HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation
2012; Springer Nature; Volume: 31; Issue: 14 Linguagem: Inglês
10.1038/emboj.2012.147
ISSN1460-2075
AutoresCecilia Johansson, Monika Somberg, Xiaoze Li, Ellenor Backström Winquist, Joanna Fay, Fergus Ryan, David Pim, Lawrence Banks, Stefan Schwartz,
Tópico(s)Viral-associated cancers and disorders
ResumoArticle22 May 2012free access Source Data HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation Cecilia Johansson Cecilia Johansson Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Monika Somberg Monika Somberg Department of BioNut, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Xiaoze Li Xiaoze Li Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Ellenor Backström Winquist Ellenor Backström Winquist Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Joanna Fay Joanna Fay National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Ireland Search for more papers by this author Fergus Ryan Fergus Ryan Dublin Institute of Technology, Dublin, Ireland Search for more papers by this author David Pim David Pim International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Lawrence Banks Lawrence Banks International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Stefan Schwartz Corresponding Author Stefan Schwartz Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Cecilia Johansson Cecilia Johansson Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Monika Somberg Monika Somberg Department of BioNut, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Xiaoze Li Xiaoze Li Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Ellenor Backström Winquist Ellenor Backström Winquist Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Joanna Fay Joanna Fay National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Ireland Search for more papers by this author Fergus Ryan Fergus Ryan Dublin Institute of Technology, Dublin, Ireland Search for more papers by this author David Pim David Pim International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Lawrence Banks Lawrence Banks International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Stefan Schwartz Corresponding Author Stefan Schwartz Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden Search for more papers by this author Author Information Cecilia Johansson1,‡, Monika Somberg2,‡, Xiaoze Li1, Ellenor Backström Winquist1, Joanna Fay3, Fergus Ryan4, David Pim5, Lawrence Banks5 and Stefan Schwartz 1 1Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, Lund, Sweden 2Department of BioNut, Karolinska Institute, Stockholm, Sweden 3National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Ireland 4Dublin Institute of Technology, Dublin, Ireland 5International Centre for Genetic Engineering and Biotechnology, Trieste, Italy ‡These authors contributed equally to this work *Corresponding author. Section of Medical Microbiology, Department of Laboratory Medicine, Lund University, BMC-B13, Lund 221 84, Sweden. Tel.:+46 46 222 0628; Fax:+46 46 130064; E-mail: [email protected] The EMBO Journal (2012)31:3212-3227https://doi.org/10.1038/emboj.2012.147 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2. Introduction Persistent infection with high-risk human papillomaviruses (HPVs), such as HPV-16, is a risk factor for the development of cervical cancer (zur Hausen, 2002; Howley and Lowy, 2006). In common with the majority of DNA viruses that establish persistence, the HPV-16 life cycle is divided into early and late phases (Doorbar, 2005; Chow et al, 2010; Bodily and Laimins, 2011). The delay in late gene expression is likely a mechanism of immune evasion that contributes to establishment of HPV persistence. None of the early HPV proteins has yet been shown to regulate HPV late gene expression. However, multiple cellular factors that regulate papillomavirus late gene expression have been identified (Zheng and Baker, 2006; Schwartz, 