title: Isolation and characterization of the herpes simplex virus 1 terminase complex.
abstract: During herpes simplex virus 1 (HSV-1) infection, empty procapsids are assembled and subsequently filled with the viral genome by means of a protein complex called the terminase, which is comprised of the HSV-1 UL15, UL28, and UL33 proteins. Biochemical studies of the terminase proteins have been hampered by the inability to purify the intact terminase complex. In this study, terminase complexes were isolated by tandem-affinity purification (TAP) using recombinant viruses expressing either a full-length NTAP-UL28 fusion protein (vFH476) or a C-terminally truncated NTAP-UL28 fusion protein (vFH499). TAP of the UL28 protein from vFH476-infected cells, followed by silver staining, Western blotting, and mass spectrometry, identified the UL15, UL28, and UL33 subunits, while TAP of vFH499-infected cells confirmed previous findings that the C terminus of UL28 is required for UL28 interaction with UL33 and UL15. Analysis of the oligomeric state of the purified complexes by sucrose density gradient ultracentrifugation revealed that the three proteins formed a complex with a molecular mass that is consistent with the formation of a UL15-UL28-UL33 heterotrimer. In order to assess the importance of conserved regions of the UL15 and UL28 proteins, recombinant NTAP-UL28 viruses with mutations of the putative UL28 metal-binding domain or within the UL15 nuclease domain were generated. TAP of UL28 complexes from cells infected with each domain mutant demonstrated that the conserved cysteine residues of the putative UL28 metal-binding domain and conserved amino acids within the UL15 nuclease domain are required for the cleavage and packaging functions of the viral terminase, but not for terminase complex assembly.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Heming
authors.forename: Jamie B
authors.initials: JB
authors.lastname: Huffman
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Jones
authors.forename: Fred L
authors.initials: FL
authors.lastname: Homa
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D004279:DNA, Viral / Q000737:chemistry; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification*; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D013058:Mass Spectrometry; D009154:Mutation; D014709:Vero Cells
chemical_list: D004279:DNA, Viral; D004706:Endodeoxyribonucleases; C030894:terminase