Severe acute respiratory syndrome coronavirus protein 6 is required for optimal replication

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes several accessory proteins of unknown function. One of these proteins, protein 6 (p6), which is encoded by ORF6, enhances virus replication when introduced into a heterologous murine coronavirus (mouse hepatitis virus [MHV]) but is not essential for optimal SARS-CoV replication after infection at a relatively high multiplicity of infection (MOI). Here, we reconcile these apparently conflicting results by showing that p6 enhances SARS-CoV replication to nearly the same extent as when expressed in the context of MHV if cells are infected at a low MOI and accelerates disease in mice transgenic for the human SARS-CoV receptor.

FIG. 1.

Time course of SARS-CoV infection and viral RNA and protein accumulations. (A) To assess p6 expression by SARS-CoVΔ6, Vero E6 cells were infected with wild-type rSARS-CoV (wt) or rSARS-CoVΔ6 (Δ6) at an MOI of 1 and analyzed by Western blot assay, using mouse anti-N monoclonal antibody (MAb; kindly provided by John Nicholls, University of Hong Kong) (N) and rat anti-p6 polyclonal antibody (P6). The latter was produced in 6-week-old female Wistar rats by intraperitoneal inoculation with 40 μg of high-pressure liquid chromatography-purified peptide 36-IVRQLFKPLTKKNYSELDDEEPM-58 coupled to Limulus polyphemus hemocyanin, followed by two boosts with peptide delivered intraperitoneally. (B to D) Vero E6 cells were infected with rSARS-CoV or rSARS-CoVΔ6 at an MOI of 0.01. (B) Cells were harvested at the indicated times, and titers on Vero E6 cells were determined. The increase in virus titers mediated by p6 is shown at each time point. (C) Total cellular RNAs were harvested from individual cultures at 5, 6, 7, and 9 h p.i. SARS-CoV N gene-specific RNA was quantified by real-time PCR, normalizing the level of N gene amplicons to that of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) amplicons, as described previously (20). *, P < 0.05 by Student's t test. (D) Infected cell cultures were harvested and analyzed by Western blot assay, using anti-N antibody. Twenty-four-hour samples were diluted eightfold to avoid overexposure (indicated by “1/8” in the figure). Data shown are representative of three independent experiments.

FIG. 2.

Complementation of rSARS-CoVΔ6 with plasmid DNA encoding p6. Vero E6 cells were grown in 24- or 6-well plates and transfected for 16 h with 1.0 or 4.0 μg pCAGGS-ORF6-HA plasmid, using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. As controls, cells were transfected with the same amount of empty vector. (A) Transfection efficiency was determined by an immunofluorescence assay using mouse anti-hemagglutinin antibody (Covance, Berkeley, CA), followed by Cy3-conjugated donkey anti-mouse antibody (Jackson Immunoresearch, West Grove, PA) (red). Nuclei were stained with Topro-3 (blue). (B) Sixteen hours after transfection, cells were infected with rSARS-CoVΔ6 at an MOI of 0.01 in serum-free Dulbecco's modified Eagle's medium. Viral titers were determined at 8 h p.i. by plaque assay on Vero E6 cells. *, P < 0.05 by Student's t test.

FIG. 3.

Weight loss, mortality, and viral titers in hACE2 transgenic mice infected with rSARS-CoV and rSARS-CoVΔ6. Mice were infected with 2.4 × 10⁴ PFU rSARS-CoV or rSARS-CoVΔ6 and monitored for weight loss (A) and mortality (B). Groups of four infected mice were analyzed in two independent experiments, and the data were combined. Virus titers in the lung (C) and brain (D) were determined on days 1 (D1) to 4 p.i. as previously described (2). Each group includes eight infected mice from two independent experiments. *, P < 0.05; **, P < 0.001 by Student's t test.

FIG. 4.

Effects of p6 on IFN-induced STAT1 nuclear translocation in SARS-CoV- or MHV-infected cells and on susceptibility to IFN. (A) Vero E6 cells were infected with rSARS-CoV or rSARS-CoVΔ6 at an MOI of 0.1 and then treated with 100 U/ml of human IFN-γ for 30 min at 12 h p.i. Cells were fixed with methanol and stained with anti-p6 followed by Cy5-conjugated donkey anti-rat antibody (Jackson ImmunoResearch) (blue), anti-SARS-CoV N followed by fluorescein isothiocyanate-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch) (green), and rabbit anti-phospho-STAT1 (Tyr701) (p-STAT1) antibody (Cell Signaling, Danvers, MA) followed by Cy3-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) (red). (B) HeLa-MHVR cells were infected with rJ2.2 or rJ2.2.6 at an MOI of 0.5 and then treated with human IFN-γ for 30 min at 14 h p.i. Cells were stained with fluorescein isothiocyanate-conjugated anti-hemagglutinin (HA) MAb (Roche, Switzerland) (green), anti-MHV N (MAb 5B188.2, kindly provided by M. Buchmeier, University of California, Irvine) followed by Cy5-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch) (blue), and anti-p-STAT1 followed by Cy3-conjugated donkey anti-rabbit antibody (red). Original magnification, ×40. (C) Vero E6 cells in triplicate were treated with the indicated concentrations of human IFN-β (PBL Biomedical Laboratories, Piscataway, NJ) 24 h prior to being infected with rSARS-CoV or rSARS-CoVΔ6 at 0.01 or 0.001 PFU/cell. Cells were then incubated for another 24 h in the presence of the same concentration of IFN-β. Samples were harvested, and virus titers were determined.