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

Tenllado, Francisco; Bol, John F.

doi:10.1006/viro.1999.0170

Cited by 62 publications

(54 citation statements)

References 37 publications

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“…In the two-hybrid system, a strong CP-CP interaction is detectable (44), resulting in fast growth on selective medium (Fig. 1, row 12; constructs pADCP and pBCP in Table 1).…”

Section: Resultsmentioning

confidence: 99%

Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

Heijden

Carette

Reinhoud

et al. 2001

J Virol

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Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1contains methyltransferase-and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.The alphavirus-like superfamily comprises a large number of animal and plant viruses sharing the properties of a capped, positive-stranded RNA genome and homologies in their RNA replication proteins. Despite homologies between the guanylytransferase/methyltransferase-like (MT), helicase-like (HEL), and polymerase-like (POL) domains, they may be expressed as parts of a single protein or as separate entities distributed over two or three proteins. Little is known about the nature of the interactions between these polypeptides and the ratio in which they are present in the viral replication complex, although purified RNA-dependent RNA polymerases (RdRp) have been used extensively to study viral replication in vitro. We have studied the interactions between the replicase proteins of Alfalfa mosaic virus (AMV) and their in situ localization to gain insight in the assembly of the replication complex.AMV is the type species of the genus Alfamovirus. It belongs to the Bromoviridae family of plant viruses, all having a tripartite RNA genome of messenger-sense polarity. RNA 3 encodes the movement protein (MP) and, via a subgenomic mRNA 4, the coat protein (CP). RNAs 1 and 2 encode the P1 and P2 replicase proteins, respectively. The N terminus of P1 contains a domain with sequence homology to the domains involved in RNA capping of the alphavirus Semliki Forest virus (SFV) nsP1 protein and the more closely related Brome mosaic virus (BMV) 1a protein (1, 2, 41). The C-terminal end of P1 has homology to the alphavirus-like supergroup of HEL domains (17,22), including the SFV nsP2 protein, for which helicase activity was recently shown in vitro (16). The P2 protein contains a central domain with sequence motifs conserved among ma...

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“…In the two-hybrid system, a strong CP-CP interaction is detectable (44), resulting in fast growth on selective medium (Fig. 1, row 12; constructs pADCP and pBCP in Table 1).…”

Section: Resultsmentioning

confidence: 99%

Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

Heijden

Carette

Reinhoud

et al. 2001

J Virol

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“…Most studies on the implication of CP in the viral life cycle have been conducted with AMV, whereas there are relatively few experimental data on PNRSV CP structural properties and biological functions (3). For instance, AMV CP is required for plus-strand RNA accumulation, encapsidation, cell-to-cell movement, and systemic spread of the virus (49). This latter function requires the establishment of a physical interaction between CP and MP.…”

mentioning

confidence: 99%

Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus

Codoñer

Fares

Elena

2006

J Virol

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The relative functional and/or structural importance of different amino acid sites in a protein can be assessed by evaluating the selective constraints to which they have been subjected during the course of evolution. Here we explore such constraints at the linear and three-dimensional levels for the movement protein (MP) and coat protein (CP) encoded by RNA 3 of prunus necrotic ringspot ilarvirus (PNRSV). By a maximum-parsimony approach, the nucleotide sequences from 46 isolates of PNRSV varying in symptomatology, host tree, and geographic origin have been analyzed and sites under different selective pressures have been identified in both proteins. We have also performed covariation analyses to explore whether changes in certain amino acid sites condition subsequent variation in other sites of the same protein or the other protein. These covariation analyses shed light on which particular amino acids should be involved in the physical and functional interaction between MP and CP. Finally, we discuss these findings in the light of what is already known about the implication of certain sites and domains in structure and protein-protein and RNA-protein interactions.

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“…The observation made in the study can be an important step toward understanding the capsid formation in case of carlavirus group, as has been suggested in case of other viruses, a head-to-tail organization of the homotypic interaction of Tomato yellow leaf curl virus [7] and TSWV N protein where N-terminal and a C-terminal regions of the protein are important for capsid assembly [28]. In case of Alfalfa mosaic virus CP, two separate regions in the C-terminus were found critical for dimer formation and assembly [2,27]. C-terminus of Cowpea chlorotic mottle virus CP is essential for dimer formation and particle assembly [32].…”

Section: Resultsmentioning

confidence: 91%

Intermolecular Interactions of Chrysanthemum virus B Coat Protein: Implications for Capsid Assembly

2011

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Chrysanthemum virus B coat protein constitutes the viral capsid which, besides other functions, encapsulates and protects the viral nucleic acid. We have demonstrated homotypic interaction of the coat protein subunits, essentially important for dimer formation, which is the first step during capsid assembly in vivo and in vitro. Interaction capacity of full length and truncated protein has been investigated and important regions have been identified through protein-protein interaction in yeast and by coimmunoprecipitation assays. Complete coat protein was found to interact strongly with similar subunits. Constructs with 102 amino acids from the N-terminal and 64 amino acids from C-terminal were found to be inconsequential for dimer formation as they did not show any interaction with similar subunits or with full length protein when analysed for b-galactosidase or histidine prototrophy. Results suggest that the region of 98-184 amino acids from the middle plays an important role in the process, probably without the involvement of N-and C-terminals.

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

Cited by 62 publications

References 37 publications

Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

Adaptive Covariation between the Coat and Movement Proteins of Prunus Necrotic Ringspot Virus

Intermolecular Interactions of Chrysanthemum virus B Coat Protein: Implications for Capsid Assembly

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