SARS coronavirus : the role of accessory proteins and nitric oxide in the replication cycle

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused the first pandemic of the 21st century. The etiological agent was identified as a novel coronavirus. Until then, human coronaviruses (HCoVs) had only been known to cause the common cold . Data indicated that the virus originated from animals. The palm civet (Paguma larvata)was identified as the source of transmitting the virus to humans, however not to be the natural host, but most likely acting as an amplifier before the virus was transmitted to humans. SARS-CoV has 14 potential open reading frames (ORFs). Eight of those are specific for SARS and encodes for the accessory proteins. The accessory proteins of SARS-CoV have no known sequence homology to any other accessory proteins found in coronaviruses.

The knowledge about the function of most of the accessory proteins are limited. In addition to the four main structural proteins; the spike (S), the envelope (E), the membrane (M) and the nucleocapsid (N) protein, the accessory protein 3a of SARS-CoV has been shown to be a minor structural protein.

SARS-CoV, as the name implies, causes severe lower respiratory syndrome. Person-to- person transmission has been through infectious droplets, and the overall mortality rate is ~10% but can vary with age. Most SARS patients were treated with antiviral drugs and glucocorticoids since no specific treatment has been available. Inhalation of nitric oxide (NO) has been given to only a few patients showed a positive effect.

In this thesis we have investigated the importance of accessory proteins 3a/3b and 7a/7b in the replication cycle. Further we compared the neutralizing properties of the endo- and the ectodomian of 3a. We also investigated the antiviral effect of NO on SARS-CoV, and a possible mechanism behind an antiviral effect.

Short interfering RNA (siRNA) was designed to specifically target sgRNA 2, 3 and 7 expressing the S, 3a/3b and 7a/7b protein respectively. The yield of progeny virus was significantly reduced for all three siRNAs. The amount of progeny virus was to some extent lower for siRNA 7, which could be due to the fact that siRNA 7 were able to silence both 7a/7b and 8a/8b protein. Cells expressing the siRNAs specifically silenced the expression of targeted proteins without affecting the infection shown by expression of the N protein. The 3a protein was further investigated, comparing neutralizing properties of antibodies towards the endodomain and the ectodomain of 3a. Antibodies towards both ends were able to detect 3a in lysate from infected Vero E6 cells. However, only antibodies against the ectodomain showed neutralizing properties in Vero E6 cells.

In order to investigate the antiviral affect of NO on SARS-CoV, both an exogenous NO donor, and endogenously produced NO was used. We showed that NO has a clear antiviral effect on SARS-CoV, inhibiting the replication cycle of the virus. To investigate the mechanism behind the antiviral effect of NO, we first confirmed that NO per se was exerting the observed antiviral effect, and not through peroxynitrite interaction. By using a cell-cell fusion assay, we showed that NO inhibits the fusion step by reducing palmitoylation of the SARS-CoV S protein.