In the X174 procapsid crystal structure, 240 external scaffolding protein D subunits form 60 pairs of asymmetric dimers, D 1 D 2 and D 3 D 4 , in a non-quasi-equivalent structure. To achieve this arrangement, ␣-helix 3 assumes two different conformations: (i) kinked 30°at glycine residue 61 in subunits D 1 and D 3 and (ii) straight in subunits D 2 and D 4 . Substitutions for G61 may inhibit viral assembly by preventing the protein from achieving its fully kinked conformation while still allowing it to interact with other scaffolding and structural proteins. Mutations designed to inhibit conformational switching in ␣-helix 3 were introduced into a cloned gene, and expression was demonstrated to inhibit wild-type morphogenesis. The severity of inhibition appears to be related to the size of the substituted amino acid. For infections in which only the mutant protein is present, morphogenesis does not proceed past the first step that requires the wild-type external scaffolding protein. Thus, mutant subunits alone appear to have little or no morphogenetic function. In contrast, assembly in the presence of wild-type and mutant subunits is blocked prematurely, before D protein is required in a wild-type infection, or channeled into an off-pathway reaction. These data suggest that the wild-type protein transports the inhibitory protein to the pathway. Viruses resistant to the lethal dominant proteins were isolated, and mutations were mapped to the coat and internal scaffolding proteins. The affected amino acids cluster in the atomic structure and may act to exclude mutant subunits from occupying particular positions atop pentamers of the viral coat protein.
Members of the Microviridae comprise two subfamilies. The microviruses (Greek for small), which infect free‐living bacteria, are among the fastest known replicating viruses. Gokushoviruses (Japanese for very small) occupy a unique niche, infecting obligate intracellular bacteria, such as Chlamydia and Bdellovibrio , or mollicutes, bacteria without a cell wall. All members of the family contain small (4000–6000 bases), circular, single‐stranded deoxyribonucleic acid (ssDNA) genomes of positive polarity, which are packaged inside small (∼25 nm diameter) T=1 icosahedral capsids. The other icosahedral, ssDNA virus families: Parvoviridae , Circoviridae , Nanoviridae and Geminiviridae ; share most of these properties, suggesting a large super family spanning several domains of life. The most well known member of the Microviridae , ϕX174, has been extensively used to study the fundamental mechanisms of DNA replication and capsid assembly. The latter is uniquely dependent on two scaffolding proteins, and has become a model system for experimental evolution. Key Concepts: Whilst overlapping reading frames increase the amount of genetic information encoded in small genomes, they do not appear to significantly impact the ability of the virus to genetically adapt to selective pressures. Due to the genome's positive polarity, DNA replication must commence before viral genes can be transcribed. Microvirus DNA replication occurs in three distinct stages: (1) ssDNA is first converted to a double‐stranded molecule, (2) amplification of the double‐stranded molecule, (3) single‐stranded genomic DNA synthesis and packaging. Genomic DNA synthesis and packaging are concurrent processes; thus, a genome is not synthesised unless there exists a capsid in which to package it. Gene expression is controlled by the finely tuned interplay of cis ‐acting genetic elements: promoters, ribosome binding sites, mRNA stability sequences and transcription terminators. Microviruses are distinguished by their two scaffolding protein system, whereas Gokushoviruses utilise a single scaffolding protein. Capsid assembly is mediated by scaffolding proteins, which induce conformational switches in the viral coat protein to control the timing and fidelity of morphogenesis. Cell lysis is achieved by inhibiting host cell wall biosynthesis, a mechanism reminiscent of some antibiotics.
