Bacteriophage phi 29 proteins required for in vitro DNA-gp3 packaging

Area of Science:

• Molecular Biology
• Virology
• Structural Biology

Background:

• Bacteriophage phi 29 assembly involves DNA-protein complex (DNA-gp3) packaging into proheads.
• This process is aided by the gene 16 product (gp16) and requires subsequent neck and tail protein assembly.

Purpose of the Study:

• To identify viral proteins essential for the DNA-gp3 encapsidation phase.
• To investigate the role of gene 16 product (gp16) in bacteriophage phi 29 DNA packaging.

Main Methods:

• Purification of biologically active proheads and DNA-gp3.
• Construction of a chimeric plasmid (pUM101) expressing only phi 29 gene 16.
• In vitro packaging assays using purified components and plasmid-expressed gp16.

Main Results:

• Plasmid-specified gp16 was necessary and sufficient for packaging 24% of added DNA-gp3 into purified proheads.
• DNA-filled heads were efficiently complemented to infectious phage with neck and tail proteins.
• A 4-fold dilution of gp16 caused a 1,000-fold reduction in phi 29 packaging, indicating a requirement for multiple gp16 copies.

Conclusions:

• The gene 16 product (gp16) is a key viral protein for bacteriophage phi 29 DNA packaging.
• Multiple copies of gp16 are likely required for efficient DNA-gp3 encapsidation.
• This study defines gp16's essential role in a critical step of viral assembly.