Engineering potent live attenuated coronavirus vaccines by targeted inactivation of the immune evasive viral deubiquitinase

Coronaviruses express a papain-like protease (PLpro) that is required for replicase polyprotein maturation and also serves as a deubiquitinating enzyme (DUB). In this study, using a Middle East respiratory syndrome virus (MERS-CoV) PLpro modified virus in which the DUB is selectively inactivated, we show that the PLpro DUB is an important MERS-CoV interferon antagonist and virulence factor. Although the DUB-negative rMERS-CoVMA replicates robustly in the lungs of human dipeptidyl peptidase 4 knock-in (hDPP4 KI) mice, it does not cause clinical symptoms. Interestingly, a single intranasal vaccination with DUB-negative rMERS-CoVMA induces strong and sustained neutralizing antibody responses and sterilizing immunity after a lethal wt virus challenge. The survival of naive animals also significantly increases when sera from animals vaccinated with the DUB-negative rMERS-CoVMA are passively transferred, prior to receiving a lethal virus dose. These data demonstrate that DUB-negative coronaviruses could be the basis of effective modified live attenuated vaccines.In this work, authors provide a proof-of-concept study showing that deubiquitinating enzyme inactivation in MERS-CoV leads to attenuation in mice, and protection against a lethal challenge.Horizon 2020 (H2020)952373Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Marvellous ubiquitin : avenging the MERS-coronavirus papain-like protease

Replication of positive-stranded RNA viruses requires the activity of proteases that cleave the viral replicase polyproteins. For Middle East respiratory coronavirus (MERS-CoV), the virus-encoded papain-like protease (PLpro) is one of such proteases. This protease also functions as a deubiquitinating enzyme (DUB) that removes ubiquitin from substrates, most likely to suppress the ubiquitin-dependent activation of the innate immune response. The work described in this thesis provides novel insights in the interaction between PLpro and ubiquitin. The crystal structure of the PLpro-ubiquitin complex facilitated the design of substitutions in PLpro that selectively disrupted its DUB activity. DUB-negative MERS-CoV induced enhanced immune responses compared to wild-type virus, while showing similar replication in infected cells. Relative to wild-type virus, the virulence of DUB-negative MERS-CoV was reduced in mice and earlier, better-regulated immune responses were measured in their lungs. In the search for novel antivirals, ubiquitin sequence variants were selected that bound with very high affinity to MERS-CoV PLpro. Expression of those ubiquitin variants affected the activity of PLpro and concomitantly inhibited virus replication resulting in severely less virus progeny. Collectively, the gained knowledge can be used to design novel coronavirus vaccines or further develop ubiquitin variants as antiviral agents against viruses that encode DUBs. LUMC / Geneeskund

Structure and Function of Viral Deubiquitinating Enzymes

Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Profiling DUBs and Ubl-specific proteases with activity-based probes

Molecular basis of virus replication, viral pathogenesis and antiviral strategie

In vivo assessment of equine arteritis virus vaccine improvement by disabling the deubiquitinase activity of papain-like protease 2

Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Crystal Structure of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Papain-like Protease Bound to Ubiquitin Facilitates Targeted Disruption of Deubiquitinating Activity to Demonstrate Its Role in Innate Immune Suppression

Molecular basis of virus replication, viral pathogenesis and antiviral strategie

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

Molecular basis of virus replication, viral pathogenesis and antiviral strategie