Magnetic fractionation and proteomic dissection of cellular organelles occupied by the late replication complexes of Semliki Forest virus
Autor(es): Varjak Margus, Saul Sirle, Arike Liisa, Lulla Aleksei, Peil Lauri, Merits Andres
Resumo: Alphavirus replicase complexes are initially formed at the plasma membrane - are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles - later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, - hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M - hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), - Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- - SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-str- RNA viruses - helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-str- RNA viral infection.
Imprenta: Journal of Virology, v. 87, n. 18, p. 10295-10312, 2013
Identificador do objeto digital: 10.1128/JVI.01105-13
Descritores: Chikungunya virus - Biochemistry ; Chikungunya virus - Cell ; Chikungunya virus - Pathogenesis ; Chikungunya virus - Protein synthesis ; Chikungunya virus - Proteins ; Chikungunya virus - Proteome ; Chikungunya virus - RNA ; Chikungunya Virus - Virus
Data de publicação: 2013
