The C-terminal 33 amino acids of the cucumber mosaic virus 3a protein affect virus movement, RNA binding and inhibition of infection and translation (original) (raw)
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Journal of General Virology
The capsid protein (CP) of Cucumber mosaic virus (CMV) is required for cell-to-cell movement, mediated by the 3a movement protein (MP). Deletion of the C-terminal 33 amino acids of the CMV 3a MP (in the mutant designated 3aDC33 MP) resulted in CP-independent cell-to-cell movement, but not long-distance movement. RNA-binding studies done in vitro using isolated bacterially expressed MP showed that the 3aDC33 MP bound RNA more strongly, with fewer regions sensitive to RNase and formed cooperatively bound complexes at lower ratios of protein : RNA than the wild-type (wt) 3a MP. Analysis of the architecture of the complexes by atomic force microscopy showed that the wt 3a MP formed a single type of complex with RNA, resembling beads on a string. By contrast, the 3aDC33 MP formed several types of complexes, including complexes with virtually no MP bound or thicker layers of MP bound to the RNA. Assays showed that protein-RNA complexes containing high levels of either MP inhibited the infectivity and in vitro translatability of viral RNAs. The 3aDC33 MP inhibited these processes at lower ratios of protein : RNA than the wt 3a MP, consistent with its stronger binding properties. The apparent contradiction between these inhibition data and the CP-independent cell-to-cell movement of CMV expressing the 3aDC33 MP is discussed.
Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein
Journal of General Virology, 1997
A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity. Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors. The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E. coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes. Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233. This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV. The cucumber mosaic virus (CMV) 3a protein has been generally considered to be the movement protein of the virus. Analysis of 3a gene deletion mutants revealed that the 3a protein is not required for replication, but is essential for the spread of infection in planta (Suzuki et al., 1991 ; Boccard & Baulcombe, 1993). Transgenic tobacco plants expressing the 3a gene have been shown to complement movement-deficient mutants (Kaplan et al., 1995). Furthermore, the CMV 3a protein shares several properties with other well-characterized viral movement proteins, such as cell-wall localization in transgenic and infected tobacco plants and the ability to modify the sizeexclusion limit of plasmodesmata (Vaquero et al., 1994, 1996) and to traffic RNA from cell-to-cell (Ding et al., 1995). The ability to bind nucleic acids in vitro is a common feature of plant virus movement proteins (Citovsky et al.,
Virology, 1998
The N-terminal basic arm of cucumber mosaic cucumovirus (CMV) coat protein (CP) contains a conserved arginine-rich motif, which is characteristic of RNA binding proteins of several plant and nonplant viruses. To identify regions of the CMV CP N-terminus that are essential for interacting with viral genomic RNA, a comprehensive set of mutations was engineered into biologically active clones of CMV RNA3 and the behavior of each variant with respect to infectivity, packaging and movement was examined. Biological assays conducted in Chenopodium quinoa (local lesion host) and Nicotiana benthamiana (systemic host) revealed that variants lacking either 12 N-proximal amino acids or a region containing four consecutive arginine residues of the CP N-terminus were competent for assembly into virions and remained infectious in plants. Interestingly, two other variants, lacking either 19 N-proximal amino acids or a domain containing a cluster of six arginines in the arginine-rich motif, were incompetent for virion assembly but retained the ability to move cell to cell. Taken together, these results indicate that a major portion of the N-terminal basic arm of CMV CP is dispensable for CP-RNA interactions and also establish that CMV can move cell to cell in a nonvirion form. The distinctive role played by the viral CP in movement and specifically, the extent to which the CP N-terminal basic arm is involved in the infection cycle of CMV are discussed.
PLoS ONE, 2014
The multifunctional 2b protein of CMV has a role in the long distance and local movement of the virus, in symptom formation, in evasion of defense mediated by salicylic acid as well as in suppression of RNA silencing. The role of conserved amino acid sequence domains were analyzed previously in the protein function, but comprehensive analysis of this protein was not carried out until recently. We have analyzed all over the 2b protein by alanine scanning mutagenesis changing three consecutive amino acids (aa) to alanine. We have identified eight aa triplets as key determinants of the 2b protein function in virus infection. Four of them (KKQ/22-24/AAA, QNR/31-33/AAA, RER/34-36/AAA, SPS/40-42/AAA) overlap with previously determined regions indispensable in gene silencing suppressor function. We have identified two additional triplets necessary for the suppressor function of the 2b protein (LPF/55-57/AAA, NVE/10-12/AAA), and two other positions were required for cell-to-cell movement of the virus (MEL/1-3/AAA, RHV/70-72/AAA), which are not essential for suppressor activity.
