SARS CORONAVIRUS THE ROLE OF ACCESSORY PROTEINS AND NITRIC OXIDE IN THE REPLICATION CYCLE

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused the first&#13;\npandemic of the 21st century. The etiological agent was identified as a&#13;\nnovel coronavirus. Until then, human coronaviruses (HCoVs) had only been&#13;\nknown to cause the common cold . Data indicated that the virus&#13;\noriginated from animals. The palm civet (Paguma larvata)was identified as&#13;\nthe source of transmitting the virus to humans, however not to be the&#13;\nnatural host, but most likely acting as an amplifier before the virus was&#13;\ntransmitted to humans. SARS-CoV has 14 potential open reading frames&#13;\n(ORFs). Eight of those are specific for SARS and encodes for the&#13;\naccessory proteins. The accessory proteins of SARS-CoV have no known&#13;\nsequence homology to any other accessory proteins found in coronaviruses.&#13;\n&#13;\n&#13;\n&#13;\nThe knowledge about the function of most of the accessory proteins are&#13;\nlimited. In addition to the four main structural proteins; the spike (S),&#13;\nthe envelope (E), the membrane (M) and the nucleocapsid (N) protein, the&#13;\naccessory protein 3a of SARS-CoV has been shown to be a minor structural&#13;\nprotein.&#13;\n&#13;\n&#13;\n&#13;\nSARS-CoV, as the name implies, causes severe lower respiratory syndrome.&#13;\nPerson-to- person transmission has been through infectious droplets, and&#13;\nthe overall mortality rate is ~10% but can vary with age. Most SARS&#13;\npatients were treated with antiviral drugs and glucocorticoids since no&#13;\nspecific treatment has been available. Inhalation of nitric oxide (NO)&#13;\nhas been given to only a few patients showed a positive effect.&#13;\n&#13;\n&#13;\n&#13;\nIn this thesis we have investigated the importance of accessory proteins&#13;\n3a/3b and 7a/7b in the replication cycle. Further we compared the&#13;\nneutralizing properties of the endo- and the ectodomian of 3a. We also&#13;\ninvestigated the antiviral effect of NO on SARS-CoV, and a possible&#13;\nmechanism behind an antiviral effect.&#13;\n&#13;\n&#13;\n&#13;\nShort interfering RNA (siRNA) was designed to specifically target sgRNA&#13;\n2, 3 and 7 expressing the S, 3a/3b and 7a/7b protein respectively. The&#13;\nyield of progeny virus was significantly reduced for all three siRNAs.&#13;\nThe amount of progeny virus was to some extent lower for siRNA 7, which&#13;\ncould be due to the fact that siRNA 7 were able to silence both 7a/7b and&#13;\n8a/8b protein. Cells expressing the siRNAs specifically silenced the&#13;\nexpression of targeted proteins without affecting the infection shown by&#13;\nexpression of the N protein. The 3a protein was further investigated,&#13;\ncomparing neutralizing properties of antibodies towards the endodomain&#13;\nand the ectodomain of 3a. Antibodies towards both ends were able to&#13;\ndetect 3a in lysate from infected Vero E6 cells. However, only antibodies&#13;\nagainst the ectodomain showed neutralizing properties in Vero E6 cells.&#13;\n&#13;\n&#13;\n&#13;\nIn order to investigate the antiviral affect of NO on SARS-CoV, both an&#13;\nexogenous NO donor, and endogenously produced NO was used. We showed that&#13;\nNO has a clear antiviral effect on SARS-CoV, inhibiting the replication&#13;\ncycle of the virus. To investigate the mechanism behind the antiviral&#13;\neffect of NO, we first confirmed that NO per se was exerting the observed&#13;\nantiviral effect, and not through peroxynitrite interaction. By using a&#13;\ncell-cell fusion assay, we showed that NO inhibits the fusion step by&#13;\nreducing palmitoylation of the SARS-CoV S protein.

Topic: SARS-CoV-2 and COVID-19 Research
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences