Labeling and Localization of the Herpes Simplex Virus Capsid Protein UL25 and Its Interaction with the Two Triplexes Closest to the Penton

Abstract

The herpes simplex virus type 1 (HSV-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 or 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.

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

“…The copy number of GFP-tagged proteins in each H-particle was measured as a proportion of the fluorescence intensity of the capsid protein, pUL25 (24,27,28). Acquisition of pUL25 during assembly is copy-controlled, and estimates of pUL25 copy number were previously obtained by cryo-EM (60 copies; 5 per vertex) and immunoblot (estimated within the range of 75 ± 15 to 82 ± 19 copies) (29)(30)(31). We refined the measurement of pUL25 by comparing the fluorescence of pUL25/GFP to rotavirus (RV) virus-like particles (VLPs), the latter of which invariantly contain 120 copies of GFP (Fig.…”

Three-Dimensional Localization of the α and β Subunits and of the II-III Loop in the Skeletal Muscle L-type Ca2+ Channel

“…However, our 3D reconstruction is incompatible with this model. First, our insertion of YFP, which, along with GFP, constitutes a commonly used method for 3D sequence localization (65,66), localizes the ␤ subunit beside Ca v 1.1. Furthermore, the core of the ␤ subunit fits very well when it is docked adjacent to Ca V 1.1 (Fig.…”

Selection of HSV capsids for envelopment involves interaction between capsid surface components pU L 31, pU L 17, and pU L 25

“…Because of its enrichment in C capsids, the U L 25/U L 17 complex was named the C capsid-specific complex or CCSC (19). The CCSC bridges pentameric vertices to the adjacent 20 planar faces on the capsid surface (10,19,20). One hypothesis to explain how C capsids are selected for envelopment is that the products of U L 25 and U L 17 bind more efficiently to the surface of type C capsids after DNA packaging is complete (19), and these capsids subsequently engage the NEC complex either directly or indirectly.…”