Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3'-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 21, pp. 13354 –13371, May 22, 2015 © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3ⴕ-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing* Received for publication, January 12, 2015, and in revised form, April 14, 2015 Published, JBC Papers in Press, April 15, 2015, DOI 10.1074/jbc.M115.638098

Soniya Dhanjal1, Naoko Kajitani1, Jacob Glahder, Ann-Kristin Mossberg, Cecilia Johansson, and Stefan Schwartz2 From the Department of Laboratory Medicine, Lund University, 221 84 Lund, Sweden Background: HPV16 late gene expression is suppressed in mitotic cells. Results: hnRNP C proteins bind to the HPV16 early, untranslated region and activate HPV16 late mRNA splicing. Conclusion: hnRNP C proteins control HPV16 late gene expression. Significance: hnRNP C proteins may contribute to the ability of HPV16 to avoid the immune system and establish persistent infections that progress to cancer.

Human papillomavirus (HPV)3 is present in 99.7% of all cervical cancers and in many other anogenital cancers as well (1). Of all cancer-associated HPV types, HPV16 is the most common HPV type (2, 3). Although the vast majority of the HPV16 infections are cleared by the immune system, HPV16 may establish persistence and cause cancer (4). These persistent

* This

work was supported by Swedish Research Council-Medicine Grant 2012-1933, Swedish Cancer Society Grant CAN 2012/542, the Gunnar Nilsson Cancer Research Trust Fund, the Hedda and John Forssmans Foundation through the Royal Physiographic Society in Lund, and the Crafoord Foundation (to S. S.). 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed: Dept. of Laboratory Medicine, Lund University, BMC-B13, Rm. B1314c, 223 62 Lund, Sweden. Tel.: 4646-2220628/29; E-mail: [email protected]. 3 The abbreviations used are: HPV, human papillomavirus; qPCR, quantitative PCR; RIPA, radioimmune precipitation assay; hnRNP, heterogeneous nuclear ribonucleoprotein; HFK, human foreskin keratinocyte; sLuc, secreted luciferase; IRES, internal ribosome entry site.

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HPV16 infections are characterized by a dysregulated HPV16 gene expression program with continuous expression of the viral oncogenes E6 and E7 and a shutdown of the late genes expressing the highly immunogenic L1 and L2 capsid proteins (5– 8). Because aberrant HPV16 gene expression may contribute to cancer progression, it is of interest to determine how HPV16 gene expression is regulated (9). Expression of the HPV16 genes is controlled by at least two promoters named p97 and p670 (10 –12), but regulation of alternative splicing and polyadenylation is just as important (9, 13, 14), and HPV16 produces a plethora of alternatively spliced and/or polyadenylated mRNAs (15, 16). The switch from the early to the late gene expression program includes a promoter switch, a poly(A) signal switch, and an activation of late splice signals in a cell differentiation-dependent manner (9). The early mRNAs produced from the HPV16 genome are polyadenylated at the early poly(A) signal pAE, which is followed by the late region encoding L1 and L2 and the late poly(A) signal pAL (Fig. 1A) (15, 16). Regulation of early polyadenylation is therefore of paramount importance because the activity of the HPV16 pAE controls the levels of both HPV16 early and late gene expression (17–19). The pAE is regulated by cellular factors and by the HPV16 E2 protein, which has been shown to perturb the conformation of the polyadenylation complex, thereby causing a read-through into the late region of the genome (20). Polyadenylation at pAE is dependent on downstream enhancer elements located within the L2 coding region and that interact with CstF-64 and hnRNP H (19, 21). The upstream, early untranslated region also enhances polyadenylation at pAE (17, 18). Efficient splicing to the HPV16 3⬘-splice SA3358 located upstream of pAE enhances polyadenylation at pAE (22), suggesting that pAE is regulated by the splicing machinery. RNA sequences downstream of SA3358 are essential for utilization of SA3358 and interact with SRSF1 (ASF/SF2), SRSF9 (SRp30c), and SRSF3 (SRp20) (23–27). The early poly(A) signal is competing with HPV16 5⬘-splice SD3632 that is used exclusively by late mRNAs and is located in between SA3358 and pAE. HPV16 late 5⬘-splice site SD3632 is used only for production of spliced late L1 mRNAs (15, 16). In undifferVOLUME 290 • NUMBER 21 • MAY 22, 2015

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In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5ⴕ-splice site SD3632; produced elevated levels of RNAbinding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the fulllength HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5ⴕsplice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

hnRNP C Family Interacts with HPV16 Early UTR entiated cells, SD3632 is suppressed by upstream sequences that encode two AUAGUA motifs and interact specifically with members of the hnRNP D family and hnRNP A2/B1 (28). HPV16 late slice site SD3632 is spliced to late 3⬘-splice site SA5639 located immediately upstream of the L1 AUG. in mitotic cells, SA5639 is suppressed. Multiple splicing silencers downstream of SA5639 interact with hnRNP A1 (29 –31). Here we have conducted a screen for proteins that are upregulated in cervical cancer cell clones that are permissive for HPV16 late gene expression. We identified one RNA-binding protein named RALYL, which belongs to the hnRNP C family of hnRNPs. We show that both RALYL and hnRNP C1 bind to the HPV16 early UTR and that they can induce HPV16 late L1 mRNA splicing in an HPV16 early 3⬘-UTR-dependent manner.

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Experimental Procedures Plasmids—The following plasmids have been described previously: pCL086 (32), pBEL (29), pBELDUTR (18), pBELsLuc (28), pBELMsLuc (25, 28), pRSVneo (25, 28), pBSpD1MCAT (28), p4xATAGTA (28), p4xMUT (28), and pHPV16ANSL (25, 28). We thank Andras Nagy (University of Toronto) for providing pCAGGS-nlscre (33). To generate pBELneo and pTEx4Mneo, a DNA fragment encoding the neoR ORF was PCR-amplified from pRSVneo and inserted into pBEL and pTEx4M (24, 25), respectively. CMV promoter-driven expression plasmids for Myc-tagged RALY (RC210723) and Myc-tagged RALYL (RC206313) were purchased from Origene. To construct RALYL deletion mutant pCRALYL-C, the N terminus was PCR-amplified with primers Sgf1C (5⬘-GCATGAGCGATCGCCATGGATTTCTACAATCGGTTATTTGAT-3⬘) and MluIC (5⬘-GCATGAACGCGTCTTTATCTGTAGAAACAGCTCATGACCCCC-3⬘) and inserted into Myc-tagged-RALYL plasmid (RC206313, Origene). pCRALYL-N was constructed by insertion of a restriction site in Myc-tagged-RALYL plasmid (RC206313, Origene) by site-directed mutagenesis followed by digestions and religation. To generate pChnRNP C1, the hnRNP C1 ORF was PCRamplified with primers hnRNPC1For (5⬘-GCATGAGCGATCGCATGGCCAGCAACG-3⬘) and hnRNPC1Rev (5⬘-GCATGAACGCGTTTAAGAGTCATCCTCGCCATTGGC-3⬘) from pHis-hnRNP C1 generously provided by Dr. LeStourgeon and inserted into Myc-tagged-RALYL (RC206313) plasmid. Transfection of HeLa, C33A, C33A2, and 293T Cells—HeLa, C33A, C33A2, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal bovine calf serum and penicillin/streptomycin. Transfections were carried out using Turbofect according to the manufacturer’s instructions (Fermentas). Briefly, the mixture of 2 ␮l of Turbofect and 100 ␮l of Dulbecco’s modified Eagle’s medium without serum was added to 1 ␮g of plasmid DNA and incubated at room temperature for 15 min prior to dropwise addition to 60-mm plates with subconfluent HeLa cells. Plasmid pCMVSEAP or pCMVsLuc was included in the transfection experiments to control for transfection efficiency. Cells were harvested at 24 h posttransfection. Each plasmid was transfected in triplicate in a minimum of two independent experiments. For stable transfections of C33A cells with pBELneo, the transfected cells were split at 24 h posttransfection and seeded into five 100-mm

