding partners, the Arabidopsis PsbP protein, which is a nuclear-encoded component of the oxygen-evolving complex of photosystem II. We validated the protein interaction in vitro with pull-down assays, in planta with bimolecular fluorescence complementation assays, and during virus infection by co-immunoprecipitations. CP interacted with the chloroplast-targeted PsbP in the cytosol and mutations that prevented the dimerization of CP abolished this interaction. Importantly, PsbP overexpression markedly reduced virus accumulation in infected leaves. Taken together, our findings demonstrate that AMV CP dimers interact with the chloroplast protein PsbP, suggesting a potential sequestration strategy that may preempt the generation of any PsbP-mediated antiviral state.

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