Viral Adaptation to an Antiviral Protein Enhances the Fitness Level to Above That of the Uninhibited Wild Type

Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks X174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. While viruses often acquire resistance to antiviral agents, resistance mutants generally exhibit lower fitness than the wildtype strain in the absence of the inhibitor Due to its rapid replication, bacteriophage X174 has become an attractive model system for evolutionary studies The inhibitory proteins most likely remove assembly intermediates by lowering the thermodynamic barriers that normally prevent off-pathway reactions (4, 5). Both off-pathway reactions and proper assembly involve D-D protein interactions across what will become the twofold axes of symmetry in the virion (8, 9). In the procapsid crystal structure, ␣-helix 3 of the D 2 , D 3 , and D 4 subunits mediates these interactions. Mutants resistant to the dominant lethal proteins were isolated in one-step genetic selections, and mutations mapped to either the coat or internal scaffolding proteins. These mutations may indirectly reinstate the avidity of the D protein electrostatic bonding partners required for productive morphogenesis (4, 5). However, the resistance phenotype is weak. To isolate a more robust phenotype, wild-type X174 was continually cultured through exponential phase cells expressing an inhibitory D protein. Results from this analysis indicate that the selection for resistance coselected for a level of dependence. The inhibitory protein stimulates resistant strain fitness significantly above the uninhibited wild-type level and appears to be required for efficient capsid nucleation. These results suggest that the virus evolved a mechanism to convert this potent antiviral agent into a beneficial factor and may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly