From Resistance to Stimulation: the Evolution of a Virus in the Presence of a Dominant Lethal Inhibitory Scaffolding Protein

Abstract

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks X174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.With developed genetics and biochemistry and solved atomic structures, bacteriophage X174 has a long and rich history as a virus assembly system (15-17, 20, 22-25). Due to its rapid replication, which allows selective pressures to be applied for hundreds of infection cycles, it recently emerged as an attractive organism for evolutionary studies (4)(5)(6)(33)(34)(35)). An evolutionary approach to assembly was used to study the evolution of resistance to a genetically engineered inhibitory protein, a dominant lethal external scaffolding protein that specifically targets procapsid morphogenesis. A multiple-mutant resistant strain was experimentally evolved by culturing X174 in exponential-phase cells while incrementally increasing the induction of the lethal dominant gene (12). Like other viruses that acquire resistance to antiviral agents (14,27,28,37), the resistant strain exhibits lower fitness than that of the uninhibited wild-type strain in the absence of the inhibitor. It is also dependent on the once inhibitory protein for optimal fitness, which is not uncommon (3, 29). But unlike similar examples, the inhibitor stimulates fitness to values equal to or above the uninhibited wild-type level, suggesting that the virus evolved a mechanism to convert the inhibitory protein into a beneficial replication factor...

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

“…D D34G and D G61D together increase the in vitro production rate and overall yield of procapsids using the evolved 12S* particle. While the mutations in the viral coat protein contribute to the resistance phenotype, they do not dramatically elevate in vivo fitness to uninhibited wild-type levels (5,6). Instead, the genome-encoded D D34G appears to stimulate fitness, but only in the presence of the dominant lethal D G61D protein and the resistant coat protein mutations.…”

“…The X174 strains and plasmids used in these study have been previously described (5,6,11,12,53). The am(C)S10 mutation, which facilitated procapsid isolation, was introduced by site-directed mutagenesis (54).…”

“…Resistance was not specific to the D G61X mutant allele to which they were selected (5,6). A strain with a robust resistance phenotype was experimentally evolved.…”