2008; Graham, 2010; Johansson et al, 2010). We have previously shown that the adenovirus E4orf4 protein can induce HPV-16 late gene expression (Somberg et al, 2009), by interfering with cellular SR proteins (Kanopka et al, 1996, 1998). HPV-5 and HPV-16 E2 interact with SR proteins (Lai et al, 1999; Bodaghi et al, 2009), HPV-16 E2 inhibits splicing in vitro (Bodaghi et al, 2009) and HPV-16 E2 activates expression of the SFRS1 gene (Mole et al, 2009). We speculated that E2 might play the role of a viral activator of HPV late gene expression. The E2 protein is modular and consists of an N-terminal transactivation domain (TAD), a hinge region, and a C-terminal DNA-binding domain (DBD; Androphy et al, 1987; Giri and Yaniv, 1988; Li et al, 1989; Bedrosian and Bastia, 1990). Binding of E2 to the viral DNA is required for replication of the viral DNA, regulation of transcription from the early promoter and for segregation of the viral episomal DNA genome at cell division (Stenlund, 2003; You et al, 2004; Baxter et al, 2005; Howley and Lowy, 2006; McBride et al, 2006; Parish et al, 2006; McBride, 2008; Kadaja et al, 2009; Thierry, 2009). Inhibition of HPV transcription from the early HPV promoter by E2 is caused by steric hindrance of cellular transcription factors (Dong et al, 1994; Thierry, 2009), but it too depends on the interaction of E2 with cellular factors, including Brd4 (Ilves et al, 2006; McPhillips et al, 2006; Wu et al, 2006; Schweiger et al, 2007; Smith et al, 2010; Yan et al, 2010), SMCX (Smith et al, 2010) and EP400 (Smith et al, 2010). In the viral life cycle, high levels of E2 protein accumulate immediately prior to late gene expression, during a stage of efficient replication of the viral DNA genome (Xue et al, 2010), and one may speculate that E2 activates HPV late gene expression. We show that HPV E2 induces HPV late gene expression by inhibiting polyadenylation at the early polyA signal on the viral genome. E2 interacts with the cellular polyadenylation factor CPSF30 in vitro and perturbs the polyadenylation complex that forms at the polyA signal. We conclude that HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation Results HPV-16 E2 induces HPV-16 late gene expression To investigate if any of the HPV-16 early proteins can induce HPV-16 late gene expression, we overexpressed HPV-16 early proteins with the subgenomic HPV-16 plasmid pBELM (Figure 1A). This plasmid produces high levels of early mRNAs, primarily E4 mRNAs upon transfection of HeLa cells. Co-transfection of plasmid pBELM with plasmids expressing each of the HPV-16 E1, E2, E4, E6 or E7 proteins revealed that the HPV-16 E2 protein induced high levels of L1 mRNAs from pBELM, whereas none of the other early HPV-16 proteins did (Figure 1B). In contrast, E4 mRNA levels were relatively unaffected by E2 (Figure 1C). Cloning and sequencing of Reverse Transcription (RT)–PCR products revealed that the L1 mRNAs induced by E2 were spliced from SD880 to SA3358 and further from SD3632 to SA5639 (Figure 1D). pBELM contains mutations that destroy splicing silencers in the L1 coding region, which allows usage of late 3′-splice site SA56329 in these plasmids. The E2 induction of L1 mRNAs from pBELM was not a result of the mutations in L1, as E2 also induced late gene expression from pBEL-OPSA (Supplementary Figure S1), in which sequences downstream of SA5639 are intact, while the 3′-splice site itself has been optimised by extension of the polypyrimidine tract upstream of SA5639. Western blot analysis of the transfected HeLa cells confirmed that E1, E2 and E4 protein production was dependent on transfection with the various expression plasmids (Figure 1E). We were unable to detect E6 and E7 expression with available sera. One cannot exclude that the lack of effect of E6 and E7 on late gene expression may be due to the relatively low expression levels of E6 and E7. Next, we tested the effect of E2 on pBEL (Figure 1A), in which splicing silencers at SA5639 are intact and late mRNA splicing is efficiently suppressed (Zhao et al, 2004). In contrast to the induction of spliced L1 mRNA from pBELM, E2 primarily induced production of the L2/L1 mRNA from pBEL (Figure 1F). This suggested that E2 did not activate HPV-16 late mRNA splicing, but rather caused a transcriptional read-through at pAE, into the late region of the HPV-16 genome (Figure 1A). Since E2 functioned on pBEL plasmids that lack the LCR, E2 acted independently of E2 binding sites located in the LCR. Figure 1.HPV E2 induces HPV-16 late gene expression. (A) Schematic representation of the HPV-16 genome and the subgenomic HPV-16 expression plasmids pBEL (Zhao et al, 2004) and pBELM (Zhao et al, 2004). Nucleotide positions refer to the HPV-16R sequence (Baker and Calef, 1997). Mut indicates a number of nucleotide substitutions that inactivated splicing silencers downstream of SA5639 in plasmid pBELM (Collier et al, 2002; Zhao et al, 2004). A subset of mRNAs that can be produced by pBEL and pBELM are indicated (Zhao et al, 2005). LCR, long control region. (B) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBELM in the absence or presence of plasmids expressing HPV-16 E1, E2, E4, E6 and E7, and probed with the L1 probe. (C) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBELM in the absence or presence of pCMV16E2, and probed with the E4 probe. (D) RT–PCR using primers 3515s and L1a or 757s and L1a on cDNA produced from cytoplasmic RNA extracted from HeLa cells transfected with pBELM in the absence or presence of pCMV16E2. MW, molecular weight marker. (E) Western blot on cell extracts from HeLa cells transfected with the expression plasmids for HPV-16 E1, E2 or E4. (F) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBEL (Zhao et al, 2004) and a two-fold serial dilution of pCMV16E2 expression plasmid, starting at 4 μg, and probed with the L1 probe. (G) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBELM (Zhao et al, 2004) and pCMV16E2 in the absence or presence of plasmids expressing HPV-16 E1 (Kadaja et al, 2007), E6, E7 (Pim et al, 2005) or L2 protein (Oberg et al, 2003), and probed with the L1 probe. (H) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBEL or pBELM (Zhao et al, 2004) in the absence or presence of plasmid pCMV11E2, and probed with the L1 probe. (I) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBELM (Zhao et al, 2004) in the absence or presence of plasmid pCMV16E2 or pCMV5E2, and probed with the L1 probe. Duplicate transfections are shown. Numbers indicate fold induction of L2/L1 or L1 mRNAs by E2. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint HPV-16 E1 inhibits induction of HPV-16 late gene expression by HPV-16 E2 The ability of E2 to induce HPV-16 late gene expression could possibly be regulated by other HPV proteins that interact with E2. To investigate this, we transfected pBELM and the E2 plasmid with a two-fold excess of expression plasmids that produce proteins that have been shown previously to interact with E2. These plasmids expressed HPV-16 E1 (Hibma et al, 1995), E6 (Grm et al, 2005), E7 (Gammoh et al, 2006) or L2 (Day et al, 1998; Heino et al, 2000; Okoye et al, 2005). As can be seen in Figure 1G, only E1 reduced the ability of E2 to induce HPV-16 late gene expression. These results demonstrated that HPV-16 E1 can inhibit E2-mediated induction of HPV-16 late gene expression, and suggested that high levels of E1 during the viral infection will favour E1- and E2-mediated genome replication over induction of HPV late gene expression by E2. As E1 and E2 are co-expressed during the HPV-16 infection, one may speculate that a high E1/E2 ratio may prevent induction of late gene expression by E2 and favour replication of the HPV genome, whereas a low E1/E2 ratio may favour induction of HPV late gene expression by E2. Induction of HPV late gene expression is