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

Self Cite

During X174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro. In previous studies, X174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. IMPORTANCE Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo. We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During X174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity.KEYWORDS bacteriophage X174, Microviridae, microvirus, scaffolding protein, virus assembly D ue to their rapid replication cycle, detailed structural information, and well developed genetic and biochemical assays, the microviruses have become an ideal model system for experimental evolution (1-4). The morphogenetic pathway can be characterized under restrictive conditions and adaptive mutations can be mapped onto X-ray structures, providing insights into evolutionary mechanisms and protein structure-function relationships (2-8).

Section: Dominant Lethal Viral Scaffolding Proteinmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Cherwa

Tyson

Bedwell

et al. 2017

Self Cite

“…Attachment and eclipse assays have been previously described (24,25). Coat protein quantification as a surrogate marker for genome piloting was performed as follows.…”

Section: Methodsmentioning

confidence: 99%

Structure-Function Analysis of the ϕX174 DNA-Piloting Protein Using Length-Altering Mutations

Roznowski

Fane

2016

Self Cite

Although the X174 H protein is monomeric during procapsid morphogenesis, 10 proteins oligomerize to form a DNA translocating conduit (H-tube) for penetration. However, the timing and location of H-tube formation are unknown. The H-tube's highly repetitive primary and quaternary structures made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein's three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis. Insertion mutants were viable. Theoretically, these proteins would produce an assembled tube exceeding the capsid's internal diameter, suggesting that virions do not contain a fully assembled tube. Lengthened proteins were also used to test the biological significance of the crystal structure. Particles containing H proteins of two different lengths were significantly less infectious than both parents, indicating an inability to pilot DNA. Shortened H proteins were not fully functional. Although they could still stimulate viral protein synthesis, they either were not incorporated into virions or, if incorporated, failed to pilot the genome. Mutant proteins that failed to incorporate contained deletions within an 85-amino-acid segment, suggesting the existence of an incorporation domain. The revertants of shortened H protein mutants fell into two classes. The first class duplicated sequences neighboring the deletion, restoring wildtype length but not wild-type sequence. The second class suppressed an incorporation defect, allowing the use of the shortened protein. IMPORTANCEThe H-tube crystal structure represents the first high-resolution structure of a virally encoded DNA-translocating conduit. It has similarities with other viral proteins through which DNA must travel, such as the ␣-helical barrel domains of P22 portal proteins and T7 proteins that form tail tube extensions during infection. Thus, the H protein serves as a paradigm for the assembly and function of long ␣-helical supramolecular structures and nanotubes. Highly repetitive in primary and quaternary structure, they are amenable to structure-function analyses using in-frame insertions and deletions as presented herein. Bacteriophages have evolved several mechanisms to transport hydrophilic genomes through cell walls containing hydrophobic membranes and peptidoglycan. Myoviruses and podoviruses carry tails that, respectively, utilize contractile sheaths (1) and form extensions within the membrane (2). In contrast, siphoviruses, filamentous phage, and other tail-less viruses co-opt host cell membrane channels for penetration (3)(4)(5). However, the tailless microvirus X174 does not require a host-provided conduit for genome transport. Instead, genome translocation is mediated by 10 to 12 copies of the DNA-piloting protein H found inside the virion (6-8).The X-ray structure of H protein's coiled-coil domain (8, 9) shows 10 parallel proteins oligomerized into a tube (Fig. 1) Th...

“…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.…”

mentioning

confidence: 99%

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

Cherwa

Fane

2011

Self Cite

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...