1.C.99 The Pore-forming Corona Viral Orf8a (Sars8a) Family

The mechanisms and functions of viral channel proteins have been reviewed by Fischer and Hsu (2011) and Fischer et al. (2012). ORF8a protein is 39 residues long and contains a single transmembrane domain. The protein has been synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers (Chen et al., 2011). It forms cation-selective ion channels with a main conductance level of 8.9±0.8pS at elevated temperature (38.5°C). Computational modeling studies including multi nanosecond molecular dynamics simulations in a hydrated POPC lipid bilayer are done with a 22 amino acid transmembrane helix to predict a putative homooligomeric helical bundle model. A structural model of a pentameric bundle was proposed by Chen et al. (2011) with cysteines, serines and threonines facing the pore. The following Corina viral proteins have been considered to be viroporins, The (1) E protein, (2) ORF7b (TC# 1.A.127), (3) ORF10 (TC# 1.A.128), (4) Orf3a (TC# 1.A.57) and (5) ORF8a (TC# 1.C.99), but some researchers have questioned some of these conclusions (Harrison et al. 2022).

As noted above, permeability results from the assembly of helical bundles. Computational models of a pentameric assembly of 8a peptides were generated using the first 22 amino acids which include the single TMS. Low energy structures reveal a hydrophilic pore mantled by residues Thr-8, and -18, Ser-11, Cys-13, and Arg-22. Potential of mean force profiles for Na+ , K+ , Cl- and Ca2+ along the pore were calculated, leading to prediction of weak cation selectivity in agreement with the experimental results (Hsu et al. 2015). 3-D structures of short homologues are available (1XAK_A of 83 aas and 1 TMS, and 1Y04_A of 87 aas and 1 TMS). Members of subfamilies 1.C.99.1 and 1.C.99.2 are similar in their first 38 aas which encompasses the first (N-terminal) TMS.


 

References:

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

Chen, C.C., J. Krüger, I. Sramala, H.J. Hsu, P. Henklein, Y.M. Chen, and W.B. Fischer. (2011). ORF8a of SARS-CoV forms an ion channel: experiments and molecular dynamics simulations. Biochim. Biophys. Acta. 1808: 572-579.

Fischer, W.B. and H.J. Hsu. (2011). Viral channel forming proteins - modeling the target. Biochim. Biophys. Acta. 1808: 561-571.

Fischer, W.B., Y.T. Wang, C. Schindler, and C.P. Chen. (2012). Mechanism of function of viral channel proteins and implications for drug development. Int Rev Cell Mol Biol 294: 259-321.

Harrison, N.L., G.W. Abbott, M. Gentzsch, A. Aleksandrov, A. Moroni, G. Thiel, S. Grant, C.G. Nichols, H.A. Lester, A. Hartel, K. Shepard, D.C. Garcia, and M. Yazawa. (2022). How many SARS-CoV-2 "viroporins" are really ion channels? Commun Biol 5: 859.

Hsu, H.J., M.H. Lin, C. Schindler, and W.B. Fischer. (2015). Structure based computational assessment of channel properties of assembled ORF-8a from SARS-CoV. Proteins 83: 300-308.

Mann, M.M., M.K. Hsieh, J.D. Tang, W.S. Hart, M.J. Lazzara, J.B. Klauda, and B.W. Berger. (2023). Understanding how transmembrane domains regulate interactions between human BST-2 and the SARS-CoV-2 accessory protein ORF7a. Biochim. Biophys. Acta. Biomembr 1865: 184174. [Epub: Ahead of Print]

Martin, S., G. Jégou, A. Nicolas, M. Le Gallo, &.#.2.0.1.;. Chevet, F. Godey, and T. Avril. (2022). A cell-based system combined with flow cytometry to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in patients with COVID-19. STAR Protoc 3: 101229.

Scott, C. and S. Griffin. (2015). Viroporins: structure, function and potential as antiviral targets. J Gen Virol 96: 2000-2027.

Examples:

TC#NameOrganismal TypeExample
1.C.99.1.1

Cation (K+, Na+)-selective pore-forming Orf8a (Sars8a) peptide of 39 aas and 1 N-terminal TMS (Hsu et al. 2015, Scott and Griffin 2015).

Virus

Orf8a of severe acute respiratory syndrome causing corona virus (SARS-CoV) (Q7TA23)

 
1.C.99.1.2

Pore-forming protein of 122 aas with 1 N-terminal TM

Viruses

Pore-former of Bat β-coronavirus

 
1.C.99.1.3

Non structural protein 8 of 121 aas and 1 N-terminal TM

Viruses

Protein 8 of Bat coronavirus 279/2005 (BtCoV)

 
1.C.99.1.4

ORF8 protein of 121 aas and 1 N-terminal TMS. Orf8 is a short 29-amino-acid single-passage transmembrane peptide that forms cation-selective channels when assembled in lipid bilayers (Barrantes 2021). A cell-based system combined with flow cytometry can be used to evaluate antibody responses against SARS-CoV-2 transmembrane proteins in patients with COVID-19 (Martin et al. 2022).

ORF8 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

 
1.C.99.1.5

Uncharaacterized protein, Sars8b of 84 aas and 0 TMSs.  This protein may be N-terminally truncated.

Sars8b of Severe acute respiratory syndrome-related coronavirus (SARS)

 
Examples:

TC#NameOrganismal TypeExample
1.C.99.2.1

Uncharacterized protein of 101 aas and 1 or 2 TMSs, with one TMS at the N-terminus, and possibly another at the C-terminus.

UP of SARS coronavirus WH20

 
1.C.99.2.2

Orf7a of 121 aas and 2 or 3 TMSs, one at the N-terminus, one at the C-terminus, and a peak of moderate hydrophobicity in the middle.  N-terminal fragments of 29 aas, 39 aas and 43 aas can be found in the NCBI protein database (Acc# QIG55990, QIS30140, and QIZ64621, respectively). Bone marrow stromal antigen 2 (BST-2; tetherin) is an antiviral response protein that inhibits transport of viral particles after budding within infected cells. RNA viruses such as SARS-CoV-2 use various strategies to disable BST-2. ORF7a TMS interactions play a key role along with extracellular and juxtamembrane domains in modulating BST-2 function (Mann et al. 2023).

Orf7a of severe acute respiratory syndrome coronavirus 2

 
1.C.99.2.3

X4-like protein of 120 aas and 2 TMSs, N- and C-terminal.

X4-like protein of Rhinolophus bat coronavirus HKU32