Inhibition of vaccinia virus DNA replication by inducible expression of nitric oxide synthase.

Nitric oxide (NO) exerts multiple biologic roles in animal cells through differential regulation of three distinct forms of NO synthase encoded by separate genes. Macrophage-inducible nitric oxide synthase (iNOS) has been correlated with inhibition of viral growth, but little is known about the mechanism of this effect. To study the antiviral role of NO, we have generated a vaccinia virus (VV) recombinant expressing iNOS under the control of Escherichia coli LacI operator/repressor elements. When cultured cells of various origins are infected with this recombinant virus, there is inducible expression of iNOS in the presence of isopropylthio-beta-galactoside, as determined by Western blot and by detection of nitrite, a NO oxidation product. The levels of nitrite increase with time after infection, correlating with marked inhibition of VV DNA and late protein synthesis. Expression of VV early proteins is not affected by NO. Inhibition of VV DNA synthesis is likely to be in part a consequence of NO-mediated inhibition of viral ribonucleotide reductase, as this inhibition can be partially overcome by addition of deoxyribonucleosides. Inhibition of the essential viral functions by NO results in a reduction of virus yields by 50 to 90%, depending on the cell line. Thus, our results demonstrate a direct antiviral effect of NO, with inhibition of VV replication occurring at the level of DNA synthesis.