In coronaviruses, the viral genome contains two large ORFs at the 5' end, ORF1a and ORF1b, which together occupy approximately two-thirds of the viral RNA. Translation of these ORFs results in the production of two large polyproteins, pp1a and pp1ab. The pp1a polyprotein is translated directly from ORF1a. However, the pp1ab polyprotein, which is significantly larger, is produced through a programmed ribosomal frameshifting event that occurs near the 3' end of ORF1a. This frameshifting allows the ribosome to bypass a stop codon and continue translation into the downstream ORF1b.
The resulting ORF1ab polyprotein (pp1ab) is then proteolytically cleaved by virally encoded proteases, primarily the main protease (Mpro, also known as 3C-like protease or 3CLpro) and the papain-like proteases (PLpro), into a set of 16 individual non-structural proteins (nsps). These nsps are essential for the viral life cycle and collectively form the viral replicase-transcriptase complex (RTC). The RTC is responsible for critical processes such as viral RNA replication and transcription of subgenomic RNAs, which are necessary for the synthesis of structural and accessory proteins.
Key non-structural proteins derived from the ORF1ab polyprotein include:
- nsp5 (Mpro/3CLpro): The main protease responsible for cleaving pp1a and pp1ab.
- nsp3 & nsp4: Components involved in forming the replication organelles (double-membrane vesicles) within infected cells.
- nsp12 (RdRp): The RNA-dependent RNA polymerase, crucial for viral RNA synthesis.
- nsp13: An RNA helicase with ATPase activity, involved in unwinding RNA structures.
- nsp14: An exoribonuclease involved in proofreading during RNA replication, and a guanine-N7 methyltransferase involved in capping viral RNA.
- nsp15: An endoribonuclease.
- nsp16: A 2'-O-methyltransferase involved in modifying the 5' cap of viral RNA.
Due to its indispensable role in viral replication, the ORF1ab polyprotein and its derived non-structural proteins are major targets for antiviral drug development.