Labeling and localization of the herpes simplex virus capsid protein UL25 and its interaction with the two triplexes closest to the penton.
The herpes simplex virus type 1 UL25 protein is one of seven viral proteins that are required for DNA cleavage and packaging. Together with UL17, UL25 forms part of an elongated molecule referred to as the C-capsid-specific component (CCSC). Five copies of the CCSC are located at each of the capsid vertices on DNA-containing capsids. To study the conformation of UL25 as it is folded on the capsid surface, we identified the sequence recognized by a UL25-specific monoclonal antibody and localized the epitope on the capsid surface by immunogold electron microscopy. The epitope mapped to amino acids 99-111 adjacent to the region of the protein (amino acids 1-50) that is required for capsid binding. In addition, cryo-EM reconstructions of C-capsids in which the green fluorescent protein (GFP) was fused within the N-terminus of UL25 localized the point of contact between UL25 and GFP. The result confirmed the modeled location of the UL25 protein in the CCSC density as the region that is distal to the penton with the N-terminus of UL25 making contact with the triplex one removed from the penton. Immunofluorescence experiments at early times during infection demonstrated that UL25-GFP was present on capsids located within the cytoplasm and adjacent to the nucleus. These results support the view that UL25 is present on incoming capsids with the capsid-binding domain of UL25 located on the surface of the mature DNA-containing capsid.
Conway
James F
JF
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Cockrell
Shelley K
SK
Copeland
Anna Maria
AM
Newcomb
William W
WW
Brown
Jay C
JC
Homa
Fred L
FL
D000911:Antibodies, Monoclonal / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins / Q000032:analysis* / Q000737:chemistry*; D020285:Cryoelectron Microscopy; D018604:Epitope Mapping; D018259:Herpesvirus 1, Human / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D017510:Protein Folding
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D036022:Capsid Proteins