plates. The day after transfer to 100-mm plates, the medium was replaced by neomycin-containing medium (1.2 mg/ml neomycin), and culturing was continued with frequent medium changes until single-cell-derived, neomycin colonies appeared. The colonies were expanded and analyzed as described below. Propagation and Transfection of Human Primary Keratinocytes—Propagation of neonatal human epidermal keratinocytes has been described previously (28). Briefly, neonatal human epidermal keratinocyte cells were purchased from Gibco and were propagated in EpiLife medium supplemented with human keratinocyte growth supplement (Gibco). For transfection, 150,000 cells were seeded per 60-mm plate and transfected with 1.5 ␮g of plasmid in Fugene 6. For analysis of episomal HPV16, plasmid pHPV16ANsL was cotransfected with plasmid pCAGGS-nlscre (generously provided by Dr. Andras Nagy, University of Toronto), which expresses the cre recombinase that releases the HPV16 genome from the plasmid at two flanking lox sites. Each plasmid was transfected in triplicates, in a minimum of two independent experiments. Medium was harvested for analysis of secreted luciferase at different time points after transfection. Secreted Luciferase Assay—The Metridia longa secreted luciferase activity in the medium of the transfected cells was monitored with the help of the Ready To Glow secreted luciferase reporter assay according to the instructions of the manufacturer (Clontech). Briefly, 50 ␮l of cell culture medium were added to 5 ␮l of 0.5⫻ secreted luciferase substrate/reaction buffer in a 96-well plate, and luminescence was monitored in a Tristar LB941 luminometer. RNA Extraction and RT-PCR—Nuclear and cytoplasmic RNA was extracted 24 h posttransfection, as described previously (28). Total RNA was extracted using an RNeasy minikit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. 200 ng of RNA were reverse transcribed in a 25-␮l reaction at 42 °C by using Superscript II and random hexamers according to the protocol of the manufacturer (Invitrogen). Two microliters of cDNA were subjected for PCR amplification of cDNA. E4 mRNAs spliced from SD880 to SA3358 were amplified with primers 757s, 773s or 880s and E4a (Fig. 1A), and L1 and L1i mRNAs were amplified with primers 757S and L1A (Fig. 1A) (25). For analysis of stably transfected, neomycin-resistant cell clones, L2 mRNAs were amplified with primers L2S (5⬘-CCTTTAGTATCAGGTCCTGATATACCC-3⬘) and L1A (5⬘GCAACATATTCATCCGTGCTTACAACC-3⬘). In transient transfections, L2 mRNAs were monitored by primers L2F (5⬘-GACCCCCTTTAACAGTAGATCC-3⬘) and L2R (5⬘-TACAGATGGGTCAGTGAAAGTG-3⬘). Spliced RALYL mRNAs were RT-PCR-amplified with primers RALYLS (5⬘-CTCCCGTGTTTTCATCGG-3⬘) and RALYLA (5⬘-AGGGGCCTCTTGTTTCCAGG-3⬘). ACTINS (5⬘-TGAGCTGCGTGTGGCTCC-3⬘) and ACTINA (5⬘-GGCATGGGGGAGGGCATACC-3⬘) were used for amplification of cDNA representing spliced actin mRNA, and ACTINS-1 (5⬘CCAGTGGCTTCCCCAGTG-3⬘) and ACTINA were used for amplification of cDNA representing unspliced actin mRNA. Real-time Quantitative PCR (qPCR) of cDNAs—qPCR was performed on 2 ␮l of cDNA prepared as described above in a

hnRNP C Family Interacts with HPV16 Early UTR


7.4, 0.5% Nonidet P-40, 0.5 M DTT, protease inhibitors). Cells were incubated on ice for 30 min with occasional vortexing to lyse cells. For immunoprecipitations, 1 ␮g of anti-hnRNP C antibody (sc-32308) or normal mouse IgG (catalog no. 12-371, Millipore) were incubated overnight at 4 °C with 0.5 mg of proteins in 0.5 ml of RIPA buffer. 20 ␮l (0.6 mg) of Dynabeads威 Protein G (catalog no. 10004D, Invitrogen) were blocked with 1% BSA/RIPA buffer for 1 h, washed five times, and then added in each tube, and incubation continued for another 1 h at 4 °C. The beads were washed twice with RIPA buffer, twice with RIPA buffer supplemented with 500 mM NaCl, and twice with RIPA buffer supplemented with 250 mM LiCl. RNA was eluted by phenol/chloroform extraction, ethanol-precipitated, and dissolved in 20 ␮l of water. 9 ␮l of immunoprecipitated RNA was reverse transcribed using M-MLV reverse transcriptase (Invitrogen) and random primers (Invitrogen) according to the protocol of the manufacturer. 2 ␮l of cDNA were subjected to PCR amplification using oligonucleotides 773s ⫹ E4a. RNA-Protein Pull-down Assay—Nuclear extracts were prepared from subconfluent HeLa cells according to the procedure described by Dignam et al. (36). Pull-down experiments were performed as described previously (28). Briefly, HeLa nuclear extract was mixed with streptavidin-coated magnetic beads (Invitrogen) bound to biotin-labeled ssDNA oligonucleotides in 500 ␮l of binding buffer (10 mM Tris, pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100). After incubation with rotation for 1 h at 4 °C, beads were washed five times in binding buffer with various concentrations of NaCl. Proteins were eluted by boiling of the beads in SDS-PAGE loading buffer, and proteins were separated on SDS-polyacrylamide gels followed by transfer to nylon membranes (Whatman) and Western blot analysis with the indicated antibodies.

Results Identification of Cervical Cancer Cells That Are Permissive for HPV16 Late Gene Expression—Our aim was to identify cellular factors that regulate HPV16 late gene expression at the level of RNA processing. To do so, we used a subgenomic expression plasmid named pBEL that consists of early and late HPV16 genes (Fig. 1A) (29) but expresses only the early genes, primarily the spliced E4 mRNA. Our experimental approach was to stably transfect this plasmid into HPV-negative C33A cells and select cell clones that were permissive for HPV16 late gene expression. Characterization of these clones might lead to the identification of cellular factors that control HPV16 late gene expression. We have shown previously that the late genes from this plasmid can be induced by overexpression of HPV16 E2 (20) but also by cellular proteins PTB (37) and SRSF9/ SRp30c (23), and we therefore speculated that rare cells exist within a population that produces high levels of factors that stimulate HPV16 late gene expression. Identification and characterization of such rare cells could identify cellular factors that control HPV16 late gene expression. To identify cells that are permissive for HPV16 late gene expression, we first modified the previously described pBEL plasmid to fit our purpose (Fig. 1A) (29, 38). A portion of the HPV16 L1 gene was replaced with the poliovirus internal ribosome entry site (IRES) followed by the G418 resistance gene VOLUME 290 • NUMBER 21 • MAY 22, 2015


