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

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

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