HPV18 Utilizes Two Alternative Branch Sites for E6~*I Splicing to Produce E7 Protein

Human papillomavirus 18(HPV18) E6 and E7 oncogenes are transcribed as a single bicistronic E6 E7 pre-mRNA. The E6 ORF region in the bicistronic E6 E7 pre-mRNA contains an intron. Splicing of this intron disrupts the E6 ORF integrity and produces a spliced E6~*I RNA for efficient E7 translation. Here we report that the E6 intron has two overlapped branch point sequences(BPS) upstream of its 30 splice site, with an identical heptamer AACUA■C, for E6~*I splicing. One heptamer has a branch site adenosine(underlined) at nt 384 and the other at nt 388. E6~*I splicing efficiency correlates to the expression level of E6 and E7 proteins and depends on the selection of which branch site. In general, E6~*I splicing prefers the 30 ss-proximal branch site at nt 388 over the distal branch site at nt 384. Inactivation of the nt 388 branch site was found to activate a cryptic acceptor site at nt 636 for aberrant RNA splicing. Together, these data suggest that HPV18 modulates its production ratio of E6 and E7 proteins by alternative selection of the two mapped branch sites for the E6~*I splicing, which could be beneficial in its productive or oncogenic infection according to the host cell environment.     

Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.     

Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16⁺ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of ⁹¹QYNK⁹⁴ to ⁹¹PSFW⁹⁴ displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.     

Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells.

We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N-terminal part of E1 and, additionally, 120 bp of the N-terminal part of L1. Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors. The expression system used provides fusions to the N-terminal part of the MS2 polymerase gene controlled by the heat-inducible lambda PL promoter. Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16. By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA. It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4-1 and the SW756 cell lines which contain HPV 18 DNA. These results were confirmed by in vitro translation of hybrid-selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells. Additionally, these experiments led to the identification of an 11-kd E6 and a 10-kd E4 protein in the CaSki cell line as well as a 70-kd E1 protein in HeLa cells.     

HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes.

The human papillomavirus types (HPVs) most often associated with cancer of the cervix, such as HPV16, have been reported previously to immortalize normal human foreskin keratinocytes in vitro, while the types that are primarily associated with benign cervical lesions failed to do so. In this study we have determined the HPV16 genes that are responsible for the immortalizing activity of the viral genome. Transfection with a plasmid in which E6 and E7 were the only intact open reading frames (ORFs) induced an indefinite life-span in the keratinocytes with an efficiency similar to that of the entire early region of the viral DNA. Mutants in the E6E7 clone with inactivating lesions in E6 or E7 failed to induce immortalization. When transfected alone, E7 could induce hyperproliferation, but these cells eventually senesced. By itself, E6 exhibited no activity, Co-transfection of a plasmid with an intact E6 ORF and a second plasmid with an intact E7 ORF generated keratinocyte lines with indefinite growth potential. The E6 and E7 proteins were detected in the lines induced by the E6E7 DNA and by co-transfection of the E6 and E7 plasmids. Therefore, we conclude that HPV16 E6 and E7 cooperative to immortalize human keratinocytes in vitro. Changes in cellular gene expression are probably also required for immortalization since all of the keratinocyte lines examined were aneuploid. Serum and calcium resistant sublines were isolated from the E6E7 induced lines, indicating that other HPV genes do not play an obligatory role in the generation of resistance to differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.     

Suppression in vivo of human papillomavirus type 18 E6-E7 gene expression in nontumorigenic HeLa X fibroblast hybrid cells.

The E6 and E7 genes of the cancer-associated human papillomavirus (HPV) types 16 (HPV16) and 18 (HPV18) can induce cell immortalization in vitro in normal human keratinocytes. This, however, is not associated with tumorigenicity in vivo. On the other hand, tumorigenicity of HPV18-positive HeLa cervical carcinoma cells can be suppressed by fusion of HeLa cells with normal human keratinocytes or fibroblasts. We have addressed the question of whether suppression of tumorigenicity in HeLa x fibroblast hybrid cells might be due to a reduced ability of these cells to express the HPV18 E6-E7 genes in vivo. Nontumorigenic hybrid cells and tumorigenic hybrid segregants were transplanted as organotypical cultures or injected subcutaneously into immunocompromised mice and were analyzed for HPV18 E6-E7 gene expression by RNA-RNA in situ hybridization. The tumorigenic hybrid cells showed a continuous and invasive growth that was associated with high levels of HPV18 E6-E7 mRNAs at all time points examined. In contrast, the nontumorigenic hybrid cells stopped cell proliferation approximately 3 days after transplantation. At this time they expressed the E6-E7 genes at low levels, whereas at day 2 high expression levels were observed. However, the mRNA levels of the cytoskeletal genes beta-actin and vimentin remained high for at least 14 days, demonstrating that inhibition of growth and of HPV18 E6-E7 gene expression was not due to cell death. These results suggest that growth inhibition of the nontumorigenic HeLa x fibroblast hybrid cells in vivo might be caused by suppression of HPV18 E6-E7 gene expression and are compatible with the idea of an intracellular surveillance mechanism for HPV gene expression existing in nontumorigenic cells.