Dual effect of nitric oxide on SARS-CoV replication: viral RNA production and palmitoylation of the S protein are affected

Abstract

Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

Fig. 1

SIN-1 treatment has no antiviral effect on SARS-CoV infected Vero E6 cells. Vero E6 cells were infected with SARS-CoV at an MOI of 1.0, at 1 hpi cells were treated with different concentrations of SIN-1 and/or MnTBAP. (A) Cells treated with different concentrations of SIN-1. 24 hpi, virus was harvested and titers determined. (B) Cells treated with 400 μM SIN-1 and different concentrations of MnTBAP. Virus was harvested 24 hpi and titers determined. (C) Cells treated with different concentrations of MnTBAP, virus was harvested 24 hpi and titers determined.

Fig. 2

Effect of SNAP on nitration and palmitoylation of S protein. Vero E6 cells were infected with rVV-L-S at an MOI of 0.1. At 1 hpi cells were treated with 400 μM SNAP or NAP. (A) At 24 h post-infection, the cells were lysed and immunoprecipitation was performed with nitrotyrosine affinity sorbent. Western blot was then performed using rabbit anti-S polyclonal antibody. (B, C) At 24 h post-infection, cells were labelled with 400 μCi of [³H]-palmitic acid for 2 h, or starved with methionine-cysteine (Met-Cys)-free medium for 30 min before being labelled with 22 μCi of [³⁵S]-methionine-cysteine for 2 h. The cells were then lysed and imunoprecipitation was performed with rabbit S polyclonal antibody to detect the amount of radiolabelled S protein.

Fig. 3

SNAP interferes with cell–cell membrane fusion. A 293T-GFP stable cell line was infected with rVV-L-S and treated with SNAP or NAP 1 h post-infection. At 24 hpi cells were trypsinized and mixed with pre-plated CHO-ACE2 cells. 6 h after mixing the cell lines, syncytium formation was observed. (A) Infected cells, mock-treated (B) Infected cells, treated with 400 μM SNAP (C) Infected cells, treated with 400 μM NAP (D) Mock-infected cells.

Fig. 4

SARS-CoV S pseudotyped virus produced in the presence of SNAP is less efficient in viral entry. Pseudotyped viruses produced under different conditions were purified and concentrated using ultra-centrifugation through a 20% sucrose bed. (A) The presence of S and HIV-1 p24 proteins in the purified viruses were determined by Western blot analysis. (B) Purified viruses were used for the transduction of CHO-ACE2 cells and the degrees of viral entry were determined by measuring the luciferase activities. Percentages of infectivity were computed by normalizing the entry of untreated S-bearing pseudotyped viruses to 100%. Means and standard deviation from duplicate readings were shown. (C) Nitric oxide or its derivatives do not exert a noticeable effect on the binding of the SARS S protein to ACE2. Cells were infected with pseudotyped virus bearing the S protein, produced under SNAP or NAP treatment. A p24 ELISA kit was used to measure p24 concentration and concentration of bound virus was derived from a standard graph.

Fig. 5

SNAP treatment causes a reduction in the production of positive-stranded viral RNA. Vero E6 cells were either treated with 400 μM SNAP, NAP or mock-treated and infected with SARS-CoV at an MOI of 1. A parallel set of cells were additionally treated an hour before infection. Cells were harvested from all sets of treatments at 3 h and 24 h post infection and subjected to reverse transcription and realtime PCR using primers and probes specific for the N gene of SARS-CoV and GAPDH. Ct values obtained for N in each sample was normalized against GAPDH control values and plotted for each time point and treatment.

Fig. 6

SNAP treatment causes a difference in observed levels of replicase polyprotein cleavage products. Lysates from either uninfected or SARS-CoV infected Vero E6 cells which had either been treated with SNAP, NAP or mock-treated harvested 24 h post-infection were subjected to Western blot analysis using a monoclonal antibody targeting the nsp8 protein.