The deubiquitinating activity of Middle East respiratory syndrome coronavirus papain-like protease delays the innate immune response and enhances virulence in a mouse model

Abstract

16Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause zoonotic infections 17 and serious disease, primarily in the Arabian Peninsula, due to repeated spill-over from dromedary 18 camels and subsequent nosocomial transmission. Approved MERS vaccines for use in animals or 19 humans are not currently available. MERS-CoV replication requires the virus-encoded papain-like 20 protease (PL pro ) to cleave multiple sites in the viral replicase polyproteins, thereby releasing 21 functional non-structural proteins. Additionally, PL pro is a deubiquitinating enzyme (DUB) that can 22 remove ubiquitin(-like) moieties from substrates, presumably to counteract host antiviral responses. 23 In previous work, we determined the crystal structure of MERS-CoV PL pro in complex with ubiquitin, 24 facilitating the design of PL pro mutations that impair DUB activity without affecting viral polyprotein 25 cleavage. Here, we introduced these DUB-inactivating mutations into the viral genome and 26 examined their impact on MERS-CoV infection both in cell culture and in a lethal mouse model. 27 Although overall replication of DUB-negative and wild-type (wt) recombinant MERS-CoV was 28 comparable in multiple cell lines, infection with DUB-negative virus markedly increased mRNA levels 29 for interferon (IFN)-β and IFN-stimulated genes. Moreover, compared to a wt virus infection, the 30 survival rate was significantly increased when DUB-negative MERS-CoV was used to infect transgenic 31 mice expressing a human MERS-CoV receptor. Interestingly, DUB-negative and wt MERS-CoV 32 replicated to the same titers in lungs of infected mice, but the DUB-negative virus was cleared faster, 33 likely due to the observed accelerated and better-regulated innate immune responses, in contrast to 34 delayed and subsequently excessive responses in wt virus-infected mice. This study provides the first 35 direct evidence that the DUB activity of a coronaviral protease contributes to innate immune evasion 36 and can profoundly enhance virulence in an animal model. Thus, reduction or removal of the innate 37 immune-suppressive DUB activity of PL pro s is a promising strategy for coronavirus attenuation in the 38 context of rational vaccine development.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response

“…DUB is another role of PLpro to help coronavirus evade attack from a host's immune response by antagonizing the IFN response. From these findings, we hypothesized that the decline in function of the two proteins may lead to the direct introduction and sticking of immune troops to continuously infected cells and prevented the replication of the virus and the life cycle [37,38]. In addition to using nonstructural proteins, SARS-CoV could utilize its protein accessories to avoid immune responses.…”

6′,6′-Difluoro-aristeromycin is a potent inhibitor of MERS-coronavirus replication

“…At the DFA dosage used, no cytotoxicity was detected in either cell line (data not shown). Replication kinetics of MERS-CoV is similar in Vero and MRC-5 cells (51,52). Thus, the different levels of progeny titer reduction observed between the two cell lines may be explained by variation in uptake or metabolic conversion of the compound (53).…”

Viral Innate Immune Evasion and the Pathogenesis of Emerging RNA Virus Infections

“…Moreover, the levels of IFNβ, IFNλ and several ISGs and pro-inflammatory cytokines were found to be upregulated at an earlier time point in the mice infected with DUB-negative MERS-CoV than in the wt virus-infected mice, while the late induction of the innate immune response in wt virus-infected mice reached an out-of-control high level. This suggests that the DUB-negative MERS-CoV is less effective in evading innate immunity and that the resulting early and balanced immune response is important for controlling the infection, while the delay in innate immune induction caused by the DUB function during wt virus infection dysregulates the response, causing more severe, immunopathological symptoms (unpublished data)[85]. In summary, this indicates that the DUB activity of nsp3 is a major contributor to the pathogenesis of coronavirus infections.…”