Uncoupling the functions of a multifunctional protein: The isolation of a DNA pilot protein mutant that affects particle morphogenesis

2014

Self Cite

. 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.

mentioning

confidence: 99%

“…The clone containing the three A or I¡D substitutions at residues 6A, 9I, and 12A within the putative transmembrane domain was generated as previously described (10), with the exception that a mutagenic 5= primer was used to amplify the gene by PCR. This primer also introduces an NcoI site, which contains an ATG start codon.…”

mentioning

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

2014

Self Cite

“…During early morphogenesis, five copies of the internal scaffolding protein B bind to the underside of the 9S coat protein F pentamer forming the 9S* particle (8). This induces the conformational changes that (i) inhibit 9S particle aggregation, (ii) facilitate DNA pilot protein H incorporation, and (iii) stimulate coat-spike protein interactions (8)(9)(10)(11)(12). During late morphogenesis, 240 copies of the external scaffolding protein D organize 12 12S* particles into procapsids (13).…”

mentioning

confidence: 99%

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

Gordon

2013

Self Cite

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.

“…H protein is involved in multiple aspects of the viral life cycle, from host attachment to intracellular assembly. Previously characterized mutants exhibited both attachment and assembly defects (27,28). Therefore, all aspects of the X174 H ϩ 14 life cycle were analyzed.…”

Section: Resultsmentioning

confidence: 99%

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

Roznowski

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...