Analysis of EV71 infection progression using triple-SILAC-based proteomics approach

Abstract

Enterovirus 71 (EV71), a member of Picornaviridae, causes severe neurological and systemic illness in children. To better understand the virus-host cell interactions, we performed a triple-SILAC-based quantitative proteomics study monitoring host cell proteome changes after EV71 infection. Based on the quantitative data for more than 4100 proteins, ∼17% of the proteins were found as significantly changed (p<0.01) at either 8 or 20 hours post infection. Five biological processes and seven protein classes showed significant differences. Functional screening of nine regulated proteins discovered the regulatory role of CHCH2, a mitochondrial protein known as a transcriptional activator for cytochrome c oxidase, in EV71 replication. Further studies showed that CHCH2 served as a negative regulator of innate immune responses. All MS data have been deposited in the ProteomeXchange with identifier PXD002483 (http://proteomecentral.proteomexchange.org/dataset/PXD002483).

Proteomics Analysis Identifies IRSp53 and Fascin as Critical for PRV Egress and Direct Cell–Cell Transmission

“…Proteins were separated by either a 10% or a 12% SDS‐PAGE gel. Western blotting was performed as previously described . The following primary antibodies were used for western blot analysis: anti‐IRSp53 rabbit polyclonal antibody at 1:1000 (Proteintech, 11 087‐2‐AP); anti‐RFP rat monoclonal antibody at 1:5000 (Chromotek, 5F8); anti‐GFP rabbit polyclonal antibody at 1:5000 (Abcam, ab290); anti‐CDC42 rabbit polyclonal antibody at 1:1000 (Proteintech, 10 155‐1‐AP); anti‐Rac1 rabbit polyclonal antibody at 1:1000 (Proteintech, 24 072‐1‐AP); and anti‐Actin mouse monoclonal antibody at 1:10 000 (CWbiotech, CW0096B).…”

Enteroviral proteases: structure, host interactions and pathogenicity

“…There are number of approaches that have been used to study the cellular targets of 2A pro and 3C pro . These include infection of cells or tissues, for example, , selective overexpression of viral proteases by transfection, for example, , transgenic techniques , a variety of in vitro assays, in which the proteases have been incubated with cell lysates, for example, , and in silico prediction of the cleavage sites based on amino acid sequences and composition of potential target proteins . To analyse whether the experimental approaches result in cleavage by enteroviral enzymes, Western blotting is a frequently used method.…”

“…They identified eight novel substrates for 3C pro , out of which they analysed the cleavage of stimulation factor 64 in more detail. Newer methodologies in quantitative proteomics have recently been used to study how enteroviruses affect the host‐cell proteome , and such methods may also be applied to identify new protease targets . A potential disadvantage with these type of analyses is that they are restricted to the proteins expressed by the infected cell, and will not provide a simultaneous analysis of the whole human proteome.…”

Proteomics progresses in microbial physiology and clinical antimicrobial therapy

“…With extensive modification of the SILAC method, a pulsed SILAC (pSILAC) has been developed to monitor modest changes of proteins during de novo protein synthesis by metabolic pulse labeling of cells using two different heavy isotopic forms of arginine and lysine [ 81 ]. Meanwhile, the triple SILAC method, accomplished by SILAC in a triple labeling format (Fig 3 ), allows to study proteins derived from three samples or the time dimension of the proteome [ 82 , 83 ]. These methods widely broaden the scope of SILAC-based proteomics.…”