High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7 oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105Rb and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.Human papillomavirus (HPV) infection of epidermal or mucosal epithelial cells causes benign and sometimes malignant neoplasms. Certain HPVs, such as HPV16, -18, -31, and -45, are detected frequently in anogenital cancers, particularly cancer of the cervix and anus, and are thus considered to be high risk or oncogenic (22,42). Among the high-risk HPVs, HPV16 and -18 cause Ͼ90% of cervical cancers (22) and Ͼ20% of oral cancers (35). High-risk HPVs encode two potent viral oncoproteins, E6 and E7, that mediate, respectively, degradation of the cellular proteins p53 and retinoblastoma protein (pRb), two tumor suppressor proteins that are essential for cell cycle control (21).In both HPV16 and HPV18, E6 and E7 are transcribed as a single bicistronic E6E7 transcript using a common promoter and a common early polyadenylation site (1). Promoter p97, upstream of the HPV16 E6 (16E6) open reading frame (ORF), and promoter p105, upstream of the HPV18 E6 (18E6) ORF, are responsible for the initiation of transcription in each virus genome. The bicistronic E6E7 pre-mRNAs of HPV16 and HPV18 contain three exons and two introns. One of the two introns, intron 1 (the cap-proximal intron), is positioned in the E6 ORF in both the HPV16 and HPV18 E6E7 pre-mRNAs ( Fig. 1A and B). Conceivably, intron 1 removal by RNA splicing would disrupt the E6 ORF and prevent full-length E6 from...

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...

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