MiniOpticon (Bio-Rad) using the Sso Advanced SYBR Green Supermix (Bio-Rad) according the manufacturer’s instructions. Primers were those described above, and all cDNA quantitations were normalized to GAPDH mRNA levels. GAPDH cDNA was amplified with primers F-GAPDH (5⬘-ACCCAGAAGACTGTGGATGG-3⬘) and R-GAPDH (5⬘-TTCTAGACGGCAGGTCAGGT-3⬘). Microarray Analysis—Total cellular RNA was extracted at two occasions from pBELneo- or pRSVneo-stably transfected cells using the RNeasy minikit (Qiagen). The RNA quality was determined using a Bioanalyzer (Agilent, Santa Clara, CA). RevertAidTM (Fermentas Inc., Glen Burnie, MD) was used for synthesis of first strand cDNA from 1 ␮g of RNA using random hexamer primers. Each sample was analyzed in triplicates on Human Gene 1.0 ST Arrays covering 36,079 transcripts. Microarray analysis was performed using the Affymetrix expression system at SCIBLU Genomics (Lund University). Basic Affymetrix chip and experimental quality analysis were performed using the Expression Console Software version 1.12 (Affymetrix, Santa Clara, CA). Robust multiarray analysis was performed for data normalization (34). Statistical analysis was performed using the TMEV version 4.0 software (35). Array results have been submitted to the Gene Expression Omnibus database repository and have received the following ID: GSE65166. Protein Extraction and Western Blotting—Western blotting was performed as described previously (20). Briefly, cells were lysed in RIPA buffer (50 mM Tris, pH 7.8, 300 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and protease inhibitors) and subjected to electrophoresis on 12% SDS-polyacrylamide gels. Proteins were transferred onto nitrocellulose membranes, blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20, and stained with the following antibody: anti-hnRNP A1 antibody (sc-10032), antihnRNP I antibody (sc-56709), anti-hnRNP C1 antibody (sc32308), anti-HuR antibody (sc-56709), anti-SRp30c antibody (sc-134036), anti-RALY antibody (ab105866), anti-RALYL antibody (ab66263), anti-SF2 antibody AK96 (Millipore), antihnRNP D antibody (Abcam 61193), anti-Myc tag antibody (sc-789), anti-CPSF30 (ab-103142), or anti-actin antibody (sc1616). Secondary antibodies conjugated with horseradish peroxidase were used, and proteins were detected using the Super Signal West Femto chemiluminescence substrate (Pierce). Immunofluorescence—Cells were seeded in 8-well chambered slides, washed with PBS, and fixed in 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were permeabilized with 0.2% Triton X-100 and subsequently washed with PBS, followed by blocking in 2% BSA in PBS. Cells were incubated overnight with primary antibody to RALYL at a 1:200 dilution in a humidified chamber. After washing in PBS, FITCconjugated secondary antibody was added (1:500), followed by washing in PBS and mounting of slides in medium containing DAPI. Intracellular UV Cross-linking and Immunoprecipitation of RNA-Protein Complexes—Transfected 293T cells in 10-cm dishes were washed with ice-cold PBS and UV-irradiated at 400 mJ/cm2 in a Bio-link cross-linker (Biometra). Cells were lysed in 1 ml of extraction buffer (0.3 M NaCl, 50 mM Tris-HCl, pH


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FIGURE 1. A, schematic representation of the HPV16 genome and subgenomic HPV16 expression plasmids. The early and late viral promoters p97 and p670 and the long control region (LCR) are indicated. Numbers indicate nucleotide positions of 5⬘- (filled circles) and 3⬘-splice sites (open circles) or the early and late poly(A) sites pAE and pAL, respectively. The major late mRNAs are indicated (15). A schematic representation of the pBELneo and pTEx4Mneo plasmids is shown. The human CMV, the poliovirus IRES, and the neomycin resistance gene (neo) are indicated. Locations of mutations that


inactivate the splicing enhancer downstream of SA3358 and the splicing silencers downstream of SA5639 are shown (24, 29). The structures of the E4 mRNAs produced by pBELneo and the E4 and L1i mRNAs produced by pTEx4Mneo are displayed below the plasmids. B, graphs display the number of neomycin-resistant colonies observed at 14 days posttransfection of pBELneo or pTEx4Mneo into C33A or HaCaT cells following selection in 1 mg/ml G418. Three different transfection reagents were used: Turbofect (T), Fugene 6 (F), and Lipofectamine (L).


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neoR, resulting in pBELneo (Fig. 1A). Transfected cells that are permissive for HPV16 late gene expression could be selected by the addition of G418 to the cell culture medium. However, first we wished to confirm that expression of neoR reflected levels of HPV16 late gene expression and therefore generated a second, mutant pBEL plasmid designed to express high levels of HPV16 late mRNAs named pTEx4Mneo (Fig. 1A) (24). In this plasmid, mutations in a splicing enhancer in the early region and in a splicing silencer in the late region were introduced (28). We have shown previously that these mutations activate HPV16 late L1 mRNA expression, and, in contrast to pBELneo, pTEx4Mneo should express high levels of neoR-encoding mRNAs. To demonstrate that pTEx4Mneo efficiently expresses HPV16 late genes and that pBELneo does not, these plasmids were transfected pairwise into C33A or HaCaT cells with three different transfection reagents (T, F, and L), followed by selection in G418 and monitoring of neomycin-resistant, single-cellderived colonies. The results revealed that pTEx4Mneo gave rise to 14 –39 times more colonies than pBELneo (Fig. 1B). Note that transfection of HaCaT with transfection reagent T did not yield any colonies with pBELneo (Fig. 1B). The results validated the use of pBELneo for the identification of cell clones that are permissive for HPV16 late gene expression. Identification of a Novel, Alternatively Spliced L1 mRNA—To identify individual cell clones that were permissive for HPV16 late gene expression, pBELneo was transfected into the HPVnegative, cervical cancer cell line C33A, followed by selection in G418. As a control, C33A cells were also transfected with RSVneo plasmid and selected in parallel. A number of clones were obtained, expanded, and subjected to RNA extraction and analysis by RT-PCR to monitor levels of early HPV16 E4 mRNAs spliced from SD880 to SA3358 (primers 880S and E4a) and late L1 mRNAs spliced between SD880-SA3358-SD3632-SA5639 or L1i mRNAs spliced from SD880 to SA5639 (primers 880S and L1A) (Fig. 2A). In addition, late L2 mRNAs were amplified with primers L2S and L1A (Fig. 2A). RT-PCR of the HPV16 E4 mRNAs identified HPV16-positive neomycin-resistant clones. Of those, one clone produced all three late mRNAs (L2, L1, and L1i) (clone 1f (Fig. 2B)), three produced L2 and L1i mRNAs (clones 1c, 1d, and 1.4a) (Fig. 2B), and three clones (clones 1.2a, 1.2c, and 1.2f) (Fig. 2C) produced L1 mRNAs that differed in size from the L1 mRNAs produced by clone 1f. The size of the L1 mRNA produced by clones 1.2a, 1.2c, and 1.2f appeared to be smaller than the L1 mRNA produced by clone 1f, which produced both L1 and L1i mRNAs of correct sizes. We therefore cloned and sequenced the L1 cDNA products of 1.2a, 1.2c, and 1.2f. The results revealed that the smaller “L1” mRNAs produced by clones 1.2a, 1.2c, and 1.2f were alternatively spliced and did not use the suppressed late splice site SD3632

