The viral early-to-late switch of papillomavirus infection is tightly linked to keratinocyte differentiation and is mediated in part by alternative mRNA splicing. Here, we report that SRp20, a cellular splicing factor, controls the early-to-late switch via interactions with A/C-rich RNA elements. An A/C-rich SE4 element regulates the selection of a bovine papillomavirus type 1 (BPV-1) late-specific splice site, and binding of SRp20 to SE4 suppresses this selection. Expression of late BPV-1 L1 or human papillomavirus (HPV) L1, the major capsid protein, inversely correlates with SRp20 levels in the terminally differentiated keratinocytes. In HPV type 16, a similar SRp20-interacting element also controls the viral early-to-late switch. Keratinocytes in raft cultures, which support L1 expression, make considerably less SRp20 than keratinocytes in monolayer cultures, which do not support L1 expression. Conversely, abundant SRp20 in cancer cells or undifferentiated keratinocytes is important for the expression of the viral early E6 and E7 by promoting the expression of cellular transcription factor SP1 for transactivation of viral early promoters.Papillomaviruses are small DNA tumor viruses that infect cutaneous or mucosal epithelial cells and cause benign tumors and sometimes malignant neoplasms, including cervical cancer in women (36). Papillomavirus infections are transmitted mainly by close skin-to-skin or mucosa-to-mucosa contact. Infecting viral particles reach the keratinocytes in the basal layer of the squamous epithelium via microwounds that expose the basal keratinocytes to incoming virus. After infection of the basal keratinocytes, viral-gene expression and replication proceed in a tightly controlled fashion regulated by keratinocyte differentiation (25, 34).Although we do not fully understand how keratinocyte differentiation regulates papillomavirus gene expression and virus production, different parts of the viral life cycle occur at different stages of keratinocyte differentiation. The early stage of virus infection takes place in undifferentiated or intermediately differentiated keratinocytes in basal or parabasal layers; at this stage, the viral early genes (E1, E2, E5, E6, and E7) are expressed from the early region of the viral genome and encode all five viral regulatory nonstructural proteins. In contrast, the expression of two structural viral capsid proteins (L1 and L2) from the late region of the virus genome at the late stage of viral infection occurs only in keratinocytes undergoing terminal differentiation in the granular and cornified layers of the epithelium (34, 41). Although the early-to-late switch of viral-gene expression involves a switch in the use of viral promoters during the viral life cycle (21, 48, 49), strict regulation of viral-RNA processing, including alternative RNA splicing and polyadenylation, is absolutely necessary for expression of the viral genes at the appropriate times (42, 57).Alternative RNA splicing and polyadenylation occur during RNA processing in most eukaryotic...