SARS-CoV-2 Accessory Proteins & ORF9b

Coronavirus SARS-CoV-2 is an enveloped virus that interacts with host cells via a protruding protein called Spike, and fuses with the cellular membrane to inject its genome into the cell. The SARS family of coronaviruses contain a large single stranded RNA genome of about 30 kb with both unique and conserved features compared to other coronaviruses [1].

The presence of a 5′ methylated cap and a 3′ poly-adenylated tail allows the positive-sense RNA to be directly translated by the cellular ribosome using two overlapping open reading frames (ORFs) termed ORF1a and ORF1b [1]. The two large translated polyproteins PP1A and PP1AB contain proteases PLpro and 3CLpro [2], which proteolytically cleave the polyproteins into 16 functional components with replicase/transcriptase activity that are essential to viral replication and maturation [3]. 

The structure of the SARS-CoV-2 genome reveals a total of 14 ORFs, with occasional overlap as is commonly observed in the coronavirus family (Figure 2, adapted from [4]). 

SARS Cov 2 Genome

ORFs 2, 4, 5, and 9a encode the four major SARS-CoV-2 structural proteins: Spike (S), Envelope (E), Membrane (M), and Nucleocapsid, respectively [3]. Other ORFs encode for accessory proteins with no known role in viral replication, such as ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, and ORF8b, some of which may or may not be present in a particular coronavirus genome. 

Indeed, a unique and particularly interesting feature of viruses is the existence of alternative sequences encoding hidden accessory proteins. With alternative ORFs, the virus maximizes the efficiency of its limited genome: translation is initiated from a start codon within an existing gene and, being out of frame, gives rise to a distinct protein product [5]. For example, coronavirus ORF3b is not functional in all genomes and is not present in SARS CoV-2. Interestingly, the SARS-CoV-2 genome of reference corresponding to the initial SARS-CoV-2 strain originating from Wuhan, China [6] does not contain overlapping ORFs within ORF9a (Nucleocapsid protein N), whereas the US strain that quickly displaced the original strain worldwide [7] contains two additional accessory genes ORF9b and ORF9c embedded within the ORF9a gene [8]. ORF9c is a 70-amino acid polypeptide, the expression of which has not yet been demonstrated. 

ORF9b [P0DTD2.1] is a short 97-amino acid protein that is expressed in host cells upon infection. The crystal structure of SARS ORF9b reveals “a novel fold, a dimeric tent-like beta structure with an amphipathic surface, and a central hydrophobic cavity that binds lipid molecules” [5].

Although accessory proteins have no known role in viral replication, they may play a role in pathogenesis [9]. ORF9b appears to be involved in the suppression of anti-viral interferon responses. Type I interferons secreted by virus-infected cells are critical to our innate immune response and constitute the first line of defense against viral infection. Suppression of the interferon response has been observed in COVID-19 patients [10]. Emerging evidence now indicates that ORF9b protein accumulates rapidly in cells of the respiratory system following SARS-CoV-2 infection, inhibiting the NF-kB pathway and subsequent production of interferon, and leading to momentary immune suppression and delayed immune response [11, 12].

Further research is needed to fully decipher the role of SARS-CoV-2 accessory proteins, including ORF9b. To support such research efforts, BPS scientists have engineered a tagged recombinant ORF9b protein that is highly purified and ready for use. Please contact us to find out more about how we can help you meet your research goals.

References

  1. Snijder EJ, Bredenbeek PJ, Dobbe JC, Thiel V, Ziebuhr J, Poon LL, Guan Y, Rozanov M, Spaan WJ, Gorbalenya AE. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol. (2003) 331: 991-1004; PMCID: PMC7159028.
  2. Ziebuhr J, Herold J, Siddell SG. Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity. J Virol. (1995) 69: 4331-4338; PMCID: PMC189173. 
  3. Yoshimoto FK. The Proteins of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2 or n-COV19), the Cause of COVID-19. The Protein Journal (2020) 39: 198–216
  4. Xianchun Tang, Gang Li, Nikos Vasilakis, Yuan Zhang, Zhengli Shi, Yang Zhong, Lin-Fa Wang, and Shuyi Zhang. Differential stepwise evolution of SARS coronavirus functional proteins in different host species. BMC Evol Biol. (2009) 9: 52; PMCID: PMC2676248. 
  5. Meier C, Aricescu AR, Assenberg R, Aplin RT, Gilbert RJ, Grimes JM, Stuart DI. The crystal structure of ORF-9b, a lipid binding protein from the SARS coronavirus. Structure. (2006) 14: 1157-65; PMCID: PMC7126280
  6. Wang C, Liu Z, Chen Z, Huang X, Xu M, He T, Zhang Z. The establishment of reference sequence for SARS-CoV-2 and variation analysis. J Med Virol. (2020) 92: 667-674; PMCID: PMC7228400.
  7. Harcourt J, Tamin A, Lu X, Kamili S, Sakthivel SK, Murray J, Queen K, Tao Y, Paden CR, Zhang J, Li Y, Uehara A, Wang H, Goldsmith C, Bullock HA, Wang L, Whitaker B, Lynch B, Gautam R, Schindewolf C, Lokugamage KG, Scharton D, Plante JA, Mirchandani D, Widen SG, Narayanan K, Makino S, Ksiazek TG, Plante KS, Weaver SC, Lindstrom S, Tong S, Menachery VD, Thornburg NJ. Severe Acute Respiratory Syndrome Coronavirus 2 from Patient with Coronavirus Disease, United States. Emerg Infect Dis. (2020) 26: 1266-1273; PMCID: PMC7258473.
  8. Michel CJ, Mayer C, Poch O, Thompson JD. Characterization of accessory genes in coronavirus genomes. Virol J. (2020) 17: 131; PMCID: PMC7450977.
  9. McBride R, Fielding BC. The role of severe acute respiratory syndrome (SARS)-coronavirus accessory proteins in virus pathogenesis. Viruses. (2012) 4: 2902-1923; PMCID: PMC3509677.
  10. Sa Ribero M, Jouvenet N, Dreux M, Nisole S. Interplay between SARS-CoV-2 and the type I interferon response. PLoS Pathog. (2020) 16: e1008737; PMCID: PMC7390284.
  11. Wu J, Shi Y, Pan X, Wu S, Hou R, Zhang Y, Zhong T, Tang H, Du W, Wang L, Wo J, Mu J, Qiu Y, Yang K, Zhang LK, Ye BC, Qi N. SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Rep. (2021) 34: 108761; PMCID: PMC7857071.
  12. Jiang HW, Zhang HN, Meng QF, Xie J, Li Y, Chen H, Zheng YX, Wang XN, Qi H, Zhang J, Wang PH, Han ZG, Tao SC. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cell Mol Immunol. (2020) 17: 998-1000; PMCID: PMC7387808.

 

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