a conserved property of HPV E2 Next, we investigated if E2 from mucosal low-risk HPV-11 as well as cutaneous HPV-5 could activate HPV-16 late gene expression. We found that E2 from HPV types 5 and 11 could induce HPV-16 late gene expression to the same extent as HPV-16 (Figure 1H and I), demonstrating that the ability of E2 to induce late gene expression is a conserved property among both cutaneous and low-risk and high-risk mucosal HPVs. To further investigate if the ability of E2 to induce late gene expression is a general property of HPV, we wished to analyse the effect of HPV-16 E2 on a distantly related, cutaneous HPV type, not associated with cancer. To this end, we generated an HPV type 1 subgenomic expression plasmid named pBEL-1, in which a CMV promoter was inserted at nucleotide position 684 of the HPV-1 genome (Figure 2A). Similarly to the HPV-16 plasmid pBEL (Figure 2B), pBEL-1 produced primarily the E4 mRNA, detected by the CMV probe that hybridised to the leader sequence in both HPV-16 pBEL and HPV-1 pBEL-1 (Figure 2B and C). Sequencing of the RT–PCR product shown in Figure 2C confirmed that it was the result of splicing from SD827 to SA3200 in HPV-1. The HPV-1 polyadenylation site was mapped to nucleotide positions 4023–25 by 3′-rapid amplification of cDNA ends (3′-RACE; Figure 2D and E). Overexpression of the HPV-16 E2 protein induced HPV-1 late gene expression (Figure 2F), while the effect of HPV-16 E2 on the HPV-1 E4 mRNA was minor (Figure 2F). We have shown previously that both adenovirus E4orf4 and PTB induce HPV-16 late gene expression (Somberg et al, 2008, 2009). Adenovirus E4orf4 induced HPV-1 late gene expression, while PTB did not (Figure 2F). High levels of PTB were produced by the pCMVPTB plasmid (Figure 2G). Therefore, HPV-1 and HPV-16 late gene expression may respond differentially to various factors, but the ability of E2 to induce HPV late gene expression is conserved. Figure 2.HPV E2 induces HPV-1 late gene expression. (A) Schematic representation of the HPV-1 genome and the subgenomic HPV-1 expression plasmid pBEL-1. (B) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with HPV-16 plasmid pBEL (Zhao et al, 2004) or HPV-1 plasmid pBEL-1. The blot was probed with the CMV probe that detects both HPV-16 mRNAs produced from pBEL (Zhao et al, 2004) and HPV-1 mRNAs produced from pBEL-1 (Collier et al, 2002; Zhao et al, 2004). (C) RT–PCR using HPV-1 primers 827s and E4a on cDNA generated from cytoplasmic RNA extracted from HeLa cells transfected with pBEL-1. (D) 3′-RACE products derived from cytoplasmic RNA extracted from HeLa cells transfected with pBEL-1. Arrow points to the band that represents cDNA synthesised from mRNAs that are polyadenylated at HPV-1 pAE. (E) Sequencing of 3′-RACE products shown in (D) demonstrated that HPV-1 early mRNAs were cut and polyadenylated at genomic nucleotide positions 4023–25. (F) Northern blot on cytoplasmic RNA extracted from HeLa cells transfected with pBEL-1 in the absence or presence of pCMV16E2 (encoding HPV-16 E2), pE4orf4 (encoding adenovirus E4orf4) (Shtrichman et al, 1999) or pCMVPTB (encoding polypyrimidine tract binding protein 1). Blots were probed with HPV-1 L1 or E4 probe. (G) Western blot on HeLa cells transfected with empty CMV vector (−) or a CMV-PTB expression plasmid (+). Figure source data can be found with the Supplementary data. Download figure Download PowerPoint HPV-16 E2 induces late gene expression from a full-length genomic clone of HPV-16 To determine if E2 could act on a full-length genomic clone of HPV-16, we transfected the E2 plasmid together with pHPV16ALuc (Figure 3A). This plasmid encodes the entire HPV-16 genome with the exception of the 3′-end of the L1 gene that has been replaced with the poliovirus 2A internal ribosome entry site (IRES) followed by the luciferase reporter gene (Figure 3A). The luciferase gene acts as a surrogate marker for HPV-16 late gene