Inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites

IFN-gamma-induced nitric oxide (NO) in the murine macrophage-derived cell line RAW 264.7 was previously shown to inhibit replication of the poxviruses ectromelia and vaccinia (VV) and HSV-1. In the current study we demonstrate that murine macrophages activated as a consequence of VV infection express inducible nitric oxide synthase. These activated macrophages were resistant to infection with VV and efficiently blocked the replication of VV and HSV-1 in infected bystander cells of epithelial and fibroblast origin. This inhibition was arginine dependent, correlated with nitrite production in cultures, and reversible by the NOS inhibitor N omega-monomethyl-L-arginine. NO-mediated inhibition of VV replication was studied by treatment of virus-infected human 293 cells with the NO donor S-nitroso-N-acetyl-penicillamine. Using a VV-specific DNA probe, antibodies specific for temporally expressed viral proteins, and transmission electron microscopy, we have shown that NO inhibited viral late gene protein synthesis, DNA replication, and virus particle formation, but not expression of the early proteins that were analyzed. Putative enzymatic targets of NO were identified by reversing the NO-mediated inhibition of VV replication in the 293 cells with exogenous ferrous sulfate and L-cysteine. Reversal of inhibition may derive from the capacity of these reagents to protect or regenerate nonheme iron or thiol groups, respectively, which are essential for the catalytic activities of enzymes susceptible to inactivation by NO.