Abstract
The herpes simplex virus type 1 (HSV-1) portal complex is a ring-shaped structure located at a single vertex in the viral capsid. Composed of 12 U(L)6 protein molecules, the portal functions as a channel through which DNA passes as it enters the capsid. The studies described here were undertaken to clarify how the portal becomes incorporated as the capsid is assembled. We tested the idea that an intact portal may be donated to the growing capsid by way of a complex with the major scaffolding protein, U(L)26.5. Soluble U(L)26.5-portal complexes were found to assemble when purified portals were mixed in vitro with U(L)26.5. The complexes, called scaffold-portal particles, were stable during purification by agarose gel electrophoresis or sucrose density gradient ultracentrifugation. Examination of the scaffold-portal particles by electron microscopy showed that they resemble the 50- to 60-nm-diameter "scaffold particles" formed from purified U(L)26.5. They differed, however, in that intact portals were observed on the surface. Analysis of the protein composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that portals and U(L)26.5 combine in various proportions, with the highest observed U(L)6 content corresponding to two or three portals per scaffold particle. Association between the portal and U(L)26.5 was antagonized by WAY-150138, a small-molecule inhibitor of HSV-1 replication. Soluble scaffold-portal particles were found to function in an in vitro capsid assembly system that also contained the major capsid (VP5) and triplex (VP19C and VP23) proteins. Capsids that formed in this system had the structure and protein composition expected of mature HSV-1 capsids, including U(L)6, at a level corresponding to approximately 1 portal complex per capsid. The results support the view that U(L)6 becomes incorporated into nascent HSV-1 capsids by way of a complex with U(L)26.5 and suggest further that U(L)6 may be introduced into the growing capsid as an intact portal.