The X174 DNA pilot protein H contains four predicted C-terminal coiled-coil domains. The region of the gene encoding these structures was cloned, expressed in vivo, and found to strongly inhibit wild-type replication. DNA and protein synthesis was investigated in the absence of de novo H protein synthesis and in wild-type-infected cells expressing the inhibitory proteins (⌬H). The expression of the ⌬H proteins interfered with early stages of DNA replication, which did not require de novo H protein synthesis, suggesting that the inhibitory proteins interfere with the wild-type H protein that enters the cell with the penetrating DNA. As transcription and protein synthesis are dependent on DNA replication in positive single-stranded DNA life cycles, viral protein synthesis was also reduced. However, unlike DNA synthesis, efficient viral protein synthesis required de novo H protein synthesis, a novel function for this protein. A single amino acid change in the C terminus of protein H was both necessary and sufficient to confer resistance to the inhibitory ⌬H proteins, restoring both DNA and protein synthesis to wild-type levels. ⌬H proteins derived from the resistant mutant did not inhibit wild-type or resistant mutant replication. The inhibitory effects of the ⌬H proteins were lessened by the coexpression of the internal scaffolding protein, which may suppress H-H protein interactions. While coexpression relieved the block in DNA biosynthesis, viral protein synthesis remained suppressed. These data indicate that protein H's role in DNA replication and stimulating viral protein synthesis can be uncoupled.
In the X174 procapsid, 240 external scaffolding proteins form a nonquasiequivalent lattice. To achieve this arrangement, the four structurally unique subunits must undergo position-dependent conformational switches. One switch is mediated by glycine residue 61, which allows a 30°kink to form in ␣-helix 3 in two subunits, whereas the helix is straight in the other two subunits. No other amino acid should be able to produce a bend of this magnitude. Accordingly, all substitutions for G61 are nonviable but mutant proteins differ vis-à-vis recessive and dominant phenotypes. As previously reported, amino acid substitutions with side chains larger than valine confer dominant lethal phenotypes. Alone, these mutant proteins appear to have little or no biological activity but rather require the wild-type protein to interact with other structural proteins. Proteins with conservative substitutions for G61, serine and alanine, have now been characterized. Unlike the dominant lethal proteins, these proteins do not require wild-type subunits to interact with other viral proteins and cause assembly defects reminiscent of those conferred by the lethal dominant proteins in concert with wild-type subunits. Although atomic structures suggest that only a glycine residue can provide the proper torsion angle for assembly, mutants that can productively utilize the altered external scaffolding proteins were isolated, and the mutations were mapped to the coat and internal scaffolding proteins. Thus, the ability to isolate strains that could utilize the single mutant D protein species would not have been predicted from past structural analyses.Proper virion assembly requires a series of precise proteinprotein interactions that proceed along an ordered morphogenetic pathway. During Tϭ1 Microvirus assembly (canonical species: X174, G4, and ␣3), early intermediates are directed into larger macromolecular structures by a class of transiently associated proteins called scaffolding proteins. These proteins mediate the conformational switching of structural proteins, assist in lowering the nucleation barrier for assembly, and ensure morphogenetic fidelity (10). While many large DNA viruses rely on a single internal scaffolding protein, the small microviruses and the satellite P4-like viruses require both internal and external scaffolding proteins (7,11,20). The atomic structures of the X174 virion, procapsid, and assembly naïve external scaffolding protein have been determined by crystallography (5, 6, 14-16). Thus, biochemical and genetic data can be interpreted within a defined structural context.The morphogenetic roles of the X174 internal and external scaffolding proteins are illustrated in Fig. 1A. The first identifiable assembly intermediates are pentamers of the viral coat F and major spike G proteins; the respective 9S and 6S particles, which form independently of both scaffolding proteins (21). Five internal scaffolding B proteins bind to the underside of the 9S particle, yielding the 9S* intermediate (3). This interaction also induces...
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...
Defective øX174 H protein-mediated DNA piloting indirectly influences the entire viral lifecycle. Faulty piloting can mask the H protein's other functions or inefficient penetration may be used to explain defects in post-piloting phenomena. For example, optimal synthesis of other viral proteins requires de novo H protein biosynthesis. As low protein concentrations affect morphogenesis, protein H's assembly functions remain obscure. An H protein mutant was isolated that allowed morphogenetic effects to be characterized independent of its other functions. The mutant protein aggregates assembly intermediates. Although excess internal scaffolding protein restores capsid assembly, the resulting mutant H protein-containing particles are less infectious. In addition, nonviable phenotypes of am(H) mutants in Su+ hosts, which insert non-wild-type amino acids, do not always correlate with a lack of missense protein function. Phenotypes are highly influenced by host and phage physiology. This phenomenon was unique to am(H) mutants, not observed with amber mutants in other genes.
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...
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