ORF9b

ORF9b (formerly sometimes called ORF13) is a gene that encodes a viral accessory protein in coronaviruses of the subgenus Sarbecovirus, including SARS-CoV and SARS-CoV-2. It is an overlapping gene whose open reading frame is entirely contained within the N gene, which encodes coronavirus nucleocapsid protein.^[2][3][4] The encoded protein is 97 amino acid residues long in SARS-CoV^[2][3] and 98 in SARS-CoV-2,^[4] in both cases forming a protein dimer.

Betacoronavirus lipid binding protein
The X-ray crystallography structure of the SARS-CoV ORF9b protein dimer, showing the lipid molecule in the central cavity (yellow). From PDB: 2CME​.[1]
Identifiers
Symbol	bCoV_lipid_BD
Pfam	PF09399
InterPro	IPR018542
Available protein structures: Pfam   structures / ECOD   PDB RCSB PDB; PDBe; PDBj PDBsum structure summary

Nomenclature

There has been inconsistency in the nomenclature used for this gene in the scientific literature. In some work on SARS-CoV, it has been referred to as ORF13. It has also sometimes been referred to as ORF9a, resulting in a downstream ORF of 76 codons in SARS-CoV, also overlapping with the N gene, being designated ORF9b. The recommended nomenclature refers to the longer ORF as 9b and the downstream, shorter ORF as ORF9c.^[5]

Structure

The ORF9b protein is 97 amino acid residues long in SARS-CoV^[2][3] and 98 in SARS-CoV-2.^[4] It forms a beta sheet-rich homodimer with a hydrophobic cavity in the center that binds lipids.^[2][3][4] The lipid-binding cavity may serve as an unusual mechanism for anchoring the protein to membranes.^[1]

A fragment of the SARS-CoV-2 ORF9b protein has been structurally characterized in a protein complex with Tom70 in which ORF9b forms an alpha helix rather than the beta-sheet structure observed in isolation.^[6] This fold switching behavior is also consistent with bioinformatics predictions and may also occur for the SARS-CoV homolog.^[7]

Expression and localization

ORF9b is one of two overlapping genes fully contained within the open reading frame of the N gene encoding coronavirus nucleocapsid protein, the other being ORF9c. ORF9b is expressed by ribosome leaky scanning from its bicistronic subgenomic RNA.^[2][3][8] Unlike its neighbor ORF9c, its length is well conserved in sarbecoviruses and there is strong evidence it is a functional protein-coding gene.^[9]

In SARS-CoV, the protein is localized to the endoplasmic reticulum (ER)^[3] and to intracellular vesicles.^[2][1] It does not have a nuclear localization sequence but can enter the cell nucleus by passive diffusion; it does however have a nuclear export sequence for exit from the nucleus.^[2][3] In SARS-CoV-2, it is reportedly associated with the mitochondrial membrane.^[4]

Function

The function of the ORF9b protein is not well characterized. It is not essential for viral replication.^[2]

Viral protein interactions

The ORF9b protein has been reported to interact with a number of other viral proteins, including ORF6, non-structural protein 5, non-structural protein 14, and coronavirus envelope protein.^[2] It has been detected in mature SARS-CoV virions and thus may be a minor viral structural protein.^[2][3][8]

Host cell effects

The ORF9b protein may be involved in modulating the host's immune system response. The SARS-CoV-2 protein has been reported to suppress interferon response via its interactions with Tom70, a component of the mitochondrial translocase of the outer membrane (TOM) complex.^[6][10]