The Expression of N-Terminal Deletion DNA Pilot Proteins Inhibits the Early Stages of φX174 Replication

2013

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C-terminal, aromatic amino acids in the X174 internal scaffolding protein B mediate conformational switches in the viral coat protein. These switches direct the coat protein through early assembly. In addition to the aromatic amino acids, two acidic residues, D111 and E113, form salt bridges with basic, coat protein side chains. Although salt bridge formation did not appear to be critical for assembly, the substitution of an aromatic amino acid for D111 produced a lethal phenotype. This side chain is uniquely oriented toward the center of the coat-scaffolding binding pocket, which is heavily dominated by aromatic ring-ring interactions. Thus, the D111Y substitution may restructure pocket contacts. Previously characterized B ؊ mutants blocked assembly before procapsid formation. However, the D111Y mutant produced an assembled particle, which contained the structural and external scaffolding proteins but lacked protein B and DNA. A suppressor within the external scaffolding protein, which mediates the later stages of particle morphogenesis, restored viability. The unique formation of a postprocapsid particle and the novel suppressor may be indicative of a novel B protein function. However, genetic data suggest that the particle represents the delayed manifestation of an early assembly error. This seemingly late-acting defect was rescued by previously characterized suppressors of early, preprocapsid, B ؊ assembly mutations, which act on the level of coat protein flexibility. Likewise, the newly isolated suppressor in the external scaffolding protein also exhibited a global suppressing phenotype. Thus, the off-pathway product isolated from infected cells may not accurately reflect the temporal nature of the initial defect.

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confidence: 99%

Effects of an Early Conformational Switch Defect during ϕX174 Morphogenesis Are Belatedly Manifested Late in the Assembly Pathway

Gordon

2013

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“…According to the current model, five B proteins bind to the underside of the 9S coat protein pentamer, forming the 9S* particle (6). B-protein binding most likely induces conformational changes that (i) inhibit 9S particle aggregation, (ii) facilitate DNA pilot protein H incorporation, and (iii) stimulate coat-6S spike protein interactions (4,6,7,32,34). The joining of the 6S complex to the top of the 9S* intermediate yields the 12S* particle.…”

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confidence: 99%

Conformational Switch-Defective ϕX174 Internal Scaffolding Proteins Kinetically Trap Assembly Intermediates before Procapsid Formation

Gordon

Knuff

2012

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Conformational switching is an overarching paradigm in which to describe scaffolding protein-mediated virus assembly. However, rapid morphogenesis with small assembly subunits hinders the isolation of early morphogenetic intermediates in most model systems. Consequently, conformational switches are often defined by comparing the structures of virions, procapsids and aberrantly assembled particles. In contrast, X174 morphogenesis proceeds through at least three preprocapsid intermediates, which can be biochemically isolated. This affords a detailed analysis of early morphogenesis and internal scaffolding protein function. Amino acid substitutions were generated for the six C-terminal, aromatic amino acids that mediate most coat-internal scaffolding protein contacts. The biochemical characterization of mutant assembly pathways revealed two classes of molecular defects, protein binding and conformational switching, a novel phenotype. The conformational switch mutations kinetically trapped assembly intermediates before procapsid formation. Although mutations trapped different particles, they shared common second-site suppressors located in the viral coat protein. This suggests a fluid assembly pathway, one in which the scaffolding protein induces a single, coat protein conformational switch and not a series of sequential reactions. In this model, an incomplete or improper switch would kinetically trap intermediates.

“…In addition to the aforementioned functions, de novo H protein synthesis is required for the efficient production of other viral proteins (16). To determine if the ⌬7, ⌬11, and ⌬14 H proteins retained the last function, lysis-resistant cells expressing the ⌬H gene constructs were infected with an am(H) mutant.…”

Section: Resultsmentioning

confidence: 99%

“…As discussed above, both conformations can be perturbed by deleting a hendecad or heptad. It was previously demonstrated that efficient viral coat protein production requires de novo H protein synthesis (16), indicating that H plays another intracellular role in the viral life cycle independent of assembly. Thus, it is likely that H has a third conformation.…”

Section: Discussionmentioning

confidence: 99%

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

Roznowski

2016

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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...