Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped respiratory β coronavirus that causes coronavirus disease (COVID-19), leading to a deadly pandemic that has claimed millions of lives worldwide. Like other coronaviruses, the SARS-CoV-2 genome also codes for non-structural proteins (NSPs). These NSPs are found within open reading frame 1a (ORF1a) and open reading frame 1ab (ORF1ab) of the SARS-CoV-2 genome and encode NSP1 to NSP11 and NSP12 to NSP16, respectively.
1. NSP1
NSP1 inhibits the translation of host proteins by binding to the mRNA binding pocket of the 40S ribosome via its C-terminal domain
[1][2]. This prevents the synthesis of important proteins involved in the innate immune response, including IFN-b, IFN-g1, IL-8, and retinoic acid-inducible gene 1, which trigger apoptosis
[3][4]. The N-terminal region of NSP1 upregulates the translation of viral mRNAs by binding to the 5′ UTR stem-loop 1 region
[3][5]. NSP1 recruits exonucleases that cleave host mRNA and uncapped mRNA transcripts at their internal ribosome entry sites
[6]. NSP1 has also been shown to block host mRNA transcripts from exiting the nucleus by binding to the NXF1-NXT1 heterodimer docking complex involved in the nuclear export of host mRNAs through the nuclear pore complex
[7]. Altogether, this virulence factor incapacitates the ability of the host cell to mount an innate immune response while enhancing viral replication
[3].
2. NSP2
Attempts to determine the function of NSP2 in the host were performed by in situ biotinylation
[8]. However, although results showed that NSP2 interacted with the host proteins prohibitin 1 (PHB1) and prohibitin 2 (PHB2), a validation of the results using immunoblotting of the immunoprecipitated proteins with an anti-NSP2 antibody (not available) could not be proven. PHB 1 and 2 are involved in different cell activities, such as the morphology of mitochondria
[9] and transcription factor regulation
[10]. Interestingly, PHB1 has been reported as a receptor facilitating the entry of Chikungunya and Dengue 2 viruses
[11][12].
3. NSP3
This papain-like protease (PLpro) protein cleaves the N-terminal region of pp1a to release NSP1 to NSP3 from the polyprotein
[13]. It is also involved in the cleavage of proteinaceous post-translational modifications on host proteins involved in the innate antiviral responses, further dampening the immune response Field
[13]. PLpro can also regulate the innate immune response by cleaving ubiquitin-like interferon-stimulated gene 15 (
ISG15) from interferon response factor 3 (IRF3) to inhibit the IFN I pathway
[13][14][15].
4. NSP4
NSP4 is a transmembrane protein that forms part of the viral replication complex within the virally infected cell
[16]. It is involved in modifying the ER membranes to form DMV in conjunction with NSP6 to compartmentalize viral replication and viral assembly, acting as an additional barrier against intracellular antiviral responses
[16].
5. NSP5
NSP5 is a 3-cysteine-like protease (3Clpro), the main viral protease of SARS-CoV-2
[17]. It cleaves at 11 specific sites after glutamine residues along pp1a and pp1ab to release NSP4 to NSP16 in their intermediate or mature forms
[17][18].
6. NSP6
NSP6 is a transmembrane protein that interacts with NSP3 and NSP4 to induce the formation of endoplasmic reticulum (ER)-derived autophagosomes and DMVs
[19]. These autophagosomes and DMVs serve as a site for replication transcription complexes (RTCs) to form, which are necessary for viral replication
[16][19]. It is also involved in modulating ER stress by binding to the host sigma receptor that is involved in limiting the production of autophagosomes and autolysosomes to disrupt viral replication
[20]. NSP6 has also been implicated in activating host autophagy pathways through the omegasome pathway to promote the assembly of viral replicase proteins and the degradation of immunomodulatory proteins
[19]. NSP6 also directly inhibits the cleavage-mediated activation of vacuolar proton pump components such as ATP6AP1 to impair lysosomal acidification and trigger inflammasome-mediated pyroptosis
[21].
7. NSP7
NSP7 is an accessory factor protein that forms a heterotetramer with NSP8 and NSP12 to assemble the RTC needed for RNA synthesis
[22].
8. NSP8
NSP8 is another accessory factor protein that forms a homodimer with itself and forms a part of the larger RTC complex alongside NSP7 and NSP12, which is necessary for RNA synthesis
[22]. It has also been shown to play a role in suppressing IFN-a signaling pathways to further progress disease pathogenesis
[23].