Host-specific encapsidation of a defective RNA 3 of Cucumber mosaic virus
Journal of General Virology, 2004
Defective (D) RNAs were generated in tobacco upon passage of two isolates of Cucumber mosaic virus (CMV) initially derived from RNA transcripts of cDNA clones. In both cases, the D RNA was derived by a single in-frame deletion of either 339 or 411 nt within the 3a gene of Fny-CMV RNA 3 or M-CMV RNA 3, respectively. The generation of D RNAs was rare and occurred with two CMV isolates, the virions of which were known to differ in physico-chemical properties. The Fny-CMV D RNA 3, designated D RNA 3-1, was maintained by passage together with Fny-CMV in tobacco, but was lost by passage in squash. D RNA 3-1 accumulated in the inoculated squash cotyledons but not in upper, systemically infected leaves. Virions purified from infected squash cotyledons or leaf mesophyll protoplasts did not contain D RNA 3-1. Therefore, the failure of D RNA 3-1 to accumulate in squash leaves systemically infected by CMV was due to a lack of encapsidation of the D RNA 3-1 and movement out of the inoculated leaves.
Journal of General Virology, 2005
The genome of Cucumber mosaic virus consists of three single-stranded RNA molecules, RNAs 1, 2 and 3. RNAs 1 and 2 encode the 1a and 2a proteins, respectively, which are necessary for replication of the viral genome and have been implicated in movement of the viral RNAs in plants. The 3a movement protein (MP), encoded by RNA 3, is essential for transferring the RNA genomes from infected cells to adjacent cells across the plasmodesmata. Far-Western analysis demonstrated that bacterially expressed 2a polymerase protein directly interacted with the MP. Interaction was confirmed in a yeast two-hybrid assay, and co-immunoprecipitation analysis showed that the MP interacted only with the 2a polymerase protein. A yeast three-hybrid assay showed that the 1a-2a protein interaction relevant for replicase complex formation was not affected by the MP. Although the MP has no affinity for the 1a protein, it interacted indirectly with the 1a protein via the 2a polymerase protein. These results suggest that the replicase complex may be involved in movement through its interaction with the MP.
Journal of General Virology, 2004
For the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protein (CP) are required. These are not reversibly exchangeable between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). The MP of CMV is able to function with the TAV CP (chimera RT), but TAV MP is unable to promote the cell-to-cell movement in the presence of CMV CP (chimera TR). To gain further insight into the non-infectious nature of the TR recombinant, RNA 3 chimeras were constructed with recombinant MPs and CPs. The chimeric MP and one of the CP recombinants were infectious. The other recombinant CP enabled virus movement only after the introduction of two point mutations (GluRLys and LysRArg at aa 62 and 65, respectively). The mutations served to correct the CP surface electrostatic potential that was altered by the recombination. The infectivity of the TR virus on different test plants was restored by replacing the sequence encoding the C-terminal 29 aa of the MP with the corresponding sequence of the CMV MP gene or by exchanging the sequence encoding the C-terminal 15 aa of the CP with the same region of TAV. The analysis of the recombinant clones suggests a requirement for compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP for cell-to-cell movement of cucumoviruses.
Molecular Plant-Microbe Interactions®, 1999
The M strain of cucumber mosaic virus (CMV) does not infect squash plants systemically and moves very slowly in inoculated cotyledons. Systemic infection and an increase in the rate of local movement were observed when amino acids 129 or 214 of the M-CMV capsid protein (CP) were altered to those present in the Fny strain of CMV. While the opposite alterations to the CP of Fny-CMV inhibited systemic infection of squash, they did not show the same effects on the rates of both cell-to-cell and long-distance movement. However, the ability of CMV to infect squash systemically was affected by the rate of cell-to-cell movement.
Journal of Virology, 2001
Plant viruses have movement protein (MP) gene(s) essential for cell-to-cell movement in hosts. Cucumber mosaic virus (CMV) requires its own coat protein (CP) in addition to the MP for intercellular movement. Our present results using variants of both CMV and a chimeric Brome mosaic virus with the CMV MP gene revealed that CMV MP truncated in its C-terminal 33 amino acids has the ability to mediate viral movement independently of CP. Coexpression of the intact and truncated CMV MPs extremely reduced movement of the chimeric viruses, suggesting that these heterogeneous CMV MPs function antagonistically. Sequential deletion analyses of the CMV MP revealed that the dispensability of CP occurred when the C-terminal deletion ranged between 31 and 36 amino acids and that shorter deletion impaired the ability of the MP to promote viral movement. This is the first report that a region of MP determines the requirement of CP in cell-to-cell movement of a plant virus.