1.A.57 The Human SARS Coronavirus Viroporin (SARS-VP) Family

The severe acute respiratory syndrome-associated coronavirus group 26 (SARS CoV) genome includes Orf3a which codes for a protein, Protein 3a (Protein IX; Protein U276; Acessory Protein 3a) of 274 aas that has 3 putative N-terminal TMSs (residues 1-130) and a C-terminal hydrophilic domain (residues 131-274). It is expressed and is found at the surface of SARS-CoV-infected or 3a-cDNA transfected cells (Lu et al, 2006). It forms a homotetrameric (composed of two homodimers) K+-sensitive channel, with intersubunit disulfide bridges. It is inhibited by Ba2+. It appears to be important for release of the virus from the cell but not for replication of the viral DNA (Lu et al, 2006). Protein 3a may be glycosylated (as in the virion) or nonglycosylated.

Accessory protein 3a interacts with several other SARS CoV proteins. These include protein E, protein-7A, the spike and M proteins and the human small glutamine-rich tetratricopeptide repeat (TPR)-containing protein A [(O43765; 313 aas)].  Viroporins have been reviewed with respect to their structures, functions, and their roles in the life cycle of SARS-CoV-2 (Breitinger et al. 2022).

The SARS CoV envelope small membrane protein E (E protein, sM protein (P59637)) of 76 aas with 1 TMS is the prototype of small homologues (70-90 aas) found in several coronaviruses specific for rats, cats, dogs, mice, people, cows, etc. These include transmissible gastroenteretis virus, mouse hepatitis virus, rat sialodacryoadenitis coronavirus, porcine hemagglutinating encephalomyelitis virus.

The Protein 7a precursor (P59635; protein X4; protein U122; accessory protein 7a) with 122 aas, has 1 TMS and is found in the endoplasmic reticulum/golgi apparatus connecting region as well as in the virion.

Fourteen ORFs are encoded in the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) genome (Lu et al. 2006). This family of proteins has been referred to as the DUF3092 family. Emodin inhibits current through SARS-associated coronavirus 3a protein (Schwarz et al. 2011). The ion channel activity of the 3a protein is linked to its pro-apoptotic function (Chan et al. 2009).

Protein 3a is a 274 amino acid polytopic channel protein with three TMSs encoded by SARS-CoV. Synthetic peptides corresponding to each of its three individual TMSs have been reconstituted into artificial lipid bilayers (Chien et al. 2013). Only TMSs 2 and 3 induced channel activity. Reconstitution of the peptides as TMS1 + TMS3 or TMS2 + TMS3 in a 1: 1 mixture induced membrane activity. In a 1: 1: 1 mixture, channel-like behavior was almost fully restored. Expression of full length 3a and reconstitution into artificial lipid bilayers revealed a weakly cation-selective (PK ≈ 2 PCl ) rectifying channel. In the presence of non-physiological concentrations of Ca2+-ions, channel activity developed.

The sequences of three polytopic viral channel proteins: (i) p7 of HCV and 2B of Polio virus (two TMSs) and (ii) 3a of SARS-CoV (three TMSs were aligned, and the region of overlap was mapped onto structural models of host channels and toxins, focusing on the pore-lining TMSs (Schindler and Fischer 2012). The analysis reveals that p7 and 2B TMSs align with the pore-facing TMS of MscL, and 3a-TMSs align with those of ligand-gated ion channels (Schindler and Fischer 2012). 

The reaction catalyzed by SARS-VP is:

Small molecules (in) small molecules (out)


 

References:

Barrantes, F.J. (2021). Structural biology of coronavirus ion channels. Acta Crystallogr D Struct Biol 77: 391-402.

Bianchi, M., A. Borsetti, M. Ciccozzi, and S. Pascarella. (2020). SARS-Cov-2 ORF3a: Mutability and function. Int J Biol Macromol. [Epub: Ahead of Print]

Breitinger, U., N.S. Farag, H. Sticht, and H.G. Breitinger. (2022). Viroporins: Structure, function, and their role in the life cycle of SARS-CoV-2. Int J Biochem. Cell Biol. 145: 106185. [Epub: Ahead of Print]

Chan, C.M., H. Tsoi, W.M. Chan, S. Zhai, C.O. Wong, X. Yao, W.Y. Chan, S.K. Tsui, and H.Y. Chan. (2009). The ion channel activity of the SARS-coronavirus 3a protein is linked to its pro-apoptotic function. Int J Biochem. Cell Biol. 41: 2232-2239.

