The rolling circle . capsid complex as an intermediate in phi X DNA replication and viral assembly

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Single-stranded DNA (ssDNA) phage phi X replicates its genome through a double-stranded replicative form.
• Late in the phage life cycle, ssDNA synthesis is coupled with virion assembly.

Purpose of the Study:

• To purify and characterize the replication-assembly complex responsible for late-stage ssDNA synthesis and virion packaging in phage phi X.
• To elucidate the mechanism of coupled DNA synthesis and viral assembly.

Main Methods:

• Purification of the replication-assembly complex to homogeneity using sedimentation velocity and density centrifugation.
• Analysis of complex components via gel electrophoresis and electron microscopy.
• Hybridization of restriction fragments to determine DNA synthesis origin and direction.

Main Results:

• The complex consists of a rolling circle ssDNA with a single-stranded tail and an intact viral capsid.
• The phi X gene A nicking protein is associated with the complex, potentially covalently linked to the DNA.
• Electron microscopy visualized the rolling circle attached to the capsid at the DNA growing point.
• The gene A endonuclease activity was identified as the termination point for rolling circle synthesis.

Conclusions:

• The purified replication-assembly complex demonstrates coupled DNA synthesis and viral packaging.
• This coupling mechanism explains the shift in DNA production from double-stranded to single-stranded forms during the phage life cycle.
• Similar complexes were observed in other ssDNA phages (G4, St-1), indicating a conserved mechanism.