hnRNP C Family Interacts with HPV16 Early UTR A

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FIGURE 2. A, schematic representation of the HPV16 subgenomic expression plasmid pBELneo. Numbers indicate the nucleotide positions of 5⬘- (filled circles) and 3⬘-splice sites (open circles) or the early and late poly(A) sites pAE and pAL, respectively, and the borders of deletions. B, RT-PCR with primers 880S and L1A (L1 and L1i mRNAs), L2S and L1A (L2 mRNAs), and 880S and E4a (E4 mRNAs) on cDNA of cytoplasmic RNA extracted from the various cell clones indicated at the top of the gels, as well as from the C33A control cells transfected with pRSVneo. C, RT-PCR with primers 880S and L1A (L1 and L1i mRNAs) on cDNA of cytoplasmic RNA extracted from clones indicated at the top of the gel. The alternatively spliced XL1 mRNA is indicated (for structure of the XL1 mRNA, see Fig. 3A). D, ratio of L1 mRNAs over E4 mRNA levels determined by RT-qPCR with primers 880S and L1A or E4a, respectively. RNA from the three cell clones 1c, 1d, and 1.4a was analyzed. E, Western blot analysis of cell extracts from the pBELneo-transfected and G418-selected cell clones indicated at the top of the gels as well as from the C33A control cells transfected with pRSVneo and selected in G418, with antibodies to SRSF1, SRSF9, hnRNP A1, hnRNP I, or HuR. Error bars, S.D.

(Fig. 3A). This mRNA was called XL1 mRNA and is displayed in Fig. 3A. It was spliced from SD880 to SA3358, as expected, and thereafter a novel 5⬘-splice site (SD3519) was used to bypass the strongly suppressed late splice site SD3632 (Fig. 3A), followed by splicing to a novel 3⬘-splice site in the L2 region (SA4980) and further from a novel 5⬘-splice site in L2 (SD5011) to L1 splice site SA5639 (Fig. 3A). The small exon in the L2 region was AU-rich (78% AU) and encompassed 32 nucleotides (Fig. 3A). This novel HPV16 L1 mRNA termed XL1 has not been detected in HPV16-infected cells to our knowledge, but a late mRNA with highly similar structure has been identified in HPV31-


infected cells (Fig. 3A) (15, 16, 39). The alternative splicing pattern of the XL1 mRNA is complex and appears to serve the purpose of bypassing the suppressed late splice site SD3632 by activating an upstream splice site (SD3519) that splices into another splice site in the L2 coding region: SA4980. In this respect, it is interesting to note that seven nucleotides upstream of SD3519 are identical to the seven nucleotides that encompass SA4980 (Fig. 3B). In HPV31, eight nucleotides upstream of the 5⬘-splice site are 75% identical to those encompassing the 3⬘-splice site in the L2 coding region (Fig. 3B), suggesting that sequence homology might contribute to pairing of the splice VOLUME 290 • NUMBER 21 • MAY 22, 2015


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35

XL1 L1

anti-HPV16 L1 ab FIGURE 3. A, schematic representation of the HPV16 genome. The early and late viral promoters p97 and p670 are indicated. Numbers indicate the nucleotide positions of 5⬘- (filled circles) and 3⬘-splice sites (open circles) or the early and late poly(A) sites pAE and pAL, respectively. LCR, long control region. HPV16 L2 and L1 mRNAs are indicated, as is the novel, alternatively spliced XL1 mRNA. Note that the E1 AUG followed by part of the E4 ORF is fused in frame with a short sequence from the L2 region (but not in the L2 reading frame) and the entire L1 ORF, creating an mRNA with the potential to produce an E4-L1 fusion protein. An mRNA with similar structure identified in HPV31-infected cells is indicated (mRNA E1∧E4*) (15, 16, 39). The E4 and L1 ORFs are not in frame on the HPV31 mRNA. B, sequences immediately upstream of the HPV16 cryptic 5⬘-splice site SD3519 are shown and aligned with sequences encompassing the downstream cryptic 3⬘-splice site SA4980. The splice sites are underlined (the first or last dinucleotides of the intron). The seven nucleotides immediately upstream of SD3519 are 100% homologues to seven nucleotides encompassing SA4980. C, Western blot analysis of extracts from cells transfected with cDNA plasmids expressing either the L1 ORF or the XL1 ORF.



13359


E4

GU

21

73

15

63

35

5 SA

42

3 SD

3 SA

09 27 SA 582 2 SA

02 13 SD 0 88 SD 2 74 SA 6 52 SA 09 4 SA 6 22 SD

E4

hnRNP C Family Interacts with HPV16 Early UTR TABLE 1 Top 20 up-regulated genes in neomycin-resistant, pBEL-neo transfected C33A cell clone 1.4a Gene

Gene accession

-Fold change

1. DSCR8 2. LGALS3BP 3. SESN3 4. PARP8 5. EMB 6. IFI35 7. CADM1 8.C4orf33 9. RALYL 10. IFIT1 11. C5orf13 12. BEX1 13. CA8 14. TINAG 15. TLR3 16. PTGIS 17. LRRC16A 18. ISL1 19. F2R 20. LPCAT3

NR_026839 NM_005567 NM_144665 NM_001178055 NM_198449 NM_005533 NM_014333 NM_173487 NM_173848 NM_001548 NM_004772 NM_018476 NM_004056 NM_014464 NM_003265 NM_000961 NM_017640 NM_002202 NM_001992 NM_005768

9.7 5.2 5.0 5.0 4.5 4.4 4.1 3.7 3.6 3.5 3.3 3.1 3.0 2.9 2.8 2.8 2.7 2.7 2.7 2.6


Down syndrome critical region 8 Lectin, galactose-binding, soluble, 3 binding protein Member of the sestrin family of stress-induced proteins Poly(ADP-ribose) polymerase (PARP) Transmembrane glycoprotein of the immunoglobulin superfamily Interferon-induced protein 35 Cell adhesion molecule 1 Chromosome 4 open reading frame 33 RALY RNA-binding protein-like (RALYL) Interferon-induced protein with tetratricopeptide repeats 1 Neuronal regeneration-related protein (NREP) Brain-expressed, X-linked 1 Carbonic anhydrase VIII, lacking activity Tubulointerstitial nephritis antigen Toll-like receptor 3 Prostaglandin I2 (prostacyclin) synthase (PTGIS) or CYP8A1 Binds CAPZA2 decreases CAPZA2 affinity for actin barbed ends. Member of the LIM/homeodomain family of transcription factors High affinity receptor for activated thrombin coupled to G proteins Acyltransferase

