A specific region of the Influenza B virus NS1 protein, which includes part of its effector domain, blocks the covalent linkage of mouse ISG15 to its target proteins both in vitroand in infected cells. Of the several hundred proteins induced by interferon (IFN) alpha/beta, the ubiquitin-like ISG15 protein is one of the most predominant. Influenza A virus employs a different strategy: its NS1 protein does not bind the ISG15 protein, but little or no ISG15 protein is produced during infection [].
The replicase polyprotein 1ab is a multifunctional protein: it contains the activities necessary for the transcription of negative stranded RNA, leader RNA, subgenomic mRNAs and progeny virion RNA as well as proteinases responsible for the cleavage of the polyprotein into functional products. Nsp1 is essential for viral subgenomic mRNA synthesis. Nsp2 cysteine proteinase which cleaves the Nsp2/Nsp3 site in the polyprotein. Also displays deubiquitinating and deISGylase activities. The deubiquitinating activity cleaves both ubiquitinated and ISGylated products and may therefore regulate ubiquitin and ISG15 dependent host innate immunity. The 3C-like serine proteinase chain (Nsp4) is responsible for the majority of cleavages as it cleaves the C terminus of the polyprotein. The helicase chain, which contains a zinc finger structure, displays RNA and DNA duplex-unwinding activities with 5' to 3' polarity [].This superfamily represents the C-terminal domain of chymotrypsin-like serine proteinase Nsp4. Nsp4 contains two β-barrels, known as N- and C-terminal barrels, as well as a unique C-terminal domain. The C-terminal domain consists of two short pairs of β-strands and two α-helices. It interacts with the C-terminal barrel through an interface consisting of conserved hydrophobic residues: Leu-105 and Leu-112 from the C-terminal β-barrel and Val-158, Leu-163, Phe-167, Ile-182, Leu-196, and Ile-197 from the C-terminal domain. There is also an exposed patch of conserved solvent-exposed hydrophobic residues that may form part of the interface with Nsp5 in the Nsp4-5 intermediate []. This hydrophobic patch may also mediate interactions with Nsp2, which associates with Nsp3-8 to induce cleavage of the Nsp4-5 site by Nsp4 []. The C-terminal domain can clearly adopt different orientations relative to the two β-barrels, which may facilitate substrate binding or autoproteolysis [].
This entry contains coronavirus (CoV) cysteine endopeptidases that belong to MEROPS peptidase family C16 (subfamilies C16A and C16B, clan CA). These peptidases are involved in viral polyprotein processing, releasing NSP1, NSP2 and NSP3 proteins []and they also function as deubiquitinating and deISG15ylating (interferon-induced gene 15) enzymes, disrupting host viral immune response to facilitate viral proliferation and replication. Therefore, this is an important target to develop antiviral treatments [].All coronaviruses encode between one and two accessory cysteine proteinases that recognise and process one or three sites in the amino-terminal half of the replicase polyprotein during assembly of the viral replication complex. MHV, HCoV and TGEV encode two accessory proteinases, called coronavirus papain-like proteinase 1 and 2 (PL1-PRO and PL2-PRO) []. IBV and SARS encodes only one called PL-PRO (PL2-PRO, conserved in all CoVs) [, , ]. The structures of both PL-PROs are similar and they also have restricted specificities. The PL1-PRO of TGEV cleaves the polyprotein between Nsp2-Nsp3 recognising the Lys-Met-Gly-Gly motif, and recognises Leu-Arg-Gly-Gly in ubiquitin (ub) which shows that it is able to accommodate residues as different as Lys and Leu. In contrast, PL-PRO from SARS-CoV recognises Leu-Xaa-Gly-Gly (Xaa could be any amino acid) and cleaves peptide bonds between Nsp1-Nsp2, Nsp2-Nsp3 and between Nsp3-Nsp4 [, , ]. In Ub and ISG15 proteins, it recognises Leu-Arg-Gly-Gly motifs. SARS-CoV and SARS-CoV-2 are closely related but exhibit different host substrate preferences: SARS-CoV-2 PL-PRO preferentially cleaves the ubiquitin-like ISG15, whereas SARS-CoV PL-PRO predominantly targets ubiquitin chains [, ].The peptidase family C16 domain is about 260 amino acids in length and the solved structures determined that it consists of thumb, palm, and fingers subdomains. The thumb is comprised of six α-helices and a small β-hairpin; the fingers subdomain is made of six β-strands and two α-helices and includes a zinc binding site, in which the zinc ion is coordinated by four cysteine residues. Zinc binding is essential for structural integrity and protease activity, with a conformation that varies most between different PL-PRO structures. The palm subdomain is comprised of six β-strands and includes the catalytic residues Cys-His-Asp, located at the interface between the thumb and palm subdomains [].
