Coat protein blocks the in vitro transcription of the virion RNAs of alfalfa mosaic virus

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.

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Houwing, C.J. and Jaspars, E.M.J., In vitro evidence that the coat protein is the programming factor in alfalfa mosaic virus-induced RNA synthesis

FEBS Letters ◽

10.1016/0014-5793(87)80336-8 ◽

1987 ◽

Vol 223 (2) ◽

pp. 424-424

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Rna Synthesis ◽

Alfalfa Mosaic Virus

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

Journal of Virology ◽

10.1128/jvi.78.15.8036-8046.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8036-8046 ◽

Cited By ~ 6

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.

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In vitro evidence that the coat protein is the programming factor in alfalfa mosaic virus-induced RNA synthesis

FEBS Letters ◽

10.1016/0014-5793(87)80952-3 ◽

1987 ◽

Vol 221 (2) ◽

pp. 337-342 ◽

Cited By ~ 8

Author(s):

Corrie J. Houwing ◽

E.M.J. Jaspars

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Rna Synthesis ◽

Alfalfa Mosaic Virus

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-02-14-0035-r ◽

2014 ◽

Vol 27 (10) ◽

pp. 1107-1118 ◽

Cited By ~ 30

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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GENETIC ENGINEERING OF THE ZUCCHINI YELLOW MOSAIC VIRUS COAT PROTEIN GENE FOR EXPRESSION IN PLANTS.

HortScience ◽

10.21273/hortsci.25.9.1130d ◽

1990 ◽

Vol 25 (9) ◽

pp. 1130d-1130

Author(s):

Guowei Fang ◽

Rebecca Grumet

Keyword(s):

Coat Protein ◽

Mosaic Virus ◽

Coat Protein Gene ◽

Protein Gene ◽

Zucchini Yellow Mosaic Virus ◽

In Vitro Transcription ◽

Yellow Mosaic Virus ◽

35S Promoter ◽

Plant Expression Vector

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.

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