Duplicate samples of RNA from C33A clone 1.4a were subjected to Human Gene 1.0 ST Array analysis in triplicates and compared with the C33A control cells, which were stably transfected with an RSV-neo plasmid. A list of the 20 most up-regulated genes in clone 1.4a compared with C33A identified one RNA-binding protein named RALY-like protein L (RALYL) (Table 1). RALYL is a member of the hnRNP C family of hnRNPs, but little is known about its function. We therefore investigated the role of RALYL in HPV16 gene expression on RALYL. We confirmed that the RALYL protein was overexpressed in cell clone 1.4a compared with C33A control cells (Fig. 4A). RT-PCR of RALYL mRNA revealed that RALYL mRNA is expressed in C33A cells, as expected, but also in human foreskin keratinocytes (HFKs) and in a previously described, in vitro HPV16-immortalized HFK cell line, C97HFK (Fig. 4B) (25). Immunofluorescence on the C33A cells showed a strong nuclear/nucleolar staining of RALYL (Fig. 4C). These results demonstrated that RALYL is expressed in normal human keratinocytes, in HPV16-immortalized keratinocytes, and in at least one cervical cancer cell line (C33A). RALYL Induces HPV16 Late Gene Expression—To investigate whether overexpression of RALYL could induce HPV16 late gene expression, a CMV-driven RALYL cDNA was cotransfected with subgenomic HPV16 expression plasmid pBELsLuc (Fig. 5A) (28). The pBELsLuc plasmid encodes the secreted luciferase (sLuc) gene as a marker for HPV16 late genes, and induction of late genes results in production of sLuc in the cell culture medium. Cotransfection of pBELsLuc with serially diluted pCRALYL revealed a RALYL protein dose-dependent induction of sLuc in 293T-, C33A-, HeLa- and HFK cells (Fig. 5, B and C). RALYL also induced late gene expression from a mutant version of pBELsLuc named pBELMsLuc, in which the splicing silencers downstream of late splice site SA5639 had been mutationally inactivated (Fig. 5, A and D) (29). These results demonstrated that RALYL did not induce HPV16 late gene expression by interfering with the splicing silencer elements in the HPV16 L1 coding region (Fig. 5D). The induction of HPV16 late gene expression required the full RALYL protein, which suggested that RALYL binds to HPV16 mRNAs (Fig. 5E). However, we do not know whether RALYL VOLUME 290 • NUMBER 21 • MAY 22, 2015


sites. In contrast to the HPV31 mRNA, the HPV16 mRNA splicing event fuses the E4 ORF in frame with the L1 ORF, thereby creating an mRNA with the potential to produce an E4-L1 fusion protein (Fig. 3A), at the same time presumably alleviating the inhibitory effect on L1 translation exerted by the strong E1 ATG. To confirm that the XL1 cDNA can express L1 protein, the XL1 cDNA was recreated and inserted after a CMV promoter. Because the L1 coding region contains cis-acting negative RNA elements, a previously described codon-modified sequence of L1 was used. The results revealed that the XL1 cDNA produced L1 protein levels that were similar to those produced by a codon-modified L1 gene described previously (Fig. 3C). We concluded that the XL1 mRNA was fully capable of expressing the novel L1 protein. Elevated Expression of the RNA-binding Protein RALYL in Cells Permissive for HPV16 Late Gene Expression—Next we focused our attention on the other neomycin-resistant and HPV16-positive cell clones 1c, 1d, and 1.4a, which produced high levels of the L1i mRNA (Fig. 2B). Because the RT-PCR shown on the gel was saturated, we first quantified E4 and L1 mRNA levels and calculated the L1/E4 ratio. The results revealed that clone 1.4a displayed the highest L1/E4 ratio, suggesting that alleviation of late gene suppression was highest in clone 1.4a (Fig. 2D). We also sought to explain the high levels of L1i mRNA production in these three clones by monitoring levels of proteins that have been shown previously to affect HPV16 late gene expression, including SRSF1 (ASF/SF2), SRSF9 (SRp30c), hnRNP A1, hnRNP I/PTB, and HuR (23, 24, 29, 31, 37). As one can see, clones 1c and 1d both expressed elevated levels of SRSF1, SRSF9, and HuR, whereas levels of hnRNP I/PTB and of the splicing inhibitory factor hnRNP A1 were relatively similar in these clones (Fig. 2E). The elevated levels of splicing factors SRSF1, SRSF9, and HuR offered a likely explanation for L1 mRNA production in clones 1c and 1d. The levels of hnRNP A1, hnRNP I/PTB, and HuR were not significantly altered in cell clone 1.4a compared with the parental C33A cells, and analysis of SRSF1 and SRSF9 revealed only a minor increase of these two proteins in clone 1.4a compared with C33A cells (Fig. 2E). Clone 1.4a was therefore investigated further.

Gene description


C

C3 3A

1.4 a

A

RALYL actin

RT:

+

-

C9 7H F

K HF

C3 3A

K

B

+

-

+ RALYL mRNA

RALYL

DAPI

OVERLAY

FIGURE 4. A, Western blot analysis of cell extracts from the pBELneo-transfected and G418-selected cell clone 1.4a and the C33A control cells transfected with pRSVneo and selected in G418 with antibodies to RALYL or actin. B, RT-PCR with primers RALYLS and RALYLA (which detect spliced RALYL mRNAs on cDNA from cytoplasmic RNA extracted from various cell lines indicated at the top of the gel). C, DAPI staining and immunofluorescence staining of C33A cells with anti-RALYL antibody and a composite image.


that RALYL and hnRNP C1 induced 3.2- and 9.1-fold higher levels of L1 mRNAs, respectively, whereas L2 mRNAs were induced 1.8- and 2.3-fold, respectively (Fig. 7F). In contrast, the early E4 mRNAs were largely unaffected (Fig. 7F). We concluded that RALYL and hnRNP C1 induced HPV16 late gene expression primarily by activating HPV16 L1 mRNA splicing. Induction of HPV16 Late Gene Expression by RALYL and hnRNP C1 Is Dependent on the HPV16 Early Untranslated Region—We have previously reported that hnRNP C1 can bind to U-rich regions in HPV1 and HPV16 UTR sequences (18, 41, 42). We therefore investigated whether induction of HPV16 late gene expression was dependent on the HPV16 early UTR. pCRALY, pCRALYL, and pChnRNPC1 were individually cotransfected into C33A cells with pBEL, which contains the entire early UTR, or with pBELDUTR (18), in which the early UTR had been deleted (Fig. 8A). RT-PCR revealed that E4 mRNA production from both pBEL and pBELDUTR was relatively unaffected by RALYL and hnRNP C1 (Fig. 8B). In contrast, both RALYL an hnRNP C1 induced production of L1 mRNAs from pBEL but had no or very little effect on pBELDUTR (Fig. 8B), indicating that the HPV16 early UTR was required for the induction of HPV16 L1 mRNA production by RALYL and hnRNP C1. The results were confirmed in a transfection experiment with 293T cells (Fig. 8C). Quantitation of the HPV16 L1 mRNAs by RT-qPCR revealed that L1 mRNA levels were 12 and 14.5 times higher in the presence of RALYL or hnRNP C1, respectively (Fig. 8D). This effect was totally dependent on the HPV16 early UTR, because L1 mRNA levels produced from pBELDUTR were unaffected by RALYL and hnRNP C1 (Fig. 8D). Furthermore, the L2 mRNA levels were also increased in an HPV16 early UTR-dependent manner by the presence of hnRNP C1, but only around 4-fold (Fig. 8D). The levels of L2 mRNAs did not change significantly in the presence of RALYL (Fig. 8D). HPV16 E4 mRNAs were unaffected by hnRNP C1, as expected (Fig. 8D), whereas RALYL appeared to cause a 3-fold increase in E4 mRNAs from both pBEL and pBELDUTR, suggesting a more general effect on mRNA synthesis or stability that was independent of the HPV16 early UTR (Fig. 8D). We concluded that hnRNP C1 and RALYL induced high levels of HPV16 late, spliced L1 mRNAs. It has recently been suggested that hnRNP C1 promotes nuclear export of cellular mRNAs (43). We therefore investigated whether overexpression of hnRNP C1 altered the nucleocytoplasmic ratio of the HPV16 L1 mRNAs. As can be seen JOURNAL OF BIOLOGICAL CHEMISTRY

