PMID- 31561412 OWN - NLM STAT- MEDLINE DCOM- 20200916 LR - 20200916 IS - 1999-4915 (Electronic) IS - 1999-4915 (Linking) VI - 11 IP - 10 DP - 2019 Sep 26 TI - Characterizing the PRRSV nsp2 Deubiquitinase Reveals Dispensability of Cis-Activity for Replication and a Link of nsp2 to Inflammation Induction. LID - 10.3390/v11100896 [doi] LID - 896 AB - 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-alpha 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. FAU - Zhou, Shaochuan AU - Zhou S AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. 15210922708@163.com. FAU - Ge, Xinna AU - Ge X AUID- ORCID: 0000-0002-5368-9687 AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. gexn@cau.edu.cn. FAU - Kong, Can AU - Kong C AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. kcaner@163.com. FAU - Liu, Teng AU - Liu T AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. Liuteng2018cau@163.com. FAU - Liu, Aijing AU - Liu A AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. laj_91@126.com. FAU - Gao, Peng AU - Gao P AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. penggao@cau.edu.cn. FAU - Song, Jiangwei AU - Song J AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. songjiangwei525@126.com. FAU - Zhou, Lei AU - Zhou L AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. leosj@cau.edu.cn. FAU - Guo, Xin AU - Guo X AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. guoxin@cau.edu.cn. FAU - Han, Jun AU - Han J AUID- ORCID: 0000-0002-7104-7223 AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. hanx0158@cau.edu.cn. FAU - Yang, Hanchun AU - Yang H AD - Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China. yanghanchun1@cau.edu.cn. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20190926 PL - Switzerland TA - Viruses JT - Viruses JID - 101509722 RN - 0 (Cytokines) RN - 0 (Ubiquitin) RN - 0 (Viral Nonstructural Proteins) RN - EC 3.4.19.12 (Deubiquitinating Enzymes) RN - EC 3.4.22.- (Cysteine Endopeptidases) SB - IM MH - Animals MH - Cells, Cultured MH - Cysteine Endopeptidases/chemistry/genetics/metabolism MH - Cytokines/metabolism MH - Deubiquitinating Enzymes/chemistry/genetics/*metabolism MH - Humans MH - Models, Molecular MH - Mutation MH - Porcine respiratory and reproductive syndrome virus/*enzymology/pathogenicity/physiology MH - Substrate Specificity MH - Swine MH - Ubiquitin/chemistry/metabolism MH - Viral Nonstructural Proteins/chemistry/genetics/*metabolism MH - Virus Replication PMC - PMC6832237 OTO - NOTNLM OT - PRRSV OT - cis-cleavage activity OT - deubiquitinase OT - deubiquitination OT - inflammation OT - trans-cleavage activity COIS- The authors declare that they have no conflicts of interest with the contents of this article. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. EDAT- 2019/09/29 06:00 MHDA- 2020/09/17 06:00 PMCR- 2019/10/01 CRDT- 2019/09/29 06:00 PHST- 2019/07/29 00:00 [received] PHST- 2019/09/14 00:00 [revised] PHST- 2019/09/19 00:00 [accepted] PHST- 2019/09/29 06:00 [entrez] PHST- 2019/09/29 06:00 [pubmed] PHST- 2020/09/17 06:00 [medline] PHST- 2019/10/01 00:00 [pmc-release] AID - v11100896 [pii] AID - viruses-11-00896 [pii] AID - 10.3390/v11100896 [doi] PST - epublish SO - Viruses. 2019 Sep 26;11(10):896. doi: 10.3390/v11100896.