Papain-like Protease

Papain-Like Proteinase of Coronavirus

Coronaviruses have large RNA genomes (27 to 32 kb), and viral replication is mediated by the viral RNA-dependent, RNA polymerase, termed the replicase^[1]. The coronavirus replicase is initially translated from the 5′-most 21 kb of the 29.7-kb SARS-CoV genomic RNA to produce two replicase polyproteins, termed pp1a and pp1ab. These polyproteins are processed by cysteine proteases to generate 16 replicase products, termed nonstructural proteins (nsp) 1 to 16.

Coronaviruses encode two types of cysteine proteases that have narrow substrate specificities: a chymotrypsin-like main protease (Mpro, contained in nsp5) and one or two papain-like proteases (PL1pro and PL2pro, residing in nsp3)^[2]. Both PLpro domains reside in the N-terminal half of nsp3 and cleave the nsp1|nsp2, nsp2|nsp3, and nsp3|nsp4 sites, while all sites downstream of nsp4 are processed by the nsp5 main protease.

The nsp3 subunits of alphacoronaviruses and subgroup 2a betacoronaviruses contain two active PLpro domains. For the alphacoronavirus HCoV-229E, it was shown previously that its papain-like proteases have overlapping specificities toward multiple sites in the replicase polyproteins and that PL1pro is dispensable for virus replication^[3]. In the case of MHV, PL1pro is responsible for the processing of the nsp1|nsp2 and nsp2|nsp3 cleavage sites, while PL2pro is dedicated to the nsp3|nsp4 cleavage site^[4]. Severe acute respiratory syndrome CoV (SARS-CoV) nsp3 contains only the PL2pro domain (often called PLpro), which cleaves all three sites in the region of nsp1 to nsp4^[5]. Gammacoronaviruses encode an active PL2pro^[6] but also carry an inactive remnant of PL1pro^[7]. Moreover, their replicase lacks an nsp1 moiety, and therefore, PL2pro cleaves only the nsp2|nsp3 and nsp3|nsp4 sites.

The key role in Replication coronavirus (CoV) replication makes the papain-like protease (PLpro) important target for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of Coronavirus (CoV). The Papain-like Protease (PLpro) of SARS-CoV has been previously described to inhibit the type I IFN signaling pathway^[8]. In addition to PLpro’s protease activity, it has been shown to have deubiquitination and de-ISGylation activities. Studies on PLpro have shown that it also inhibits host innate immune signaling by inhibiting phosphorylation, dimerization and nuclear import of IRF3^[9].

A recent report demonstrated that PLpro interacts with stimulator of IFN genes (STING), a scaffolding protein associated with the mitochondrial membrane that interacts with IRF3, RIG-I, IKKi and TBK1^[10]. By blocking phosphorylation of IKKi and TBK1, PLpro interaction with STING prevents the sensing of SARS-CoV RNA in the cell, and subsequent induction of IFNβ.

Organization of the SARS-CoV genome^[11]. The location of the different nsps in ORF1a/1ab and the ORFs for structural and accessory proteins are marked. PLpro and 3CLpro cleavage sites are indicated by red and black vertical lines, respectively. (Inset) Arrangement of different functional subdomains of nsp3. The location of PLpro is highlighted in red. The N- and C-terminal cleavage sites that define the boundaries of nsp3 are indicated by ↓.

Reference

1.Ziebuhr, J. 2005. The coronavirus replicase. Curr. Top. Microbiol. Immunol. 287:57-94.
2.Ziebuhr, J., E. J. Snijder, and A. E. Gorbalenya. 2000. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J. Gen. Virol. 81:853-879.
3.Jayaram, H.; Fan, H.; Bowman, B.R.; Ooi, A.; Jayaram, J.; Collisson, E.W.; Lescar, J.; Prasad, B.V.X-ray structures of the N-and C-terminal domains of a coronavirus nucleocapsid protein: Implications for nucleocapsid formation. J. Virol.2006,80, 6612–6620.
3.Ziebuhr, J., B. Schelle, N. Karl, E. Minskaia, et al. 2007. Human coronavirus 229E papain-like proteases have overlapping specificities but distinct functions in viral replication. J. Virol. 81:3922-3932.
4.Graham, R. L., and M. R. Denison. 2006. Replication of murine hepatitis virus is regulated by papain-like proteinase 1 processing of nonstructural proteins 1, 2, and 3. J. Virol. 80:11610-11620.
5.Han, Y. S., G. G. Chang, C. G. Juo, et al. 2005. Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition. Biochemistry 44:10349-10359.
6.Liu, D. X., I. Brierley, and T. D. Brown. 1995. Identification of a trypsin-like serine proteinase domain encoded by ORF 1a of the coronavirus IBV. Adv. Exp. Med. Biol. 380:405-411.
7.Ziebuhr, J., V. Thiel, and A. E. Gorbalenya. 2001. The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond. J. Biol. Chem. 276:33220-33232.
8.Frieman M, Ratia K, Johnston RE, Mesecar AD, Baric RS. 2009. Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-kappaB signaling. J Virol, 83: 6689-6705.
9.Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar AD, Baker SC. 2005. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol , 79: 15189-15198.
10.Sun L, Xing Y, Chen X, et al. 2012. Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling. PLoS One, 7: e30802.
11.Ratia, K., Saikatendu, K. S., et al. 2006. Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proceedings of the National Academy of Sciences , 103 (15), 5717-5722.