13361


binds RNA directly. RNA analysis of C33A cells cotransfected with pBELsLuc and pCRALYL revealed that pBELsLuc produced ⬃7-fold higher L1 mRNA levels and 3-fold higher L2 mRNA levels in the presence of RALYL (Fig. 5, F and G). We concluded that RALYL induced HPV16 late gene expression primarily by activating production of spliced L1 mRNA. hnRNP C1 Induces HPV16 Late Gene Expression—To determine whether RALY could induce HPV16 late gene expression also from a full-length HPV16 genome, plasmid pHPV16ANsL (Fig. 6A) (25, 28) was cotransfected with pCRALYL. To create episomal structures of the HPV16 genomes, the HPV16 plasmids were cotransfected with a cre-expressing plasmid that efficiently excises the HPV16 genome at two lox sites flanking the HPV16 genome, as described previously. The results revealed that RALYL induced HPV16 late gene expression from the episomal form of the HPV16 genome cre-loxed from the same plasmid (Fig. 6B). Similar results were obtained in primary HFKs (Fig. 6B). In conclusion, RALYL induced HPV16 late gene expression from both full-length HPV16 genomes and from subgenomic HPV16 expression plasmids. The cellular hnRNP proteins can be divided into various families, and RALYL belongs to the hnRNP C family, which consists of RALY, RALYL, hnRNP C, and hnRNP CL1 (40). We therefore investigated whether RALY and hnRNP C1 could also activate HPV16 late gene expression. Although RALY could not induce HPV16 late gene expression from pBELsLuc (Fig. 7B), it activated late gene expression from pBELMsLuc in a dose-dependent manner (Fig. 7, A and C), suggesting that RALY could overcome suppression of late gene expression only if the inhibitory sequences at late splice site SA5639 were inactivated. These results further supported the idea that the hnRNP C family of proteins induced HPV16 late gene expression independently of the splicing silencers in the L1 coding region. In contrast to RALY, hnRNP C1 induced HPV16 late gene expression from both pBELsLuc and pBELMsLuc and to a higher extent than both RALY and RALYL (Fig. 7, B and C). We also transfected pCRALYL and pChnRNPC1 into a previously described reporter cell line, C33A2 (28), which contains pBELsLuc stably integrated into its genome. The results confirmed that both RALYL and hnRNP C1 could induce HPV16 late gene expression from pBELsLuc and that hnRNP C1 did so more efficiently than RALY and RALYL (Fig. 7D). RT-PCR revealed that both proteins induced L1 mRNA production (Fig. 7E). Quantitation of the HPV16 mRNA levels by RT-qPCR showed


pB

LCR

0

L1

E5

10

E1

E7

pCRALYL (ng)

L2

0 30 0

E2 E4

10

p97 E6

pAL

pAE

20

p670

EL sLu c mo ck

B

HPV-16 genome

50

A

7372

E4a

pAL

pAE E2 E4

E1

CMV 88 13 0 02

25 27 82 09

33

L2 E5

pBELMsLuc E2 E4

E1 88 13 0 02

33

Mut IRES sLuc 42

36

58

15

32

()

56

73

39

21

pBELsLuc

50 0 0

10

50 10 2 3 0 00 00

35

5 4

30 25 20 15

2

0

50 10 2 3 0 00 00

0

E

600 80

200

sLuc x 10

4

200 50

100 0

0

0

RT:

-

+

-

G

L

LY

RA

pC

L2 L1i

L 86 LY L0 CRA p

pC 8

+

L1

E4

L LY A -R L LY

L08

pC

L

6 LY L08 CRA p pC

L

6

L 6 LY L08 CRA p pC

L LY 86 L0 CRA p

7 6 5 4 3 2 1 pBELsLuc


aa291

-C

0 30 0 pCRALYL

LY

0 30 0 pCRALYL

aa134

0 A -R

0 pCRALYL

C-RALYL

20

-N

30

aa133

pC

0

aa1

40

RA

0 pCRALYL

aa291

N-RALYL

60

pC

30

20

aa1

80

pC

0

25 20 15 10 5 0

RALYL

100

2

40

45 40 35 30

&

100

120

1 P-

300

60

sLuc x 10

400

150

0

pCRALYL

RN

sLuc x 10

4

4

4

500

30

0

HFK

4

100

HeLa

sLuc x 10

250

0

50 10 2 3 0 00 00

pCRALYL

pBELMsLuc 700

10

pCRALYL

C33A

3

1

5 10

pCRALYL

293T

4

10

0

D

HFK

L2 mRNA (fold induction)

100

6

sLuc x 10

150

40

L1/L1i mRNA (fold induction)

sLuc x 10

4

200

HeLa

4

250

50 45 40 35 30 25 20 15 10 5 0

sLuc x 10

300

C33A

6

pC

5 4 3 2 1 pBELsLuc

VOLUME 290 • NUMBER 21 • MAY 22, 2015


25 27 82 09

L2 E5

293T

4

21

pAL

C sLuc x 10

73

39

pAE

CMV

M

()

56

15

32

IRES sLuc

L1 42

36

58

actin

L1as

L2s

pBELsLuc

sLuc x 10

21 73

39 56

15 42

32 36

58 33

09 27 2 8 25

02 13 0

6

2

88

74

22

6 52 9

40

L2 L1 L1i 880s

F

RALYL

E4

Major late mRNAs:

hnRNP C Family Interacts with HPV16 Early UTR loxP p97

A

pAL p97 p670

16S

B

16A

100

16A sLuc

16S

pHPV16ANsL

60

S IR E

+

pAE

4

4

HPV-16

+pCAGSS-nlscre

60 40

30 20 10

0

0

30 0

0 pCRALYL (ng)

0

0

RSV-neo SV40

40

20

30

pAE

L1

HFK

50

80 sLuc x 10

pAL

loxP

HeLa

sLuc x 10

loxP

p670

pCRALYL (ng)

L2

from the RT-PCR, hnRNP C1 induced high levels of L1 mRNAs both in the nucleus and in the cytoplasm, whereas E4 mRNA levels were unaffected (Fig. 8E). Analysis of unspliced and spliced actin mRNAs confirmed proper cell fractionation (Fig. 8E). Furthermore, the nuclear levels of L1 mRNAs produced from pBELDUTR were unaffected by hnRNP C1 (Fig. 8F), confirming that the effect of hnRNP C1 on HPV16 late gene expression was dependent on the HPV16 early UTR and extending these observations to include nuclear events. These results demonstrated that hnRNP C1 acted on the HPV16 mRNAs in the nucleus, prior to nuclear export, consistent with an enhancing effect of hnRNP C1 on HPV16 late L1 mRNA splicing and an inhibitory effect on HPV16 early polyadenylation. We speculated that hnRNP C1 induced L1 mRNA splicing by alleviating inhibition of HPV16 late 5⬘-splice site SD3632, thereby enhancing HPV16 L1 mRNA splicing. hnRNP C1 Enhances Splicing from HPV16 Late 5⬘-Splice Site SD3632—To test the idea that hnRNP C1 enhances splicing from HPV16 late 5⬘-splice site SD3632, we transfected hnRNP C1 with HPV16 reporter plasmid pBSpD1MCAT (Fig. 9A) (28), which contains only two HPV16 splice sites: late splice sites SD3632 and SA5639. In pBSpD1MCAT, splicing silencers in the L1 coding region at SA5639 were mutationally inactivated (L1M), and late gene expression is suppressed primarily by splicing silencers at SD3632, as reported previously (29).

