Complete Virion Assembly with Scaffolding Proteins Altered in the Ability To Perform a Critical Conformational Switch

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. However, monomers are incorporated into 12 pentameric assembly intermediates, which become the capsid's icosahedral vertices. The protein's N terminus, a predicted transmembrane helix, is not represented in the crystal structure. To investigate its functions, a series of absolute and conditional lethal mutations were generated. The absolute lethal proteins, a deletion and a triple substitution, were efficiently incorporated into virus-like particles lacking infectivity. The conditional lethal mutants, bearing cold-sensitive (cs) and temperature-sensitive (ts) point mutations, were more amenable to further analyses. Viable particles containing the mutant protein can be generated at the permissive temperature and subsequently analyzed at the restrictive temperature. The characterized cs defect directly affected host cell attachment. In contrast, ts defects were manifested during morphogenesis. Particles synthesized at permissive temperature were indistinguishable from wild-type particles in their ability to recognize host cells and deliver DNA. One mutation conferred an atypical ts synthesis phenotype. Although the mutant protein was efficiently incorporated into virus-like particles at elevated temperature, the progeny appeared to be kinetically trapped in a temperature-independent, uninfectious state. Thus, substitutions in the N terminus can lead to H protein misincorporation, albeit at wild-type levels, and subsequently affect particle function. All mutants exhibited recessive phenotypes, i.e., rescued by the presence of the wild-type H protein. Thus, mixed H protein oligomers are functional during DNA delivery. Recessive and dominant phenotypes may temporally approximate H protein functions, occurring before or after oligomerization has gone to completion.

Section: Discussionmentioning

confidence: 99%

Mutations in the N Terminus of the øX174 DNA Pilot Protein H Confer Defects in both Assembly and Host Cell Attachment

Young

Hockenberry

Fane

2014

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“…The first resistant strain isolated contained three mutations in the coat protein F (F D44H , F D205N , and F S227P ), which were identical or similar to resistance mutations isolated in single-step selections. In general, all of these mutations cluster underneath the D 3 subunit in the X-ray structure (12,16). There was also a mutation in the DNA pilot protein H (H D136G ).…”

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confidence: 99%

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

Cherwa

Tyson

Bedwell

et al. 2017

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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).

“…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.…”

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confidence: 99%

“…All amino acid substitutions for G61 inhibit the ability to undergo the requisite conformational switch. The severity of the conferred dominant lethal phenotypes directly correlates with the side chain sizes of the substituted amino acids (4,5).…”

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confidence: 99%

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

Cherwa

Sanchez-Soria

Wichman

et al. 2009

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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 (6,16,17,25) and can develop a dependency on the antiviral agent (1, 19). However, the molecular mechanism of dependency rarely, if ever, involves the productive use of the antiviral agent to elevate fitness above the uninhibited wild-type level. Many studies are conducted with animal viruses, often in clinical settings, which can impose restraints on the experimental durations. Thus, prolonged exposure to antiviral agents may be required for the emergence of a multiply mutant strain that has evolved mechanisms to productively utilize inhibitors.Due to its rapid replication, bacteriophage X174 has become an attractive model system for evolutionary studies (2,3,23,24). Selective pressures can be applied for hundreds of infection cycles in a relatively short period of time. Using the atomic structure of assembly intermediates as a guide (8, 9, 15), viral scaffolding proteins that inhibit virion assembly have been designed (4). The molecular mechanism of inhibition was characterized, and resistance mutants were isolated via onestep genetic selections (4). In this study we report the isolation of a more robust resistant mutant. The quintuple mutant was generated by propagating X174 for approximately 150 life cycles in the presence of increasing concentrations of the inhibitory protein, which was derived from the external scaffolding D protein. This protein forms asymmetric dimers that direct procapsid assembly. A...