Isolation and characterization of the herpes simplex virus 1 terminase complex

Area of Science:

• Virology
• Molecular Biology
• Protein Biochemistry

Background:

• Herpes simplex virus 1 (HSV-1) infection involves procapsid assembly and genome packaging by the terminase complex.
• The terminase complex consists of HSV-1 UL15, UL28, and UL33 proteins.
• Previous studies faced challenges in purifying the intact terminase complex for biochemical analysis.

Purpose of the Study:

• To isolate and characterize the intact HSV-1 terminase complex.
• To investigate the roles of specific protein domains in terminase function.
• To elucidate the assembly and functional requirements of the viral terminase.

Main Methods:

• Tandem-affinity purification (TAP) of terminase complexes using recombinant viruses expressing NTAP-UL28 fusion proteins.
• Mass spectrometry, Western blotting, and silver staining for protein identification.
• Sucrose density gradient ultracentrifugation to determine complex oligomeric state.
• Generation and analysis of recombinant viruses with mutations in conserved UL15 and UL28 domains.

Main Results:

• The intact UL15-UL28-UL33 heterotrimer was successfully isolated and identified.
• The C terminus of UL28 is crucial for interaction with UL15 and UL33.
• Conserved residues in the UL28 metal-binding domain and UL15 nuclease domain are vital for cleavage and packaging functions.
• These conserved domains are not required for the initial assembly of the terminase complex.

Conclusions:

• The study successfully purified and characterized the HSV-1 terminase complex, revealing its heterotrimeric structure.
• Specific conserved domains within UL15 and UL28 are critical for the catalytic functions of DNA cleavage and packaging.
• Terminase complex assembly is independent of the functional domains required for cleavage and packaging, suggesting distinct regulatory mechanisms.