Enhancement of HPV16 late L1 mRNA splicing from pBSpD1MCAT therefore monitors the activity of HPV16 late 5⬘-splice site SD3632. Cotransfection of pBSpD1MCAT with hnRNP C1 resulted in a small but significant effect on HPV16 late L1 mRNA production (Fig. 9B), confirming that hnRNP C1 can activate SD3632. We have previously reported that HPV16 late 5⬘-splice site SD3632 is suppressed by an adjacent splicing silencer located upstream of SD3632 and consisting of two ATAGTA motifs (28). Replacing this sequence upstream of SD3632 in pBSpD1MCAT with four copies of ATAGTA, as in p4xATAGTA, or four copies of ACACTG, a mutant-inactive sequence, as in p4xMUT (Fig. 9A) (28), revealed a 23-fold difference in L1 mRNA production between the two plasmids (Fig. 9C). Overexpression of hnRNP C1 induced HPV16 L1 mRNA production from p4xATAGTA but not from p4xMUT (Fig. 9D), suggesting either that hnRNP C1 acted on the splicing silencer directly or that suppression of HPV16 SD3632 is required for induction of HPV16 late mRNA splicing by hnRNP C1. RALYL also induced HPV16 L1 mRNA production from p4xATAGTA but not from p4xMUT (Fig. 9E). The effect of RALYL was lower than the effect of hnRNP C1. We concluded that hnRNP C1 and RALYL can induce HPV16 late L1 mRNA production by derepressing the HPV16 late 5⬘-splice site SD3632.

FIGURE 5. A, schematic representation of the HPV16 genome and subgenomic HPV16 expression plasmids. The early and late viral promoters p97 and p670 and the long control region (LCR) are indicated. Numbers indicate nucleotide positions of 5⬘- (filled circles) and 3⬘-splice sites (open circles) or the early and late poly(A) sites pAE and pAL, respectively. The major late mRNAs are indicated. A schematic representation of the pBELsLuc and pBELMsLuc plasmids is shown. The human CMV, the poliovirus IRES, and the sLuc gene are indicated. Locations of mutations that inactivate the splicing silencers downstream of SA5639 are shown (29). B, Western blot analysis with antibodies to RALYL or actin of cell extracts from C33A cells cotransfected with pBELsLuc and serially diluted pCRALYL. C, sLuc enzyme activity produced by 293T, C33A, HeLa, or HFK cells transfected with pBELsLuc (28) in the absence or presence of serially diluted pCRALYL plasmid. Mean values and S.D. (error bars) are shown. D, sLuc enzyme activity produced by 293T, C33A, HeLa, or HFK cells transfected with pBELMsLuc (28) in the absence or presence of serially diluted pCRALYL plasmid. Mean values and S.D. are shown. E, sLuc enzyme activity produced by C33A cells transfected with pBELsLuc (28) and pCRALYL or the various RALYL deletion mutants indicated to the right. Mean values and S.D. are shown. The indicated RNA-binding domains RNP-1 and -2 are predicted, and RNA-binding assays have not been performed. F, RT-PCR on cytoplasmic RNA extracted from C33A cells transfected with pBELsLuc and empty CMV promoter plasmid (pCL086) (32) or pCRALYL. RT-PCR was performed with primers 880S and E4a to detect spliced E4 mRNAs, with 880S and L1A to detect L1 and L1I mRNAs, and with L2S and L1A to detect L2 mRNAs. Because the latter did not span a splice junction, RT-PCR was performed in the absence (⫺RT) or presence of RT (⫹RT). G, RT-qPCR on the HPV16 L1 and L2 mRNAs produced by pBELsLuc cotransfected with either empty vector (pC) or pCRALYL.



13363


FIGURE 6. A, schematic representation of genomic HPV16 plasmid pHPV16ANSL (25). LoxP sites and HPV16 early (p97) and late (p670) promoters and early (pAE) and late (pAL) poly(A) signals are indicated. The arrows denote positions of PCR primers 16S and 16A. The cassette encoding the Rous sarcoma virus long terminal repeat promoter driving the neomycin resistance gene, followed by the simian virus 40 poly(A) signal, is indicated in black. The effect of the cre recombinase on these plasmids is illustrated, as described previously (25). L1 and L2, late HPV16 genes L1 and L2. B, sLuc activity produced by HeLa cells or HFK cells transfected with pHPV16ANSL in the absence or presence of the RALYL expression plasmid pCRALYL. Error bars, S.D.

hnRNP C Family Interacts with HPV16 Early UTR B 50

60

4

40

sLuc x 10

sLuc x 10

4

50

30

pBELsLuc

pBELMsLuc 500

45

450

40

400

35

350

30

300

4

pBELMsLuc 70

sLuc x 10

A

25 20

250 200

20

15

150

10

10

100

5

50

0 pCRALY (ng):

0

50

0

10 20 30 0 0 0

-

0

pC

p p RA CRA Chn LY LY RN L

-

pC

p p RA CRA Chn LY LY RN L

PC

1

oc m

0 -

20

0

pChnRNPC1(ng):

10

RALY

50

k

10

0 20 0 30 0 pB EL M m s oc Luc k

1

hnRNP C1 actin

M

70 60 50 sLuc x 10

3

E4

40 30 L1

20

L1i

10 0

pC p L0 CRA hn 86 LY RN L P

C33A2 reporter cell line

pC

C1

F Fold difference of L1/L1i, L2 or E4 mRNA levels

80

pC

C33A2 reporter cell line

L0 pC 86 RA L pC YL hn RN PC

1

E

D

C33A2 reporter cell line 10

L1mRNA L2 mRNA E4 mRNA

8

6

4

2 1

pC pC pC pC p RA hn RA Chn RA hn R L LY RN Y NP LYL RNP L PC L C1 C1 1

pC

FIGURE 7. A, sLuc enzyme activity produced by C33A cells transfected with subgenomic HPV16 plasmid pBELMsLuc (28) in the absence or presence of serially diluted pCRALYL plasmid. Mean values and S.D. (error bars) are shown. B, sLuc enzyme activity produced by C33A cells transfected with pBELsLuc or pBELMsLuc (28) in the absence or presence of CMV promoter-driven plasmid pCRALY, pCRALYL, or pChnRNPC1. Mean values and S.D. are shown. C, Western blot analysis with antibodies to RALY, hnRNP C1, or actin of cell extracts from C33A cells cotransfected with pBELMsLuc and serially diluted pCRALY or pChnRNPC1. Note that also endogenous hnRNP C1 protein is detected by the hnRNP C1 antibody. D, sLuc enzyme activity produced by the previously described C33A2 reporter cell line containing stably integrated copies of the pBELsLuc plasmid (28) and transiently transfected with empty plasmid pCL086 (32) or with RALY or hnRNP C1 expression plasmid pCRALY or pChnRNPC1. E, RT-PCR on cytoplasmic RNA extracted from the C33A2 reporter cell line transfected with empty plasmid pCL086, pCRALYL, or pChnRNPC1. RT-PCR was performed with primers 880S and E4a to detect spliced E4 mRNAs and with 880S and L1A to detect L1 and L1i mRNAs. F, -fold induction of HPV16 L1, L2, or E4 mRNAs in the C33A2 reporter cell line (28) transfected with pCRALYL or pChnRNPC1 over reporter cell C33A2 transfected with empty vector (pCL086) (32). RT-qPCR was performed with primers 880S and E4a to detect spliced E4 mRNAs, with 880S and L1A to detect L1 and L1i mRNAs, and with L2S and L1A to detect L2 mRNAs. Error bars, S.D.

