Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus

Abstract

Nitric oxide (NO) is an important signaling molecule between cells which has been shown to have an inhibitory effect on some virus infections. The purpose of this study was to examine whether NO inhibits the replication cycle of the severe acute respiratory syndrome coronavirus (SARS CoV) in vitro. We found that an organic NO donor, S-nitroso-N-acetylpenicillamine, significantly inhibited the replication cycle of SARS CoV in a concentration-dependent manner. We also show here that NO inhibits viral protein and RNA synthesis. Furthermore, we demonstrate that NO generated by inducible nitric oxide synthase, an enzyme that produces NO, inhibits the SARS CoV replication cycle.

FIG. 1.

NO has an antiviral effect on SARS CoV infection. Vero E6 cells were infected with SARS CoV at an MOI of 1.0. At 1 hpi, the cells were treated with different concentrations of SNAP (•) and NAP (○). (A) Viruses was harvested at 24 hpi and titers were determined. (B) Nitrite concentrations produced at 24 h posttreatment with different concentrations of SNAP and NAP. (C) Cell viability, as determined by MTT assays. The mean values from two experiments are indicated.

FIG. 2.

Immunofluorescence assays and Western blot analyses revealed that SNAP inhibits the SARS CoV replication cycle. Vero E6 cells were infected with SARS CoV at an MOI of 1.0 and then treated with 400 μM SNAP or NAP at 1 hpi. Vero E6 cells were fixed with 80% acetone at 24 hpi and then analyzed by an immunofluorescence assay (A), lysed at 12 hpi and then analyzed by Western blotting (B), or pulsed with 250 μCi of radiation/ml for 1 h, lysed, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (C).

FIG. 3.

SNAP blocks viral RNA replication of SARS CoV. Vero E6 cells were infected at an MOI of 0.01 and then treated with 400 μM SNAP (•) or NAP (○) at 1 hpi. Treated cells were lysed by the use of Trizol at different times, and viral RNAs were quantified as described in the text.

FIG. 4.

iNOS has an inhibitory effect on the replication cycle of SARS CoV. Vero E6 cells were mock treated or treated with IL-1β and IFN-γ, with or without the iNOS inhibitor l-NMMA. At 48 h posttreatment, the cells were infected with SARS CoV at an MOI of 0.1. Progeny virus was harvested at 24 hpi, and the titer was deduced by calculating the TCID₅₀ (A). Nitrite concentrations were determined by the use of Griess reagent (B). The mean values from three separate experiments are given, and error bars indicate standard deviations.