Integrative Multi-omics Landscape of Non-structural Protein 3 of Severe Acute Respiratory Syndrome Coronaviruses.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is currently a global pandemic. Extensive investigations have been performed to study the clinical and cellular effects of SARS-CoV-2 infection. Mass spectrometry-based proteomics studies have revealed the cellular changes due to the infection and identified a plethora of interactors for all SARS-CoV-2 components, except for the longest non-structural protein 3 (NSP3). Here, we expressed the full-length NSP3 proteins of SARS-CoV and SARS-CoV-2 to investigate their unique and shared functions using multi-omics methods. We conducted interactome, phosphoproteome, ubiquitylome, transcriptome, and proteome analyses of NSP3-expressing cells. We found that NSP3 plays essential roles in cellular functions such as RNA metabolism and immune response (e.g., NF-κB signal transduction). Interestingly, we showed that SARS-CoV-2 NSP3 has both endoplasmic reticulum and mitochondrial localizations. In addition, SARS-CoV-2 NSP3 is more closely related to mitochondrial ribosomal proteins, whereas SARS-CoV NSP3 is related to the cytosolic ribosomal proteins. In summary, our integrative multi-omics study of NSP3 improves the understanding of the functions of NSP3 and offers potential targets for the development of anti-SARS strategies.

Structure of the multiple functional domains from coronavirus nonstructural protein 3.

Coronaviruses (CoVs) are potential pandemic pathogens that can infect a variety of hosts and cause respiratory, enteric, hepatic and neurological diseases. Nonstructural protein 3 (nsp3), an essential component of the replication/transcription complex, is one of the most important antiviral targets. Here, we report the first crystal structure of multiple functional domains from porcine delta-coronavirus (PDCoV) nsp3, including the macro domain (Macro), ubiquitin-like domain 2 (Ubl2) and papain-like protease (PLpro) catalytic domain. In the asymmetric unit, two of the subunits form the head-to-tail homodimer with an interaction interface between Macro and PLpro. However, PDCoV Macro-Ubl2-PLpro mainly exists as a monomer in solution. Then, we conducted fluorescent resonance energy transfer-based protease assays and found that PDCoV PLpro can cleave a peptide by mimicking the cognate nsp2/nsp3 cleavage site in peptide substrates and exhibits deubiquitinating and de-interferon stimulated gene(deISGylating) activities by hydrolysing ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and ISG15-AMC substrates. Moreover, the deletion of Macro or Macro-Ubl2 decreased the enzyme activity of PLpro, indicating that Macro and Ubl2 play important roles in maintaining the stability of the PLpro domain. Two active sites of PLpro, Cys260 and His398, were determined; unexpectedly, the conserved site Asp412 was not the third active site. Furthermore, the motif "NGYDT" (amino acids 409-413) was important for stabilizing the enzyme activity of PLpro, and the N409A mutant significantly decreased the enzyme activity of PLpro. These results provide novel insights into the replication mechanism of CoV and new clues for future drug design.

Spatial and temporal roles of SARS-CoV PLpro -A snapshot.

SARS-CoV and SARS-CoV-2 encode four structural and accessory proteins (spike, envelope, membrane and nucleocapsid proteins) and two polyproteins (pp1a and pp1ab). The polyproteins are further cleaved by 3C-like cysteine protease (3CLpro ) and papain-like protease (PLpro ) into 16 nonstructural proteins (nsps). PLpro is released from nsp3 through autocleavage, and then it cleaves the sites between nsp1/2, between nsp2/3 and between nsp3/4 with recognition motif of LXGG, and the sites in the C-terminus of ubiquitin and of protein interferon-stimulated gene 15 (ISG15) with recognition motif of RLRGG. Alone or together with SARS unique domain (SUD), PLpro can stabilize an E3 ubiquitin ligase, the ring-finger, and CHY zinc-finger domain-containing 1 (RCHY1), through domain interaction, and thus, promote RCHY1 to ubiquitinate its target proteins including p53. However, a dilemma appears in terms of PLpro roles. On the one hand, the ubiquitination of p53 is good for SARS-CoV because the ubiquitinated p53 cannot inhibit SARS-CoV replication. On the other hand, the ubiquitination of NF-κB inhibitor (IκBα), TNF receptor-associated factors (TRAFs), and stimulator of interferon gene (STING), and the ISGylation of targeted proteins are bad for SARS-CoV because these ubiquitination and ISGylation initiate the innate immune response and antiviral state. This mini-review analyzes the dilemma and provides a snapshot on how the viral PLpro smartly manages its roles to avoid its simultaneously contradictory actions, which could shed lights on possible strategies to deal with SARS-CoV-2 infections.