expression. As expected, this plasmid produced only low levels of luciferase upon transfection of C33A cells (Figure 3B). Co-transfection of a serial dilution of the E2 plasmid induced luciferase expression from this construct in a plasmid dose-dependent manner (Figure 3C). Analysis of L1 mRNA levels by real-time RT–PCR confirmed that E2 induced production of late mRNAs from pHPV16ALuc (Figure 3D). To determine if E2 could induce HPV-16 late gene expression from episomal copies of the genome, the HPV-16 genome flanked by loxP sites in pHPV16ANsL (Supplementary Figure S2A) was released by the cre recombinase, as recently described for HPV-18 (Wang et al, 2009). Overexpression of HPV-16 E2 induced late gene expression (Supplementary Figure S2C). Only episomal copies of the HPV-16 genome could be detected (Supplementary Figure S2B). We concluded that HPV-16 E2 could induce late gene expression from a full-length HPV-16 genome. Figure 3.HPV-16 E2 induces late gene expression from the HPV genome. (A) Schematic representation of the HPV-16 genome and genomic HPV-16 expression plasmid pHPV16ALuc. Poliovirus IRES followed by the luciferase coding sequence was inserted between BamHI in HPV-16 L1 and the L1 stop codon. The most common E6/E7 mRNA is indicated. In addition, the read-through mRNAs induced from this plasmid by E2 overexpression are indicated (late/Luc mRNA). (B) Luciferase expression in C33A cells transfected with pHPV16ALuc or a CMV promoter-driven luciferase expression plasmid (pLuc). (C) Relative luciferase levels monitored in C33A cells transfected with pHPV16ALuc and a 10-fold serial dilution of the pCMV16E2 expression plasmid, starting at 1 μg. Bars represent fold increase in luciferase production from pHPV16ALuc in the presence of increasing concentrations of transfected pCMV16E2 expression plasmid. Error bars show standard deviation from triplicate transfections. (D) Real-time PCR on total RNA extracted from the C33A cells shown in (C). Primers used for PCR of spliced L1 mRNA are shown in (A). Mean values and standard deviation from two parallel transfection experiments, each performed in triplicates, are shown. The qPCR was normalised to qPCR on the GAPDH mRNAs in the same samples. Download figure Download PowerPoint Both the transactivation and hinge domains of HPV-16 E2 are required for induction of HPV-16 late gene expression Next, we investigated the ability of various E2 mutants to induce HPV-16 late gene expression (Figure 4A). These mutants were efficiently expressed in transfected cells, with the exception of the hinge domain (Figure 4B). The mutational analyses of E2 showed that both the N-terminal TAD and hinge domains of HPV-16 E2 were required for induction of late gene expression from pBELM (Figure 4C). We also tested two previously described and characterised E2 mutants. M11 has an amino-acid substitution that makes E2 unable to support replication of viral DNA, but still able to activate transcription. M12 has a four amino-acid deletion and was shown to be defective in transcription regulation, but could replicate viral DNA (M12) (Piccini et al, 1997; Figure 4A). The M11 mutant activated HPV-16 late gene expression, whereas M12 did not (Figure 4D). These results demonstrated that the ability of HPV-16 E2 to induce HPV-16 late gene expression was distinct from its effects on viral DNA replication, but might be coupled to the effects on transcription. Figure 4.The E2 N-terminus and hinge are required to induce HPV-16 late gene expression. (A) Schematic representation of the HPV-16 E2 protein. The transactivation domain (TAD), hinge domain and DNA-binding domain (DBD) are indicated. Numbers represent amino-acid positions at the borders of the E2 protein domains. Various E2 mutants are shown. (B) Western blot on cell extracts from HeLa cells transfected with the indicated plasmids. The E2HC protein also migrates as a dimer (*). (C, D) Northern blots