Alanine Scanning of Cucumber Mosaic Virus (CMV) 2B Protein Identifies Different Positions for Cell-To-Cell Movement and Gene Silencing Suppressor Activity

Abstract

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.

Phosphorylation regulates the subcellular localization of Cucumber Mosaic Virus 2b protein

“…The infection was followed by visual observation and northern blot analysis. Infections of N. benthamiana with Rs-SPS/40-42/APA and Rs-SPS/40-42/DPD were asymptomatic similarly to the previously described Rs-SPS/40-42/AAA 23 during the monitored period (six weeks). Infection with Rs-SPS/40-42/SPD and Rs-SPS/40-42/DPS induced mild symptoms: mild leaf distortion, mild systemic mosaic, and mild stunting (Fig.…”

“…We found that mutants 2b/40-42/SAS, 2b/40-42/APS and 2b/40-42/SPA retained partial suppressor activity. However, mutants 2b/40-42/DPS, 2b/40-42/SPD, 2b/40-42/DPD and 2b/40-42/APA failed to suppress GFP activity similarly to the previously described 2b/40-42/AAA 23 . The accumulation of the mutant proteins were analysed by western-blot using penta-his antibody to ascertain that the different GFP levels were caused by the different suppressor activities not by the instability of the proteins (Fig.…”

“…Since antibodies for 2b protein detection were not available, the coding region of histidine tagged 2b protein was cloned into the infectious cDNA clone of Rs-CMV RNA 2. It was previously found that attachment of a hexahistidine sequence did not alter the subcellular localization and the ability to bind sRNAs 23 , 28 . Nicotiana benthamiana plants were infected with the chimaera Rs-2bHis in vitro transcripts in the presence of the Rs-CMV RNA 1 and 3 transcripts.…”

“…Nicotiana benthamiana plants were infected with the chimaera Rs-2bHis in vitro transcripts in the presence of the Rs-CMV RNA 1 and 3 transcripts. Parallel to the infection, N. benthamiana plants were infiltrated with the binary vector expressing the previously described 2bHis protein 23 . Ten days after inoculation (symptoms appeared on the upper non-inoculated leaves) and 4 days post infiltration, the accumulation and the phosphorylation of 2b protein were analysed by Western-blot using penta-his and phosphoserine antibodies (Fig.…”

“…It is one of the first described suppressors of antiviral RNA silencing, recognizing and binding not only RNA (siRNA, miRNA) but the protein components (AGO1 and AGO4) of the defense machinery as well 19 , 20 . It has also roles in symptom induction, evasion of the defense mechanism mediated by salicylic and jasmonic acid 21 , 22 as well as in cell-to-cell movement 23 . Cell fractionation studies suggested earlier that during viral infection CMV 2b protein accumulated mainly within the cell nucleus, apparently in insoluble form 24 ; tagged 2b protein with reporter markers confirmed its predominant nuclear localization in a CMV-free environment 25 , 26 .…”