A splicing enhancer in the E4 coding region of human papillomavirus type 16 is required for early mRNA splicing and polyadenylation as well as inhibition of premature late gene expression

Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3' splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3' splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3' splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3' splice site at position 5639 in the late region. Optimization of the position 3358 3' splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3' splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression.     

Activation of human papillomavirus type 18 gene expression by herpes simplex virus type 1 viral transactivators and a phorbol ester.

Several viral trans-activators and a tumor promoter were examined for the ability to activate human papillomavirus type 18 (HPV-18) gene expression. A plasmid containing the HPV-18 noncoding region placed upstream of the chloramphenicol acetyltransferase reporter gene was cotransfected with different herpes simplex virus type 1 (HSV-1) genes into several cell lines. Both HSV-1 TIF and ICP0 activated HPV-18 expression; however, activation by TIF was observed only in epithelial cells, while ICP0 stimulated expression in a wide variety of cells. The element activated by both TIF and ICP0 was mapped to a 229-base-pair fragment which also contains an HPV-18 epithelial cell-preferred enhancer. The inclusion of a papillomavirus E2 trans-activator with TIF and ICP0 further increased HPV-18 expression. In contrast, the HSV-1 ICP4 and ICP27 genes, as well as the human T-cell lymphotropic virus type I and human immunodeficiency virus type 1 tat genes, were found to have no effect on HPV-18 expression. In transient assays, the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also activated HPV-18 expression. The region of HPV-18 activated by TPA was localized to a sequence which is homologous to other TPA-responsive elements.     

The cell-specific enhancer of the mouse transthyretin (prealbumin) gene binds a common factor at one site and a liver-specific factor(s) at two other sites.

We previously defined two distinct cell-specific DNA elements controlling the transient expression of the transthyretin gene in Hep G2 (human hepatoma) cells: a proximal promoter region (-202 base pairs [bp] to the cap site), and a far-upstream cell-specific enhancer located between 1.6 and 2.15 kilobases (kb) 5' of the cap site (R. H. Costa, E. Lai, and J. E. Darnell, Jr., Mol. Cell. Biol. 6:4697-4708, 1986). In this report, we located the effective transthyretin enhancer element within a 100-bp region between 1.96 and 1.86 kb 5' to the mRNA cap site. In Hep G2 nuclear extracts, three protein-binding sites within this minimal enhancer element were identified by gel mobility and methylation protection experiments. Each binding site was required for full enhancer activity in Hep G2 transient expression assays. Competition experiments in protein-binding assays suggested that two of the three sites were recognized by a similar factor and that the protein interaction with the third site was different. The nuclear protein(s) which bound to the two homologous sites was found mainly or only in cells of hepatic origin, suggesting an involvement of this region in the cell-specific function of this enhancer. The nuclear protein(s) recognizing the third enhancer region was also found in HeLa and spleen cells.     

Leaky scanning is the predominant mechanism for translation of human papillomavirus type 16 E7 oncoprotein from E6/E7 bicistronic mRNA

Human papillomaviruses (HPV) are unique in that they generate mRNAs that apparently can express multiple proteins from tandemly arranged open reading frames. The mechanisms by which this is achieved are uncertain and are at odds with the basic predictions of the scanning model for translation initiation. We investigated the unorthodox mechanism by which the E6 and E7 oncoproteins from human papillomavirus type 16 (HPV-16) can be translated from a single, bicistronic mRNA. The short E6 5' untranslated region (UTR) was shown to promote translation as efficiently as a UTR from Xenopus beta-globin. Insertion of a secondary structural element into the UTR inhibited both E6 and E7 expression, suggesting that E7 expression depends on ribosomal scanning from the 5' end of the mRNA. E7 translation was found to be cap dependent, but E6 was more dependent on capping and eIF4F activity than E7. Insertion of secondary structural elements at various points in the region upstream of E7 profoundly inhibited translation, indicating that scanning was probably continuous. Insertion of the E6 region between Renilla and firefly luciferase genes revealed little or no internal ribosomal entry site activity. However when E6 was located at the 5' end of the mRNA, it permitted over 100-fold-higher levels of downstream cistron translation than did the Renilla open reading frame. Internal AUGs in the E6 region with strong or intermediate Kozak sequence contexts were unable to inhibit E7 translation, but initiation at the E7 AUG was efficient and accurate. These data support a model in which E7 translation is facilitated by an extreme degree of leaky scanning, requiring the negotiation of 13 upstream AUGs. Ribosomal initiation complexes which fail to initiate at the E6 start codon can scan through to the E7 AUG without initiating translation, but competence to initiate is achieved once the E7 AUG is reached. These findings suggest that the E6 region of HPV-16 comprises features that sponsor both translation of the E6 protein and enhancement of translation at a downstream site.     

In vitro biological activities of the E6 and E7 genes vary among human papillomaviruses of different oncogenic potential.

Human papillomavirus type 16 (HPV-16) and HPV-18 are often detected in cervical carcinomas, while HPV-6, although frequently present in benign genital lesions, is only rarely present in cancers of the cervix. Therefore, infections with HPV-16 and HPV-18 are considered high risk and infection with HPV-6 is considered low risk. We found, by using a heterologous promoter system, that expression of the E7 transforming protein differs between high- and low-risk HPVs. In high-risk HPV-16, E7 is expressed from constructs containing the complete upstream E6 open reading frame. In contrast, HPV-6 E7 was efficiently translated only when E6 was deleted. By using clones in which the coding regions of HPV-6, HPV-16, and HPV-18 E7s were preceded by identical leader sequences, we found that the ability of the E7 gene products to induce anchorage-independent growth in rodent fibroblasts correlated directly with the oncogenic association of the HPV types. By using an immortalization assay of normal human keratinocytes that requires complementation of E6 and E7, we found that both E6 and E7 of HPV-18 could complement the corresponding gene from HPV-16. However, neither E6 nor E7 from HPV-6 was able to substitute for the corresponding gene of HPV-16 or HPV-18. Our results suggest that multiple factors, including lower intrinsic biological activity of E6 and E7 and differences in the regulation of their expression, account for the low activity of HPV-6, in comparison with HPV-16 and HPV-18, in in vitro assays. These same factors may, in part, account for the apparent difference in oncogenic potential between these viruses.