SARS coronavirus accessory proteins

Abstract

The emergence of the severe acute respiratory syndrome coronavirus (SARS-CoV) has led to a renewed interest in studying the role of accessory proteins in regulating coronavirus infections in the natural host. A significant body of evidence has accumulated in the area of SARS-CoV and host interactions that indicate that the accessory proteins might play an important role in modulating the host response to virus infection and thereby, contribute to pathogenesis. In this review, we have compiled the current knowledge about SARS-CoV accessory proteins, obtained from studies in cell culture systems, reverse genetics and animal models, to shed some light into the possible role of these proteins in the propagation and virulence of SARS-CoV in its natural host. We conclude by providing some questions for future studies that will greatly advance our knowledge about the biological significance and contributions of the accessory proteins in the development of SARS in humans.

Fig. 1

Genome organization of SARS coronavirus accessory genes. The accessory genes are shown as gray boxes. The ORFs 1a and 1b comprise the SARS-CoV replicase genes. The figure is not drawn to scale. RFS: ribosomal frameshift.

Fig. 2

3a protein enhances dsRNA-mediated activation of NF-κB-regulated promoters. Human embryonic kidney (HEK) 293 cells were transfected with an NF-κB-driven luciferase (luc) reporter plasmid (A) or a RANTES-luc reporter plasmid (B) for the reporter assays. These cells were co-transfected with one of the indicated SCoV accessory protein expression plasmids, or a control empty vector, pcDNA 3.1 (EV) and a plasmid constitutively expressing β-galactosidase (β-gal) as an internal control. After 24 h post transfection, cells were mock-transfected, or transfected with dsRNA, poly I:C (10 μg/ml), for 6 h. Cell extracts were analyzed for luciferase activities and normalized to β-gal activity to obtain the relative luciferase activity. Triplicate samples were analyzed for each experimental group.

Fig. 3

3a protein-mediated enhancement of NF-κB promoter activation requires intracellular dsRNA. HEK 293 cells were co-transfected with an NF-κB-luc reporter plasmid, β-gal plasmid along with 3a-expression plasmid or empty vector (EV). After 24 h post transfection, the cells were either mock-treated, treated with poly I:C (50 μg/ml) in the media, mock-transfected or transfected with poly I:C (10 μg/ml), for 6 h. Cell extracts were analyzed for luciferase activities and normalized to β-gal activity to obtain the relative luciferase activity. Note that 3a protein enhances the dsRNA-mediated activation of NF-κB promoter only in the dsRNA-transfected samples.

Fig. 4

SARS-CoV infection induces the activation of NF-κB-regulated promoters. 293/ACE2 cells, stably expressing the SARS-CoV receptor, human angiotensin converting enzyme 2 (ACE2), were transfected with NF-κB-luc reporter plasmid (A and B) or RANTES-luc reporter plasmid (C). After 9 h post transfection, the cells were mock-infected or infected with the Urbani strain of SARS-CoV [multiplicity of infection (MOI) = 3] or the control virus, Sendai virus (100 HA units/ml). After 18 h post-infection, RNA was extracted from the cells and luciferase mRNA levels were measured by real-time PCR analysis. The fold change in expression level, compared to mock-infected cells, is shown. The expression levels are normalized to 18S ribosomal RNA. Note the delayed kinetics in up-regulation of NF-κB and RANTES-promoter-driven luc mRNA in SARS-CoV-infected cells compared to Sendai virus infection.