Enteroviruses encode
proteinases that are essential for processing of the translated viral
polyprotein. In addition, viral
proteinases also target host
proteins to manipulate cellular processes and evade innate
antiviral responses to promote replication and
infection. Although some host
protein substrates of enterovirus
proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal
amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host
protein targets of poliovirus and coxsackievirus B3 (CVB3)
3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cproin vitro including three common
protein targets,
phosphoribosylformylglycinamidine synthetase (PFAS),
hnRNP K, and
hnRNP M, of both
proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable
mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target
proteins modulated
infection both negatively and positively, suggesting that cleavage by 3Cpro promotes
infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering
protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral
proteinases in a nonbiased manner.IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode
proteases that cleave the viral
polyprotein into the individual mature
viral proteins. In addition,
viral proteases target host
proteins in order to modulate cellular pathways and block
antiviral responses in order to facilitate
virus infection. Although several host
protein targets have been identified, the entire list of
proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C
protease, thus providing further insights into the network of cellular pathways that are modulated to promote
virus infection.