On the Sensitivity of the Virion Envelope to Lipid Peroxidation

Emerging viruses are a public health threat best managed with broad spectrum antivirals. Common viral structures, like capsids or virion envelopes, have been proposed as targets for broadly active antiviral drugs. For example, a number of lipoperoxidators have been proposed to preferentially affect viral infectivity by targeting metabolically inactive enveloped virions while sparing metabolically active cells. However, this presumed preferential virion sensitivity to lipoperoxidation remains untested. To test whether virions are indeed more sensitive to lipoperoxidation than are cells, we analyzed the effects of two classic generic lipoperoxidators: lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) and hydrophilic 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) on Vero and human foreskin fibroblasts (HFF) cell viability, HSV-1 plaquing efficiency, and virion and cell lipoperoxidation. Cells or virions were incubated with the lipoperoxidators at 37°C for 2 h or incubated in atmospheric O₂, and dose responses (half maximal cytotoxic and effective concentration [CC₅₀ and EC₅₀]) were evaluated by three or four parameter regression. The HSV-1 virions were slightly more sensitive to lipoperoxidators than were the cells (selectivity index [SI], 3.3 to 7.4). The effects of the lipophilic AMVN on both cell and virion viability directly correlated with the extent of membrane lipoperoxidation as evaluated by two different probes, C11-Bodipy and liperfluo. Moreover, the hydrophilic AAPH-induced virion inactivation at lower concentrations than did lipoperoxidation. Known lipoperoxidators inhibit infectivity via lipoperoxidation-independent mechanisms. Antioxidants protected against a loss of viral infectivity by less than 5-fold. Carrier bovine serum albumin (BSA) protected against both peroxidators to a similar extent when present together with the lipoperoxidating agents, suggesting that BSA quenches them as expected. Virions incubated in atmospheric oxidative conditions suffered losses of infectivity that were similar to those of chemically peroxidated virions, and they were protected by water soluble vitamin C and BSA with no evident lipoperoxidation, indicating predominant peroxidative damage to nonlipid virion components. Thus, lipoperoxidation is not a mechanism by which to specifically inhibit the infectivity of enveloped viruses, and the effects of known lipoperoxidators on virion infectivity are not solely mediated by lipoperoxidation. IMPORTANCE Small molecules that induce lipoperoxidation have been proposed repeatedly as potential antiviral drugs based on a presumed unique sensitivity of virions to this type of damage. Several small molecules that inactivate virions without affecting cells have been proposed to act primarily by inducing lipoperoxidation. However, the preferential sensitivity of virions to lipoperoxidators had not been experimentally evaluated. Using two of the best characterized small molecule lipoperoxidators, which are widely considered to be the prototypical water soluble and liposoluble lipoperoxidators, we show that lipoperoxidators have no preference for virions over cells. Moreover, they also inactivate virions by mechanisms other than the induction of lipoperoxidation. Therefore, the general induction of lipoperoxidation is not a path by which to develop antivirals. Moreover, molecules with specific antiviral activity which are not cytotoxic and have no preference to localize to virions over cells are unlikely to act primarily by inducing lipoperoxidation.