Structural and Functional Dissection of SARS-CoV-2 Proteins

Genomic Structure and Viral Entry

The SARS-CoV-2 genome features the replicase gene ORF1ab and structural protein genes S, E, M, N after its 5' UTR followed by accessory protein genes ORF3a and ORF6.

1. ORF1ab: Two-thirds of the genome region produces polyproteins pp1a and pp1ab through a -1 ribosomal frameshift (-1 PRF) process. Through proteolytic cleavage scientists transform polyproteins into 16 separate nonstructural proteins (nsp1-16) which assemble into the replication-transcription complex (RTC) that carries out viral RNA synthesis and processing.
2. Structural Proteins: The structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N) which aid in viral assembly and host recognition while helping to dodge immune detection are produced by the 3' terminal ORFs.
3. Accessory Proteins: Such as ORF3a and ORF7b, these may modulate host cell cycles or suppress interferon responses, though their precise functional roles require further investigation.

Viral entry, involving host cell membrane attachment and fusion, is mediated by the S protein. Coronaviral entry typically requires two spike (S) protein cleavage events: The first cleavage event occurs at the S1-S2 subunit junction while the second takes place at the S2' site inside the S2 subunit. Viral maturation in infected cells triggers cleavage of the polybasic motif at the S1-S2 boundary in SARS-CoV-2 but the S2' site cleavage occurs after the spike protein binds to ACE2 on target cells. The S1 subunit undergoes conformational changes upon ACE2 binding which exposes the S2' cleavage site located in the S2 subunit. Depending on the entry pathway, the S2' site is cleaved by distinct proteases. If target cells lack sufficient transmembrane protease serine 2 (TMPRSS2) or the virus-ACE2 complex does not encounter TMPRSS2, the complex is internalized via clathrin-mediated endocytosis into endolysosomes. In the presence of TMPRSS2, S2' cleavage occurs at the cell surface. In both pathways, S2' cleavage exposes the FP, and S1-S2 dissociation triggers large conformational rearrangements in the S2 subunit—particularly in HR1—driving FP insertion into the target membrane to initiate fusion. The merger of viral and cellular membranes forms a fusion pore through which viral RNA is released into the host cytoplasm.

Figure 2. Two distinct SARS-CoV-2 entry pathways
(Source: Jackson CB, et al. 2022)

Core Roles of Nonstructural Protein Complexes

The nsps encoded by ORF1ab assemble into the RTC, which exhibits extensive intermolecular interactions. The viral replication machinery is anchored to convoluted membranes via the transmembrane proteins nsp3, nsp4, and nsp6, shielding dsRNAs from immune degradation.

Key components include:

1. nsp3 (PLpro): Possesses deubiquitinase activity and suppresses host innate immune signaling pathways, such as the NF-κB cascade.
2. nsp5 (3CLpro): The primary protease that breaks down polyproteins into functional units remains the focal target for antiviral medications including Paxlovid.
3. nsp12 (RdRp): The core replicase consists of RNA-dependent RNA polymerase and nsp7 and nsp8 while the fidelity of this complex is improved through the proofreading function of nsp14 (ExoN).
4. nsp16: The protein nsp16 functions as a 2'-O-methyltransferase which modifies the 5' end of viral RNA to avoid detection by host pattern recognition receptors including MDA5.

• Nsp3

Nsp3 is a multi-domain membrane-associated protein and the largest protein encoded by the coronavirus genome. It acts as a membrane-anchored scaffold, interacting with other nsps and host proteins to form the RTC. Nsp3 comprises the N-terminal Nsp3a domain, Macrodomain-X, SUD, PLpro, RBD, marker domain (MR), transmembrane domain (TM), and Y domain.

The PLpro enzyme processes viral polyprotein precursors pp1a and pp1ab through proteolytic cleavage at three exact positions resulting in the generation of nonstructural proteins Nsp1, Nsp2, and Nsp3. The PLpro enzyme of SARS-CoV-2 starts with an N-terminal ubiquitin-like domain that stands apart from its catalytic core domain while SARS-CoV PLpro remains unchanged. The catalytic core adopts an open right-handed architecture containing thumb, palm, and finger subdomains. PLpro functions as a galactose protease, with its active site located in a cleft between the thumb and palm subdomains. In unliganded PLpro structures, the flap adopts an open conformation. Upon inhibitor binding, the flap closes over the catalytic cleft entrance via an induced-fit mechanism, forming intermolecular interactions with the ligand. The loop can assume multiple conformations depending on the size and type of the inhibitor molecule.

• Nsp5

3CLpro, which is also called Nsp 5 (3C-like protease), functions as a 33.8 kDa cysteine protease that processes the viral polyproteins pp1a (486 kDa) and pp1ab (790 kDa). The 3CLpro monomer comprises three domains: The 3CLpro protein consists of Domain I which spans residues 8–101, Domain II which spans residues 101–184, and Domain III which spans residues 201–303 along with a long connecting loop between Domains II and III which spans residues 185–200. The active site of 3CLpro emerges from the space between Domains I and II and consists of a Cys-His catalytic dyad made up of Cys145 and His41. The nsp5 protein from SARS-CoV-2 demonstrates 96% sequence homology compared to the SARS-CoV version of this protein. 3CLpro plays an essential role in the viral life cycle through its direct acceleration of nsp maturation. Research into its structure and catalytic mechanism has demonstrated that this protein represents a valuable target for the development of drugs against coronavirus.

• Nsp12

The 932 amino acid long RNA-dependent RNA polymerase SARS-CoV-2 nsp12 includes a polymerase domain structured as a "right-hand grip" and an N-terminal domain. The finger domain of SARS-CoV-2 nsp12 includes three helices and a helix-loop-helix motif typical for RdRp along with two-stranded β-sheets as structural components while the palm domain contains two helices and a β-hairpin structure that holds catalytic aspartates for nucleotide transfer functions. The binding site for the nsp7-nsp8 heterodimer exists at the interaction site between the thumb and index finger subdomains while nsp12 and nsp7 maintain most of these contacts. The coronavirus family shows high conservation levels in core functional regions including the template entry site, template-primer exit site, nucleoside triphosphate (NTP) tunnels and the polymerase active site.

The addition of nsp7 and nsp8 cofactors significantly enhances nsp12 activity. Although other viral factors are required, the nsp12-nsp7-nsp8 complex is indispensable for nucleotide polymerization. Similar to the SARS-CoV polymerase complex, the nsp7-nsp8 heterodimer interacts above the thumb subdomain of nsp12. This interaction is primarily mediated by nsp7, while nsp8 exhibits limited contact with the polymerase subunit nsp12. Another nsp8 subunit engages the top region of the finger domain and forms additional interactions with the interface domain. However, the RNA synthesis efficiency of the SARS-CoV-2 nsp12-nsp7-nsp8 complex (~35%) is lower than that of the SARS-CoV counterpart due to alterations in the nsp8 subunit. Additionally, residue substitutions in SARS-CoV-2 nsps result in relatively lower thermal stability for both nsp8 and nsp12.