on cytoplasmic RNA extracted from HeLa cells transfected with pBELM (Zhao et al, 2004) in the absence or presence of plasmids expressing HPV-16 E2 or E2 mutants shown in (A). The blot was probed with L1 probe (Figure 1A). Fold induction of L1 mRNA by E2 is indicated below the gels. (E) Schematic representation of the HPV-16 subgenomic expression plasmids pBELMCAT, pBELLuc and pBELMLuc. The IRES is followed by the CAT gene or the luciferase gene (Luc). A subset of mRNAs produced by these plasmids is indicated. (F, G) Levels of CAT protein produced from pBELMCAT transfected into HeLa cells in the absence or presence of plasmids expressing HPV-16 E2, or E2 mutants. Arbitrary CAT units monitored by CAT ELISA are shown. (H) Relative levels of luciferase activity produced from pBELLuc or pBELMLuc transfected into primary human keratinocytes in the absence or presence of E2 expression plasmids. Error bars represent standard deviations from triplicate transfections. Download figure Download PowerPoint The effects of the E2 mutants were also analysed using a reporter plasmid named pBELMCAT, in which the poliovirus 2A IRES element followed by the CAT reporter gene had been inserted in the 3′-end of the L1 gene (Figure 4E). CAT protein levels serve as a surrogate marker for HPV-16 late gene expression. Co-transfection of pBELMCAT with the various E2 mutants confirmed the northern blot results, in that E2, E2NH and M11 induced CAT production, while E2HC and M12 did not (Figure 4F and G). To confirm these results in human primary keratinocytes, the natural target cell of HPV-16, we replaced the CAT reporter gene with the gene for luciferase, for which there is a more sensitive assay, resulting in pBELLuc and pBELMLuc (Figure 4E). Co-transfection of these plasmids with pCMV16E2, or plasmids expressing E2 mutants, into human primary keratinocytes, confirmed the results obtained in HeLa cells (Figure 4H, left and right panel). However, in keratinocytes, the levels of induction were generally higher and the E2NH mutant induced higher levels of late gene expression than the full-length E2 protein (Figure 4H). We concluded that the hinge domain and the N-terminal TAD were required for induction of HPV-16 late gene expression, both in human primary keratinocytes and in cervical cancer cells, while the C-terminal DBD was not. HPV-16 E2 acts on a 300-nucleotide fragment of the HPV-16 genome that encodes the early polyA signal pAE Data shown above indicated that HPV-16 E2 caused a read-through into the late region of the HPV-16 genome, indicating that E2 inhibited the HPV-16 early polyA signal pAE. To narrow-down the target sequence of HPV-16 E2 on the HPV-16 genome, a 520- or 300-nucleotide sequence encompassing the HPV-16 pAE was inserted between the CMV promoter and the CAT gene in the pM2-CAT plasmid, generating pM2A-CAT and pM2ADE-CAT (Figure 5A), respectively. These insertions both inhibited expression of CAT mRNA (Figure 5B). Overexpression of HPV-16 E2 induced production of the CAT mRNA from both pM2A-CAT and pM2ADE-CAT (Figure 5B), whereas CAT expression from pM2-CAT, which lacks HPV sequences, was unaffected by HPV-16 E2 (Figure 5B). These results demonstrated that a 300-nucleotide sequence encompassing HPV-16 pAE was sufficient to subject the CAT gene to E2 regulation. We subjected RNA from the pM2A-CAT or pM2ADE-CAT transfected cells to 3′-RACE, which confirmed that pAE was efficiently utilised (Figure 5C). Cloning and sequencing of the 3′-RACE product mapped the cleavage and polyadenylation site to position 4232/4233/4234, as previously described (Baker and Calef, 1997; Zhao et al, 2005). In addition to the CAT reporter plasmid, we generated the luciferase reporter plasmid pE5Luc (Figure 5D), in which an HPV-16 sequence encoding the entire HPV-16 E5 open reading frame, the HPV-16 early untranslated region (UTR) and the pAE was inserted upstream of the poliovirus 2A IRES and