Inhibition of influenza virus replication by nitric oxide

Abstract

Nitric oxide (NO) has been shown to contribute to the pathogenesis of influenza virus-induced pneumonia in mouse models. Here we show that replication of influenza A and B viruses in Mabin Darby canine kidney cells is severely impaired by the NO donor, S-nitroso-N-acetylpenicillamine. Reduction of productively infected cells and virus production proved to correlate with inhibition of viral RNA synthesis, indicating that NO affects an early step in the replication cycle of influenza viruses.

FIG. 1

Addition of the NO donor SNAP inhibits the replication of influenza A and B viruses in a dose-dependent manner. MDCK cells were infected with influenza virus A/Netherlands/202/95 at an MOI of 0.3 (A). The culture supernatants were assayed for hemagglutination activity 24 h postinfection. MDCK cells were also infected with influenza virus B/Netherlands/22/95 with an MOI of 0.03 (B). The culture supernatants were analyzed for HA 48 h postinfection. Using Griess reagent, the release of NO from SNAP was demonstrated by detection of its metabolite nitrite (C). The infected cells were treated with the indicated concentrations of SNAP (○) or NAP (●).

FIG. 2

The addition of the NO donor SNAP inhibits the release of infectious virus into the culture supernatant. MDCK cells were infected with influenza virus A/Netherlands/202/95 at the indicated MOI and subsequently treated with 400 μM SNAP or NAP or left untreated. The measurement of infectious virus in culture supernatants of the cells was performed by limiting dilution analysis and expressed as 50% tissue culture infective doses per milliliter (TCID₅₀/ml) as previously described (30).

FIG. 3

The number of influenza virus-infected MDCK cells is affected by the addition of SNAP to the cultures before or after infection. SNAP or NAP was added at the indicated concentrations 3 h before infection or 1 h after infection of the cells with influenza virus A/Netherlands/18/94 at an MOI of 1.0. Twelve hours postinfection, the cells were fixed with acetone and incubated with a monoclonal antibody specific for the nucleoprotein.

FIG. 4

The synthesis of virus-specific positive strand (mRNA and cRNA) and negative strand (vRNA) is impaired by the addition of SNAP to the cultures. SNAP (S) or NAP (N) was added to the cultures at the indicated concentrations 3 h before or immediately after infection with influenza virus A/Netherlands/18/94. Untreated cultures were included as controls (C). MDCK cells were infected at an MOI of 1.0 and RNA was extracted 12 h postinfection. Threefold serial dilutions of the RNA preparations were transferred to the membranes and subsequently hybridized to DIG-labelled RNA probes specific for positive-sense HA or NP mRNA/cRNA, negative-sense HA and NP vRNA, and positive-sense β-actin RNA, which was included as a control for cellular mRNA, as described previously (30).