NSP1 inhibits the translation of host proteins by binding to the mRNA binding pocket of the 40S ribosome via its C-terminal domain [36,37]. 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 [21,38]. The N-terminal region of NSP1 upregulates the translation of viral mRNAs by binding to the 5′ UTR stem-loop 1 region [21,39]. NSP1 recruits exonucleases that cleave host mRNA and uncapped mRNA transcripts at their internal ribosome entry sites [40]. 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 [41]. Altogether, this virulence factor incapacitates the ability of the host cell to mount an innate immune response while enhancing viral replication [21].

Attempts to determine the function of NSP2 in the host were performed by in situ biotinylation [24]. 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 [42] and transcription factor regulation [43]. Interestingly, PHB1 has been reported as a receptor facilitating the entry of Chikungunya and Dengue 2 viruses [44,45].

This papain-like protease (PLpro) protein cleaves the N-terminal region of pp1a to release NSP1 to NSP3 from the polyprotein [14]. 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 [14]. 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 [14,18,19].

NSP4 is a transmembrane protein that forms part of the viral replication complex within the virally infected cell [25]. 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 [25].

NSP5 is a 3-cysteine-like protease (3Clpro), the main viral protease of SARS-CoV-2 [26]. It cleaves at 11 specific sites after glutamine residues along pp1a and pp1ab to release NSP4 to NSP16 in their intermediate or mature forms [26,46].

NSP6 is a transmembrane protein that interacts with NSP3 and NSP4 to induce the formation of endoplasmic reticulum (ER)-derived autophagosomes and DMVs [47]. These autophagosomes and DMVs serve as a site for replication transcription complexes (RTCs) to form, which are necessary for viral replication [25,47]. 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 [48]. 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 [47]. 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 [49].

NSP7 is an accessory factor protein that forms a heterotetramer with NSP8 and NSP12 to assemble the RTC needed for RNA synthesis [50].

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 [50]. It has also been shown to play a role in suppressing IFN-a signaling pathways to further progress disease pathogenesis [51].

NSP9 is a single-stranded RNA-binding dimeric replicase protein that binds to and activates NSP8 as a cofactor [7]. 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 [7,9]. 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 [22]. This mislocalization of p65 leads to decreased NF-kb-regulated gene expression, resulting in a dampened immune response [22].

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 [52]. 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 [29]. 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 [29,53].

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].

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 [54], allowing viral replication to proceed [55]. The primary RNA polymerase produces nascent ssRNA from the viral RNA template [56]. 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 [29].

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 [57,58]. 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 [59].

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 [29,53,60]. This exoribonuclease domain is stabilized by the zinc-finger domain of NSP10, which increases its ability to excise nucleotides [61]. 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 [13].

NSP15 is an Mn²⁺-dependent nidoviral RNA uridylate-specific endoribonuclease (NendoU) with 3 domains: an N-terminal oligomerization domain, a central domain, and a C-terminal catalytic domain [62,63]. 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 [62,63]. 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 [62,64]. 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 [65].

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 [13,14,15]. 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 [66]. This allows the viral mRNA to evade being detected by host MDA5 and RIG-I receptors that recognize and degrade unprocessed mRNA transcripts.