the luciferase reporter gene (Figure 5D; Backström Winquist et al, 2012). Cloning and sequencing of 3′-RACE products revealed that pAE was used in pE5Luc (Figure 5E). Overexpression of HPV-16 E2 or E2NH significantly enhanced production of luciferase activity from pE5Luc while E2HC had no detectable effect (Figure 5F). Similar results were obtained by transfection of primary human keratinocytes (HEKn; Figure 5F), with the exception that E2NH induced higher levels of luciferase than E2 in primary keratinocytes (Figure 5F). These results supported the conclusion that HPV-16 E2 induced late gene expression by inhibiting the early polyA site pAE. Figure 5.HPV-16 E2 targets the early polyA signal. (A) Schematic representation of the HPV-16 genome and the pM2A-CAT, pM2ADE-CAT and pM2-CAT expression plasmids. The HPV-16 sequences present in the CAT plasmids are indicated. IRES, poliovirus 2A internal ribosome entry site. (B) Northern blots on cytoplasmic RNA extracted from HeLa cells transfected with pM2A-CAT and pM2ADE-CAT, or pM2-CAT in the absence or presence of pCMV16E2, probed with CAT probe. RNAs from duplicate transfections are shown. (C) 3′-RACE products derived from cytoplasmic RNA extracted from HeLa cells transfected with the indicated plasmids. Arrow points to the band that represents cDNA synthesised from mRNAs that are polyadenylated at HPV-16 pAE. (D) Schematic representation of the HPV-16 genome and the pE5Luc expression plasmid. Luc, luciferase gene. The borders of the HPV-16 sequences present in the pE5Luc plasmid are indicated. The E5 and Luc mRNAs that can be produced by pE5Luc are indicated. (E) 3′-RACE products derived from cytoplasmic RNA extracted from A549 cells transfected with pE5Luc. Arrow points to the band that represents cDNA synthesised from mRNAs that are polyadenylated at HPV-16 pAE. (F) Relative luciferase activity in A549 cells (left panel) or human primary keratinocytes (HEKn) transfected with plasmid pE5Luc in the absence or presence of plasmids encoding HPV-16 E2, E2NH or E2HC. Error bars show standard deviation from at least three independent transfection experiments. Figure source data can be found with the Supplementary data. Download figure Download PowerPoint Inactivation of HPV-16 pAE by nucleotide substitutions or overexpression of known cellular, polyadenylation inhibitory factors mimics the effect of HPV-16 E2 on HPV-16 late gene expression If E2 inhibits pAE, then mutational inactivation of pAE should induce L2/L1 mRNAs from pBEL, and L1 mRNAs from pBELM, as E2 did (Figure 1B, C and F). Inactivation of pAE with six nucleotide substitutions in either pBEL or pBELM, resulting in plasmids pBELDP and pBELDPM (Zhao et al, 2005), respectively, caused an induction of L2/L1 or L1 mRNA (Supplementary Figure S3A), similarly to the effects of E2 on pBEL and pBELM (Supplementary Figure S3B). In addition, the HuR and the influenza virus NS1 proteins with documented inhibitory effect on polyadenylation (Nemeroff et al, 1998; Zhu et al, 2007; Dai et al, 2012) exhibited the same effect on HPV-16 late gene expression as HPV-16 E2 (Supplementary Figure S3C and D), while the SR protein 9G8 had no detectable effect on HPV-16 late gene expression (Supplementary Figure S3C). In addition, NS1 induced luciferase production from the reporter plasmid pE5Luc (Supplementary Figure S3E) in a manner similar to HPV-16 E2 (Figure 5F). Collectively, these results strengthened the conclusion that HPV-16 E2 inhibits HPV-16 early polyadenylation. HPV-16 E2 inhibits HPV-16 pAE more efficiently than pAL Since our results indicated that E2 induced late gene expression by inhibiting the HPV-16 early polyA signal, we speculated that E2 exerted a greater inhibitory effect on pAE than on pAL, as that would cause a higher induction of late gene expression, than if E2 inhibited both polyA signals to the same exten
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