The life cycle of human papillomavirus type 16 (HPV16) is intimately linked to differentiation of the epithelium it infects, and late events in the life cycle are restricted to the suprabasal layers. Here we have used 5′RACE of polyadenylated RNA isolated from differentiated W12 cells (cervical epithelial cells containing episomal copies of the HPV16 genome) that express virus late proteins to map virus late mRNAs. Thirteen different transcripts were identified. Extensive alternative splicing and use of two late polyadenylation sites were noted. A novel promoter located in the long control region was detected as well as P97 and Plate. Promoters in the E4 and E5 open reading frames were active yielding transcripts where L1 or L2 respectively are the first open reading frames. Finally, mRNAs that could encode novel proteins E6*^*E7, E6*^E4, E1^*E4 and E1^E2C (putative repressor E2) were identified, indicating that HPV16 may encode more late proteins than previously accepted.

Pre-mRNA splicing occurs in the spliceosome, which is composed of small ribonucleoprotein particles (snRNPs) and many non-snRNP components. SR proteins, so called because of their C-terminal arginine-and serine-rich domains (RS domains), are essential members of this class. Recruitment of snRNPs to 5 and 3 splice sites is mediated and promoted by SR proteins. SR proteins also bridge splicing factors across exons to help to define these units and have a central role in alternative and enhancer-dependent splicing. Here, we show that the SR protein SF2/ASF is part of a complex that forms upon the 79-nucleotide negative regulatory element (NRE) that is thought to be pivotal in posttranscriptional regulation of late gene expression in human papillomavirus type 16 (HPV-16). However, the NRE does not contain any active splice sites, is located in the viral late 3 untranslated region, and regulates RNA-processing events other than splicing. The level of expression and extent of phosphorylation of SF2/ASF are upregulated with epithelial differentiation, as is subcellular distribution, specifically in HPV-16-infected epithelial cells, and expression levels are controlled, at least in part, by the virus transcription regulator E2.Human papillomaviruses (HPVs) are a family of epitheliotropic viruses that infect both cutaneous and mucosal epithelia. HPV infection most commonly results in benign papillomas or warts; however, on rare occasions, malignant lesions can develop following infection with a high-risk HPV type and integration of the virus genome into the host genome (18). HPV-16 is the most significant member of this high-risk subgroup, being associated with approximately 60% of cervical carcinoma cases worldwide (52).Transcription of the 8.0-kb virus genome generates a number of transcripts as a result of a complex program of alternative splicing and polyadenylation (44). Viral mRNAs are translated to yield six early proteins, expressed throughout the virus life cycle (primarily involved in episomal maintenance of the genome, transcriptional regulation, and cell transformation) (52) and two late proteins, the capsid proteins L1 and L2. Expression of the capsid proteins is restricted to cells undergoing terminal differentiation in the uppermost layers of the stratified epithelium (31) but because late transcripts are expressed in less-differentiated epithelial cells (43), control of late-gene expression is largely attributed to posttranscriptional mechanisms. cis-acting inhibitory elements identified within the late region of several papillomaviruses (40) appear to regulate gene expression via distinct RNA-based mechanisms. However, the overall effect is the same in that each element probably acts in a position-and orientation-dependent manner through interactions with cellular factors (40) to reduce the levels of polyadenylated viral late mRNAs in the cytoplasm of undifferentiated cells. The best-understood example is bovine papillomavirus type 1 (BPV-1), in which regulation of lategene expression by a short negative...

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the squamous epithelia that it infects. Capsid proteins, and hence mature virions, are produced in the outermost layer of differentiated cells. As late gene transcripts are produced in the lower layers, posttranscriptional mechanisms likely prevent capsid protein production in less differentiated cells. For HPV type 16 (HPV-16), a 79-nucleotide (nt) negative regulatory element (NRE) inhibits gene expression in basal epithelial cells. To identify key NRE sequences, we carried out transient transfection in basal epithelial cells with reporter constructs containing the HPV-16 late 3 untranslated region with deletions and mutations of the NRE. Reporter gene expression was increased over 40-fold by deletion of the entire element, 10-fold by deletion of the 5 portion of the NRE that contains four weak consensus 5 splice sites, and only 3-fold by deletion of the 3 GU-rich region. Both portions of the element appear to be necessary for full repression. Inactivating mutations in the 5 splice sites in the 5 NRE partially alleviated repression in the context of the 79-nt NRE but caused full derepression when assayed in a construct with the 3 NRE deleted. All four contribute to the inhibitory effect, though the second splice site is most inhibitory. Sm proteins, U1A and U1 snRNA, but not U1 70K, could be affinity purified with the wild-type NRE but not with the NRE containing mutations in the 5 splice sites, indicating that a U1 snRNP-like complex forms upon the element.Human papillomaviruses (HPVs) are small DNA viruses that specifically infect squamous epithelial cells, giving rise to warts or papillomas (16). They may be divided into mucosal or cutaneous types and also into high-or low-risk types, depending on the probability of the lesions that they cause becoming malignant. High-risk mucosal types, which include HPV type 16 (HPV-16), HPV-18, HPV-31, HPV-33, and HPV-45, may give rise to cervical intraepithelial neoplasias and cervical carcinomas. HPV-16 is the most clinically significant of these, being found in over 50% of such cervical lesions (36).The early genes of the 8-kb HPV-16 genome encode proteins involved in the regulation of DNA replication, episomal maintenance of the genome, and control of host cell division. The late region encodes the major and minor capsid proteins, L1 and L2. The noncoding region contains the late gene 3Ј untranslated region (late 3Ј UTR) at its 5Ј end and regulatory sequences controlling activity of the viral promoter toward its 3Ј end (Fig. 1A). The major promoter in HPV-16, P 97 , is constitutively active from early in the viral life cycle (25). A second promoter, P 670 , also becomes active in differentiated cells (12). The genome consists of a single transcription unit, and alternative splicing generates individual mRNAs (7,17,23). Transcripts may be polyadenylated at the early polyadenylation [poly(A)] site; alternatively one of the tandem late poly(A) sites downstream of the L1 coding region may be...

HPV-16 (human papillomavirus type 16) is a small dsDNA (double-stranded DNA) virus which infects mucosal epithelial tissue of the cervix. Epithelial tissue is composed of a basal layer of cells, capable of division, and a number of suprabasal layers, wherein the cells become more differentiated the closer to the surface of the epithelium they become. Expression of viral proteins is dependent upon epithelial differentiation status, and, within the HPV-16 genome, several elements have been found which control expression both transcriptionally and post-transcriptionally. Expression of the highly immunogenic capsid proteins, L1 and L2, is restricted to only the most differentiated cells, where immune surveillance is limited. However, L1 and L2 transcripts can be detected in less differentiated cells, suggesting post-transcriptional mechanisms exist to prevent their expression in these cells. Indeed, a number of cis-acting RNA elements have been observed within the HPV-16 late region which may be involved in control of capsid gene expression. Mechanisms controlling HPV-16 capsid gene expression and the cellular RNA-processing factors involved will be the focus of this article.

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