9. NSP9
NSP9 is a single-stranded RNA-binding dimeric replicase protein that binds to and activates NSP8 as a cofactor
[24]. NSP9 also plays a significant role in RNA synthesis by acting as a primer for NSP12 after being modified by the NiRAN domain of NSP12 by transferring a nucleotide monophosphate to the N-terminus of NSP9
[24][25]. In the host cell, NSP9 localizes around the ER membrane, which associates with the nucleoporin 62 (NUP62), a structural protein that forms part of the nuclear pore complex, to prevent the import of p65 into the nucleus
[26]. This mislocalization of p65 leads to decreased NF-kb-regulated gene expression, resulting in a dampened immune response
[26].
10. NSP10
NSP10 is a growth-factor-like protein with two zinc-binding motifs that allow it to act as a cofactor for NSP16 to aid in capping viral mRNA transcripts
[27]. This capping of viral mRNA transcripts increases the rate of replication by the RTC and allows the transcript to evade the host RNases that cleave uncapped mRNA transcripts
[28]. NSP10 also acts as a costimulatory factor to NSP14 to enhance its exonuclease activity to remove mismatched bases, and it also allosterically activates NSP14 to aid in mRNA capping
[28][29].
11. NSP11
NSP11 is a short protein that shares identical homology to the first segment of NSP12
[30]. It is an intrinsically disordered protein that sits near the junction of pp1a and pp1b. It may be involved in host cytosolic membrane affinity interactions
[30]. It is also suggested that NSP11 may play a role in ribosomal frameshifting to adjust the reading frame by −1 as the reading frame for the NSPs in pp1a and pp1ab are different
[30]. NSP11 is also shown to regulate endoribonuclease activity and is necessary for viral replication
[30].
12. NSP12
NSP12 is an RNA-dependent RNA polymerase (RdRP) protein that acts as the core of the RTC, which binds one NSP7 and two NSP8 molecules to stabilize the RNA binding region
[31], allowing viral replication to proceed
[32]. The primary RNA polymerase produces nascent ssRNA from the viral RNA template
[33]. NSP12 also possesses an N-terminal nidovirus RdRP-associated nucleotidyltransferase (NiRAN) domain that transfers a GMP moiety to the 5′ pp-RNA to form a 5′ Gppp-RNA cap that NSP14 can methylate in the second step of mRNA capping
[28].
13. NSP13
NSP13 is an ATP-dependent RdRP containing an ATP-binding helicase core domain and a zinc-binding domain involved in unwinding the RNA during replication and the transcription of complex RNAs with secondary and tertiary structures
[34][35]. It also acts as an RTPase that removes the 5′ gamma phosphate from the mRNA to produce a 5′ pp-RNA in the first step of mRNA capping
[36].
14. NSP14
NSP14 is a Zn-dependent exoribonuclease that proofreads and removes mismatched nucleotides in a 3′-5′ direction incorrectly added by the RNA polymerase during genome replication
[28][29][37]. This exoribonuclease domain is stabilized by the zinc-finger domain of NSP10, which increases its ability to excise nucleotides
[38]. NSP14 also possesses an N7-guanine methyltransferase domain that adds a methyl group to the N7 position of the 5′ Gppp-RNA intermediate in the presence of an S-adenosylmethionine methyl donor during the second step of mRNA capping
[39].
15. NSP15
NSP15 is an Mn
2+-dependent nidoviral RNA uridylate-specific endoribonuclease (NendoU) with 3 domains: an N-terminal oligomerization domain, a central domain, and a C-terminal catalytic domain
[40][41]. The catalytic domain cleaves the nucleotides of single-stranded and double-stranded RNA molecules at the 3′ end of uridylates to produce 2′-3′ phosphodiester and 5′-hydroxyl termini
[40][41]. NendoUs are highly conserved in coronaviruses, and they avoid immune detection by cleaving the poly-U sequences on their negative-sense viral RNA templates to avoid detection by MDA5 pattern recognition receptors
[40][42]. They also act against the innate immune system by suppressing IFN-IFN-α/β-associated pathways that lead to the formation of certain cytoplasmic stress granules
[43].
16. NSP16
NSP16 is a 2′-O-ribose methyltransferase that requires an S-adenosylmethionine (SAM) methyl donor that promotes the association of NSP16 to its cofactor, NSP10
[13][39][44]. This increases the affinity of the NSP16-NSP10 complex to mRNA transcripts and allows the complex to add a methyl group to the 2′-O-ribose of the 5′ cap in the final step of mRNA capping
[45]. This allows the viral mRNA to evade being detected by host MDA5 and RIG-I receptors that recognize and degrade unprocessed mRNA transcripts.