Proteomics Study On CMV Replication-associated Host Factors

Cucumber mosaic virus(CMV)is one of the most economically endangered plant viruses.CMV is the typical member of the genus Cucumovirus family Bromoviridae.CMV genome is composed of three single-strand,positive-sense RNAs.Replicase components 1a encoded by RNA1 and RdRp encoded by RNA2,recruit host factors to form viral replication complexes on tonoplast for replication.Recently,host factors involved in the regulation of CMV replication is poorly understood.Therefore,identification and analysis of sort kind of host factors is certainly significant for uncovering the molecular mechanism of CMV replication.Based on the characteristics of CMV replication on the vacuolar membrane,healthy and CMV-infected tobacco protoplasts were successfully prepared from tobacco plants,then vacuoles were isolated from the protoplasts.Vacuolar membrane was enriched by ultracentrifugation and the vacuolar membrane proteins were identified by LC / MS-MS.The results showed that 2 and 35 host proteins were detected to be specifically present in the Mock samples and CMV-infected samples,respectively.The increased species of protein is likely associated with viral replication on the vacuolar membrane.By comparing the protein species and analyzing the difference in pipetide number,31 host protein factors probably associated with CMV replication were selected.Based on this finding,the differential protein factors were classified using GO analysis.The results showed that there were 15 proteins associated with biological process,14 proteins were related to cellular components,and 15 proteins were related to molecular function.Analysis of subcellular localization was performed by the predicted bioinformatic tools.The results showed that 8 proteins located in the chloroplast and 1 protein located in the vacuole,indicating that the remaining 22 proteins were recruited to tonoplast due to CMV infection.To analyze the correlation between the identified proteins and CMV replication,we selected glycine-rich RNA-binding protein(GRP),calmodulin(CAM)and temperature-induced lipocalin(TIP)for further study.The three genes were silenced individually using Tobacco rattle virus(TRV)vector in the Nicotiana benthamiana plants.Subsequently,the effects of gene silencing on accumulation of CMV expressing green fluorescent protein(GFP)were tested via fluorescence observation and Northern blotting.Observation of fluorescence showed that downregulation of all these genes had a varied reduction of GFP fluorescence.RNA blotting showed that the knockdown of GRP or CAM decreased the accumulation of viral positive and minus RNAs and knockdown of TIP increased levels of RNA1,RNA2 and RNA5.These results suggest that GRP,CAM and TIP are involved in virus replication.In summary,31 protein species that may be involved in CMV replication were screeened by comparing tonoplast proteasomes.Furthermore,the roles of three protein species were determined in virus replication.This demonstrates that this method is reliable to screen and identify host factors associated with CMV replication,and laid a good foundation for further study of CMV replication.