SARS-CoV-2 enzyme Papain-like

Papain-like proteinase

Responsible for the cleavages located at the N-terminus of the replicase polyprotein. In addition, PL-PRO possesses a deubiquitinating/deISGylating activity and processes both 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains from cellular substrates. Participates together with nsp4 in the assembly of virally-induced cytoplasmic double-membrane vesicles necessary for viral replication. Antagonizes innate immune induction of type I interferon by blocking the phosphorylation, dimerization and subsequent nuclear translocation of host IRF3. Prevents also host NF-kappa-B signaling.^[1][2]

Function & Structure of the Non-structural Protein 3

The SARS-CoV-2 non-structural protein nsp3 contains 1945 amino acids and is therefore the largest of the viruses proteins^[3]. Like its SARS-CoV counterpart, the nsp3 consists of several domains. The nomenclature as well as the counting of the domains is not always consistent. These domains are described in more detail below in order of their position within the protein, from the N- to the C-terminus:

Ubiquitin-like domain1 (Ubl1) and the Glu-rich acidic domain (AC domain)/hypervariable region (HVR)

The ubiquitin-like domain exists in all coronaviruses. It is located at the N-terminus of nsp3 and together with the Glu-rich acidic region and it is often named nsp3a. In general the Ubl1 in coronaviruses is associated to single stranded RNA (ssRNA) binding and interacting with the nucleocapsid protein^[4].

In addition the Ubl1 is suggested to reside in the cytosol directed prongs of a molecular pore on double membrane vesicles (also see double-pass transmembrane domains below) ^[5]. The Glu-rich acidic region is present in all coronaviruses, but with a non-conserved sequence and currently unknown function^[4].

Macro domain (MD)/X domain/Mac1/ADP-ribose-100-phosphatase (ADRP)

The Macrodomain, also called nsp3b, is a conserved macrodomain occurring in all coronaviruses, following the HVR or papain-like proteinase (PLpro) domain^[4]. It is ADP-binding and suggested to enzymatically remove ADP-ribose. This action could protect the virus from the anti-viral ADP-ribosylation activated by the innate immune system^[6]. The domain exhibits a three-layer α/β/α sandwich fold^[7].

SARS Unique domains (SUD N, SUD M)/ Mac2, Mac3

The SARS unique domain (SUD), or nsp3c, is mainly but not exclusively found in SARS-CoV and SARS-CoV-2. After being found outside of the SARS coronaviruses, the domains were renamed into Mac2 (SUD N) and Mac3 (SUD M) ^[4]. Mac2 and Mac3, similar to the macro domain, fold into a three-layer α/β/α sandwich. It is believed the Mac-2/3 polypeptide binds to oligo(G)-nucleotides that are able to form G-quadruplexes. Because of this it is suggested they might be involved in the formation of the replication/transcription complex and also in interactions with other viral components^[7].

Domain preceding Ubl2 and PL2pro (DPUP)/ SUD C

The domain preceding Ubl2 and PL2pro (DPUP), is located between the Mac3 domain and the Ubiquitin-like domain 2. In SARS-CoV a deletion of this domain led to a strong reduction in RNA synthesis. Its exact function or its influence on the other domains of nsp3 in SARS-CoV-2 are not yet known^[4].

Ubiquitin-like domain 2 (Ubl2)

The ubiquitin-like domain 2 (Ubl2) is sometimes seen as a subdomain of the papain-like protease (PLpro) ^[8]. Within different coronaviruses the Ubl2 is structurally more conserved than the Ubl1 domain in nsp3. In some other ubiquitin-specific proteases with a similar fold to the coronaviral PLpro, the Ubl domains can interact with other partners and regulate the catalytic activity of the protein^[4].

Papain-like protease (PLpro)

Like SARS-CoV and MERS-CoV, the SARS-CoV-2 only encodes one papain-like protease (PLpro) domain^[9], also called nsp3d^[10]. It plays an important role in the viral replication, being responsible for the cleavage of the polyproteins pp1a and pp1ab^[9]. The PLpro can recognize the tetrapeptide LXGG motif to release the nsp1, nsp2 and nsp3, which are critical for the replication of the virus^[11].

Another important characteristic of the SARS-CoV-2 PLpro is the interference with the innate immune system and inflammatory signalling pathways by its deubiquitinase and DelSGylase activities^[12]. These mechanisms rely partly on ubiquitin (Ub) and ubiquitin-like (Ubl) modifications (like ISG15), of which both exhibit the LXGG motif at their C-terminus. The PLpro is able to hydrolyse these proteins^[13].

The crystal structure of the SARS-CoV-2 PLpro comprises of four subdomains. These are the N-terminal ubiquitin-like (Ubl2, β1-3), the Thumb (α2-7), the Finger (β4-7) and the Palm subdomain (β8-13). Furthermore a zinc finger of the zinc ribbon fold group consisting of the conserved C189, C192, C224 and C226 cysteines exists in the Finger subdomain^[8]. The conserved catalytic triad is given by the residues C111, H272 and D286^[8] and is located between the Thumb and Palm subdomain.

