Nitric oxide inhibits Epstein-Barr virus DNA replication and activation of latent EBV

Nitric oxide (NO), a mediator of biological functions, has antimicrobial activity against a variety of pathogens including viruses. Effects of NO donors on EBV replication in two EBV lytic systems, Raji cells infected with P3HR-1 virus and P3HR-1 cells activated with TPA plus n-butyrate, were studied. S-nitroso-N-acetylpenicillamine (SNAP), which generates NO when placed in an aqueous solution, and 3-morpholinosydnonimine (SIN-1), which liberates NO and O2-, resulting in the formation of peroxynitrite, were used as NO donors. Immunoprecipitation analysis showed that in superinfected Raji cells, SNAP inhibited EBV late protein synthesis but not EBV early protein expression. Analysis of the structure of EBV DNA termini demonstrated that SNAP suppressed the amplification of EBV DNA in superinfected Raji cells at a dose which did not affect synthesis of EBV early proteins required for EBV DNA replication. In TPA plus n-butyrate-treated P3HR-1 cells, SNAP inhibited synthesis of both early and late proteins of EBV. Northern blot analysis of RNA expressed in TPA plus n-butyrate-treated P3HR-1 cells demonstrated that expression of EBV immediate-early mRNAs coded from BZLF1 and BRLF1 genes was inhibited by SNAP. SIN-1 showed no or little effect on EBV replication in both cell systems. Cell viability and cellular protein synthesis were not affected by either NO donor under the conditions used. These findings suggest that NO prevents EBV replication by inhibiting EBV DNA amplification during the lytic phase of the life cycle as well as by blocking activation of the latent EBV genome. The mechanism for inhibiting of EBV replication by NO was discussed in relation to the role of NO in EBV latency in vivo.