Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis

• Published in: PLoS pathogens Vol. 19; no. 12; p. e1011872
• Main Authors: Bailey-Elkin, Ben A, Knaap, Robert C M, De Silva, Anuradha, Boekhoud, Ilse M, Mous, Sandra, van Vught, Niek, Khajehpour, Mazdak, van den Born, Erwin, Kikkert, Marjolein, Mark, Brian L
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 14-12-2023; Public Library of Science (PLoS)
• Subjects: 60 APPLIED LIFE SCIENCES; Animal diseases; Animals; Biological response modifiers; Biology and Life Sciences; Care and treatment; Cellular manufacture; Cellular structure; Coronaviruses; Crystal structure; Crystals; Diagnosis; Dosage and administration; Down-regulation; Equartevirus; Health aspects; Horses; Humans; Interferon; Interferons - genetics; Medicine and Health Sciences; Mutagenesis; Papain; Papain - chemistry; Papain - genetics; Papain - metabolism; Peptide Hydrolases - genetics; Physical Sciences; Porcine reproductive and respiratory syndrome; Porcine respiratory and reproductive syndrome virus - genetics; Porcine respiratory and reproductive syndrome virus - metabolism; Protease; Proteins; Replication; Research and Analysis Methods; Respiratory diseases; Risk factors; Structure; Substrates; Swine; Ubiquitin; Vaccines; Viral diseases; Viral Nonstructural Proteins - metabolism; Viral vaccines; Virus Replication; Viruses; United States; United States > US
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1011872

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.