Furthermore, the PLpro domain exhibits three binding sites: S1, S1’ and S2. The S1 Ub/Ubl-binding site is able to distinctly interact with ubiquitin and ISG15. Ubiquitin sits on the Palm subdomain and held by the Fingers subdomain in an open hand architecture with the C-terminus reaching into the catalytic center. The ISG15 also sits on the Palm subdomain but interacts with the Thumb subdomain. Studies have shown a strong preference of SARS-CoV-2 for ISG15 by the S1 Ub/Ubl-binding site as compared to ubiquitin^[12].

The conserved α2 with Phe69 provides the S2 Ub-binding site, which exhibits a specificity for Lys48-linked polyubiquitin. A contribution of the chain length on the activity is indicated^[12]. Between the PLpros of SARS-CoV and SARS-CoV-2 occur 54 differences in the amino acid sequence, of which six sites are located at the ubiquitin interacting motif (UIM). This motif can accommodate the ISG15 and the Ub. These mutations (especially K232(Q)) suggest a different enzymatic activity compared to SARS-CoV-2^[9]. When comparing the enzymatic activities and efficiency of SARS-CoV and SARS-CoV-2, substantial differences become visible. The SARS-CoV-2 PLpro shows a high substrate affinity but a low turnover as a deubiquitinase, contrary to SARS-CoV. Even though the SARS-CoV-2 PLpro has a strong preference for ISG15, the SARS-CoV PLpro still happens to be a more robust deISGylase with a 3 times higher efficiency^[9]. Compared to SARS-CoV, SARS-CoV-2 additionally lost its interferon-antagonizing function, with the deubiquitinating activity being suggested as requirement for it^[14]. Other functions of the PLpro might be the stabilization of the replicase complex and keeping the viruses replicase free of Lys48-polyubiquitin^[12].

Nucleic acid-binding domain (NAB) and the Group 2 specific marker (G2M)/ Betacoronavirus specific marker domain (βSM)

Together with the group 2 specific marker (G2M), the nucleic acid-binding domain (NAB) forms the nsp3e, which is unique to the betacoronaviruses. In SARS-CoV the NAB was shown to perform G-rich ssRNA binding and DNA-unwinding. G2M is predicted to be intrinsically disordered^[4][10].

Two double-pass transmembrane domains (TM1, TM2)

The two double-pass transmembrane domains are located around the 3Ecto domain^[4].

Coronaviruses can transform membranes of the endoplasmic reticulum into viral replication organelles, including double membrane vesicles (DMVs). The DMVs might offer a good microenvironment for the viral RNA synthesis while shielding the virus’s RNA from innate immune sensors. As the mRNAs have to be released from the DMVs into the cytosol, this can happen through molecular pores embedded in the DMV membranes, which have already been observed in SARS-CoV-2. In the murine hepatitis coronavirus (MHV), used as a model for betacoronaviruses, these pores exhibited an overall sixfold symmetry. Furthermore, the MHV nsp3 has been identified as a major constituent of the pore complex with the Ubl1 domain residing in the prongs directed to the cytosol. It is suggested that six copies of nsp3 form the pore together with other nsps. Nsp4 and nsp6 are the most promising candidates, as they are, like nsp3, transmembrane proteins. On both sides of the membrane the pore appears to dynamically interact with other not further specified macromolecules^[5].

Ectodomain (3ecto)/Putative metal binding region/Zinc finger domain (ZN)

As located between the two transmembrane domains, the ectodomain is the only domain of nsp3 located on the lumenal side of the endoplasmic reticulum. This domain is unlikely to be a zinc-finger, as the metal binding cluster is not conserved within all coronaviruses and has therefore been renamed into 3ecto^[4].

Y1 domain/ Nidovirus-conserved domain of unknown function (Y1)

Within the viruses of Nidovirales, the Y1-domain is conserved, but its function is still unclear^[4].

Coronavirus specific carboxyl-terminal domain (CoV-Y)

The CoV-Y domain is conserved within coronaviruses, but its specific function is unclear^[4].

The domains of SARS-CoV-2 nsp3.

Disease

The global COVID-19 pandemic, which started in 2019, is caused by the SARS-CoV-2.

Variations

The amino acid sequence of SARS-CoV and SARS-CoV-2 of nsp3 have a sequence identity of 76.0% and a sequence similarity of 91.8%^[3].

Relevance

The multi-domain protein nsp3 is a good targets for the antiviral drug development, as it performs many different functions, including essential roles in the replication of the virus as well as the interference with the innate immune system^[11].

See also

Coronavirus_Disease 2019 (COVID-19)