Chen, I.Y., M. Moriyama, M.F. Chang, and T. Ichinohe. (2019). Severe Acute Respiratory Syndrome Coronavirus Viroporin 3a Activates the NLRP3 Inflammasome. Front Microbiol 10: 50.

Chien, T.H., Y.L. Chiang, C.P. Chen, P. Henklein, K. Hänel, I.S. Hwang, D. Willbold, and W.B. Fischer. (2013). Assembling an ion channel: ORF 3a from SARS-CoV. Biopolymers 99: 628-635.

Kern, D.M., B. Sorum, S.S. Mali, C.M. Hoel, S. Sridharan, J.P. Remis, D.B. Toso, A. Kotecha, D.M. Bautista, and S.G. Brohawn. (2021). Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs. Nat Struct Mol Biol 28: 573-582.

Lu, W., B.J. Zheng, K. Xu, W. Schwarz, L. Du, C.K. Wong, J. Chen, S. Duan, V. Deubel, and B. Sun. (2006). Severe acute respiratory syndrome-associated coronavirus 3a protein forms an ion channel and modulates virus release. Proc. Natl. Acad. Sci. USA 103: 12540-12545.

Schindler, C. and W.B. Fischer. (2012). Sequence alignment of viral channel proteins with cellular ion channels. J Comput Biol 19: 1060-1072.

Schwarz, S., K. Wang, W. Yu, B. Sun, and W. Schwarz. (2011). Emodin inhibits current through SARS-associated coronavirus 3a protein. Antiviral Res 90: 64-69.

Tee, W.V., Z.W. Tan, K. Lee, E. Guarnera, and I.N. Berezovsky. (2021). Exploring the Allosteric Territory of Protein Function. J Phys Chem B 125: 3763-3780.

Zhang, B., Y. Jin, L. Zhang, H. Wang, and X. Wang. (2022). Pentamidine Ninety Years on: the Development and Applications of Pentamidine and its Analogs. Curr. Med. Chem. [Epub: Ahead of Print]

Examples:

TC#NameOrganismal TypeExample
1.A.57.1.1

SARS-CoV Viroporin tetrameric ion channel. Protein 3a is of 274 aas and 3 TMSs. It activates the Nod-like receptor family members which are pyrin domain-containing 3 (NLRP3)proteins that regulate the secretion of proinflammatory cytokines such as interleukin 1 beta (IL-1beta) and IL-18. K+ efflux and mitochondrial reactive oxygen species are important for SARS-CoV 3a-induced NLRP3 inflammasome activation (Chen et al. 2019). Viroporin 3a exhibits allosteric properties (Tee et al. 2021).

Animal Viruses

SARS-Caronavirus
Viroporin
(Protein 3a)
(P59632)

 
1.A.57.1.2

Orf3 of 249 aas and 3 putative TMSs

Viruses

Orf3 of Zaria bat coronavirus (F1BYM0)

 
1.A.57.1.3

The NS3 protein of 230aas and 3 N-terminal TMSs as well as 3 potential C-terminal TMSs of low hydrophobicity.

Viruses

NS3 of Bat coronavirus HKU9-5-1 (E0ZN37)

 
1.A.57.1.4

Orf3 of 238aas and 3 putative TMSs

Viruses

Orf3 of Eidolon bat coronavirus (F1DAZ2)

 
1.A.57.1.5

Orf3a of 275 aas and 3 central TMSs. Orf3a forms homodimers and homotetramers and is a non-selective catioin channel that is blocked by polycation channel inhibitors (Kern et al. 2021). They carry a PDZ-binding domain, lending them the versatility to interact with more than 400 target proteins in infected host cells. Structural considerations have been discussed (Barrantes 2021). Naturally occurring mutations in ORF3a are common and have been analyzed, and 28 fully concerved residues in 70,000 sequences that probably have structural or functional roles were also identified (Bianchi et al. 2020). Viroporin 3a exhibits allosteric properties (Tee et al. 2021). It has been implicated in apoptosis and inhibition of autophagy. The structure of the dimer has been determined at 2.1 Å resolution by cryoEM (Kern et al. 2021). Pentamidine is a channel blocker of Orf3a (Zhang et al. 2022).

Orf3a of severe acute respiratory syndrome (SARS) coronavirus 2

 
Examples:

TC#NameOrganismal TypeExample