Abstract
The papain-like cysteine protease 2 (PLP2) within the N-terminus of the porcine reproductive and respiratory syndrome virus (PRRSV) nsp2 replicase protein specifies a deubiquitinating enzyme (DUB), but its biochemical properties and the role in infection have remained poorly defined. By using in vitro assays, we found that the purified PLP2 could efficiently cleave K63 and K48 linked polyubiquitin chains Ub3-7 in vitro although displaying a differential activity in converting the respective ubiquitin dimers to monomer. The subsequent mutagenesis analyses revealed that the requirement for PLP2 DUB activity surprisingly resembled that for cis-cleavage activity, as several mutations (e.g., D91R, D85R, etc.) that largely ablated the DUB function also blocked the cis- but not trans-proteolytic cleavage of nsp2/3 polyprotein. Moreover, the analyses identified key mutations that could differentiate DUB from PLP2 cis- and trans-cleavage activities. Further reverse genetics analyses revealed the following findings: (i) mutations that largely blocked the DUB activity were all lethal to the virus, (ii) a point mutation T88G that selectively blocked the cis-cleavage activity of PLP2 did not affect viral viability in cell culture, and (iii) an E90Q mutation that did not affect either of the PLP2 activities led to rescue of WT-like virus but displayed significantly reduced ability to induce TNF-α production. Our findings support the possibility that the PLP2 DUB activity, but not cis-cleavage activity, is essential for PRRSV replication. The data also establish a strong link of nsp2 to pro-inflammatory cytokine induction during infection that operates in a manner independent of PLP2 DUB activity.
Highlights
Protein ubiquitination is an important post-translational modification that regulates a variety of cellular biological processes [1,2,3], i.e., signaling transduction [4], protein turnover [5], cell cycle progression [6], and the immune and inflammatory responses [4,7,8], etc
To identify critical residues that are potentially important for papain-like cysteine protease 2 (PLP2) deubiquitinating enzyme (DUB) activity, we modeled the structure of PLP2 core domain with the online program I-TASSER [45] by using equine arteritis virus (EAV) PLP2
The results showed that the mutations of the residues D84 and E90 (e.g., D84N, D84R, E90R, E90Q, etc.) did not have much effect on PLP2 DUB activity, as the corresponding mutants could efficiently cleave K63 or K48 polyubiquitin chains into monomers (Figure 4A, lanes 3, 4, 7, and 8)
Summary
Protein ubiquitination is an important post-translational modification that regulates a variety of cellular biological processes [1,2,3], i.e., signaling transduction [4], protein turnover [5], cell cycle progression [6], and the immune and inflammatory responses [4,7,8], etc. This modification, can often be reversed by deubiquitinases (DUBs) through removing various ubiquitin molecules from substrates [9]. This report concerns the deubiquitinase of porcine reproductive and respiratory syndrome virus (PRRSV), a positive-stranded RNA virus in the family Arteriviridae of the order Nidovirales [25,26] and a major pathogen of swine that causes colossal annual economic losses to the worldwide swine industry [27,28,29,30,31]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
