Recruitment of polymerase to herpes simplex virus type 1 replication foci in cells expressing mutant primase (UL52) proteins.

The ordered assembly of the herpes simplex virus (HSV) type 1 replication apparatus leading to replication compartments likely involves the initial assembly of five viral replication proteins, ICP8, UL9, and the heterotrimeric helicase-primase complex (UL5-UL8-UL52), into replication foci. The polymerase and polymerase accessory protein are subsequently recruited to these foci. Four stages of viral infection (stages I to IV) have been described previously (J. Burkham, D. M. Coen, and S. K. Weller, J. Virol. 72:10100-10107, 1998). Of these, stage III foci are equivalent to the previously described promyelocytic leukemia protein (PML)-associated prereplicative sites and contain all seven replication proteins. We constructed a series of mutations in the putative primase subunit, UL52, of the helicase-primase and have analyzed the mutant proteins for their abilities to form intermediates leading to the formation of replication compartments. The results shown in this paper are consistent with the model that the five proteins, ICP8, UL5, UL8, UL9, and UL52, form a scaffold and that formation of this scaffold does not rely on enzymatic functions of the helicase and primase. Furthermore, we demonstrate that recruitment of polymerase to this scaffold requires the presence of an active primase subunit. These results suggest that polymerase recruitment to replication foci requires primer synthesis. Furthermore, they support the existence of two types of stage III intermediates in the formation of replication compartments: stage IIIa foci, which form the scaffold, and stage IIIb foci, which contain, in addition, HSV polymerase, the polymerase accessory subunit, and cellular factors such as PML.