Mutagenic Analysis of a DNA Translocating Tube's Interior Surface

Area of Science:

• Microbiology
• Structural Biology
• Virology

Background:

• Bacteriophage ϕX174 employs a DNA piloting protein tube for host cell penetration.
• The tube's inner surface features specific amino acid residues, with a conserved C-terminal region crucial for genome delivery.

Purpose of the Study:

• To investigate the role of conserved residues in the ϕX174 DNA piloting tube's C-terminus during genome delivery.
• To elucidate the mechanism by which the viral genome is released into the host cell.

Main Methods:

• Site-directed mutagenesis of non-glutamine and glutamine residues in the conserved C-terminal region of the DNA piloting tube.
• Phenotypic analysis of mutant phages, including assessment of virion assembly, host attachment, eclipse, and genome delivery.
• Temperature-dependent assays to evaluate mutant phenotypes.

Main Results:

• Mutations in the conserved C-terminal region resulted in temperature-dependent inhibition of genome delivery.
• Virion assembly, host attachment, and eclipse were unaffected by the mutations.
• The results suggest that inward-facing glutamine residues in this region play a critical role in controlling genome release.

Conclusions:

• A balance of forces, including genome pressure and frictional drag from tube residues, governs phage genome delivery.
• Conserved glutamine residues in the ϕX174 DNA piloting tube act as a braking mechanism, controlling the rate of genome release into the host.