Papovaviruses are valuable models for the study of DNA replication in higher eukaryotic organisms, as they depend on host factors for replication of their DNA. In this study we investigate the interactions between the human papillomavirus type 11 (HPV-11) origin recognition and initiator protein E1 and human polymerase ␣/primase (pol ␣/primase) subunits. By using a variety of physical assays, we show that both 180-(p180) and 70-kDa (p70) subunits of pol ␣/primase interact with HPV-11 E1. The interactions of E1 with p180 and p70 are functionally different in cell-free replication of an HPV-11 origin-containing plasmid. Exogenously added p180 inhibits both E2-dependent and E2-independent cell-free replication of HPV-11, whereas p70 inhibits E2-dependent but stimulates E2-independent replication. Our experiments indicate that p70 does not physically interact with E2 and suggest that it may compete with E2 for binding to E1. A model of how E2 and p70 sequentially interact with E1 during initiation of viral DNA replication is proposed.Papillomaviruses are members of the small DNA tumor virus family. They cause papillomas in a wide variety of hosts and certain high risk human papillomavirus types are strongly linked to the development of cervical or penile cancer in humans (1). The mode of viral replication is closely coupled to the differentiation status of the infected squamous epithelium (for review see Ref.2). In the basal and parabasal cells of the squamous epithelium, the virus is maintained as a low copy number extra-chromosomal episome and undergoes regulated DNA replication modulated by both viral and host proteins. As cells undergo progressive differentiation, vegetative viral replication is triggered, "late" viral genes are expressed, and progeny virions are produced in a fraction of the terminally differentiated cells in papillomas or condylomas. The latent stage of papillomaviral replication provides an ideal system for the study of regulated eukaryotic DNA replication.We and others (3, 4) have previously reported a cell-free replication system for bovine papillomavirus type 1 (BPV-1) 1 (3) and human papillomavirus type 11 (HPV-11) (4). Papillomaviral replication requires the viral proteins E1 and E2 (5, 6), as well as the full complement of host replication proteins that have previously been identified in SV40 in vitro replication, including DNA polymerases ␣ and ␦ (7, 8). It is therefore probable that physical interactions between the host initiation enzymes and the papillomaviral initiation proteins E1 or E2 occur during initiation of viral DNA replication.The papillomaviral E1 protein is a functional homolog of SV40 large T antigen, with origin binding activity as well as ATPase and helicase activity (9 -15). It associates as a trimer or a hexamer on its cognate E1-binding site in the viral origin (ori) with relatively low affinity and low sequence specificity (3, 4, 16 -23). As a result, high concentrations of E1 can bind DNA nonspecifically and initiate ori-independent replication at low efficiency ...

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