RALYL and hnRNP C1 Interact with the HPV16 Early Untranslated Region—Because the results suggested that RALYL and hnRNP C1 interacted directly with the HPV16 early UTR to activate mRNA splicing, we performed an RNA CLIP analysis on either pBEL- or pBELDUTR-transfected cells in the absence or presence of RALYL or hnRNP C1. We monitored levels of immunoprecipitated HPV16 E4 mRNAs because they contain the HPV16 early UTR. As can be seen from Fig.


10A, HPV16 E4 mRNA could be immunoprecipitated with antibodies to the Myc tag on the RALYL protein (Fig. 10A), demonstrating that RALY binds to these mRNAs in the living cells. The levels of E4 mRNAs were ⬎100-fold higher than those pulled down by the IgG negative control (Fig. 10B). Furthermore, the anti-Myc tag antibodies failed to immunoprecipitate HPV16 E4 mRNAs from cells transfected with the HPV16 early UTR-less plasmid pBELDUTR (Fig. 10C), strongly VOLUME 290 • NUMBER 21 • MAY 22, 2015


C pCRALY (ng):

50

PC

hnRNP C Family Interacts with HPV16 Early UTR A pBEL

E2 E4

E1

CMV

SD SD 88 13 0 02

pAE

pAL

L2

E5

SA SA SA 33 25 27 58 82 09

L1

SD

()

SA

73

56

36

21

39

32

pAE

E5

pAE

)

(

4212

4105

E4 mRNAs

EL

1 hn

RA

pB

LY L

RN PC

1 LY

LY L

RN PC

RA

RA

mo ck

EL D pB

LY

UT

EL pB

RN PC

LY L

hn

RA

RA

hn

LY

RN PC

LY L

LY

RA

RA

M

R

1

L1 mRNAs

1

B

hn

E5

RA

pBEL: pBELDUTR:

E4

* pBEL

20

-

hn

pBELDUTR

D

RN

PC RN -

hn

-

LY L

M

-

RA

-

-

M

RA

LY L

C

PC

1

1

pBELDUTR

+RT

E4 actin

-RT

L1/L1i mRNA

16

12

12

8

8

4

4

1 RALYL:

L1 L1i

+RT

M pBEL

pBELDUTR

pBEL

mRNA (fold induction)

M

pBELDUTR

R

UT

E

D EL EL pB pB

F

pBEL

N

C

+ - + hnRNPC1: pBEL pBELDUTR

L1

L1

N

E4

N

actin-U

C

actin-U

N

actin-S

C

actin-S

L2 mRNA

8

6

6

4

4

2 1

2 1

RALYL:

L1/L1i mRNA

- + pBEL

10

8

-

N

1

-

10

-RT

20

16


pBEL

L2 mRNA

- + - + hnRNPC1: - + pBEL pBEL pBELDUTR

10

E4 mRNA

- + pBELDUTR

10

- + pBELDUTR E4 mRNA

E4

actin-U

actin-S

-

-

+

+

-

-

+

8

8

6

6

4

4

2

2 1

1 RALYL:

hn

- + pBEL

hnRNPC1: - + pBELDUTR

- + pBEL

- + pBELDUTR

C1

P RN

C1

P RN

1

C1

PC

RN

P RN

hn

hn

hn


+


L1i


13365


L1 L1i

hnRNP C Family Interacts with HPV16 Early UTR A p4xATAGTA: AATAGTAATAGTAATAGTAATAGTAATTTAAAAGT p4xMUT: ACACTGCCACTGCCACTGCCACTGCATTTAAAAGT

pAL

pAE L2 E4

pBSpD1MCAT:

L1M IRES

E5

CAT

LCR

CMV 73

21

39

32

56 SD

36 SD

99

15 42 TR eU

35

7372

mRNAs:

E4 U L1

-

RALYL:

-

+

UT xM

xM

+

p4

UT

A TA xA

+

A

-

+

1

GT

:

pB

p4

TA p4

xA

-

hnRNPC1:

GT

GT

A:

+

CA M

1

1

Sp

D1

2

TA

4

4

2

xA

8

Fold induction of L1 mRNA levels

12

L1 mRNA

3

p4

16

6

UT

-

20

T

hnRNPC1:

24

E

L1 mRNA

8 Fold induction of L1 mRNA levels


1

D

FIGURE 9. A, schematic representation of the subgenomic HPV16 plasmid p4xATAGTA, p4xMUT, and pBSpD1MCAT (28). The 5⬘- (filled circles) and 3⬘-splice sites (open circles) and the early and late poly(A) sites pAE and pAL are indicated. pBEL is driven by the human CMV promoter. The four copies of the wild type active or mutant inactive motifs of the splicing silencer inserted upstream SD3632 are shown. SD3632 is indicated by a filled triangle. mRNAs produced by the plasmids are indicated. Nucleotide positions refer to the HPV16R sequence. B, RT-qPCR on the HPV16 L1 mRNAs produced in C33A cells transfected with pBSpD1MCAT (28) in the absence or presence of pChnRNPC1. C, RT-qPCR on the HPV16 L1 mRNAs produced in C33A cells transfected with p4xATAGTA or p4xMUT (28). D, RT-qPCR on the HPV16 L1 mRNAs produced in C33A cells transfected with p4xATAGTA or p4xMUT (28) in the absence or presence of pChnRNPC1 or RALYL (E). Error bars, S.D.

suggesting that RALYL binds directly to the HPV16 early UTR in the living cells. Similar experiments were performed with hnRNP C1, with the exception that an antibody to the native hnRNP C1 protein was used that also detected endogenous hnRNP C1. Therefore, the hnRNP C1 antibody pulled down HPV16 E4 mRNAs both in the absence and in the presence of cotransfected hnRNP C1 plasmid, but with different efficiency (Fig. 10D), whereas plain IgG did not (Fig. 10D). However, the HPV16 E4 mRNAs could not be efficiently immunoprecipitated with anti-hnRNP C1 antibody from cells transfected with pChnRNP C1 and

pBELDUTR, which lacks the HPV16 early UTR (Fig. 10E). The difference in immunoprecipitation of E4 mRNA was seen only when hnRNP C1 was overexpressed (Fig. 10F), which is in line with the increase in HPV16 late gene expression from pBEL, and not from pBELDUTR, in the presence of exogenous hnRNP C1 (Fig. (8, B–D and F). Quantitation of immunoprecipitated E4 mRNA levels from pBEL and pBELDUTR was determined in the absence of exogenous hnRNP C1. The results revealed that levels of immunoprecipitated E4 mRNAs produced from pBEL plasmid increased by 2.5-fold as a result of hnRNP C1 overexpression, whereas the levels of E4 mRNAs immunoprecipitated

FIGURE 8. A, schematic representation of the subgenomic HPV16 plasmid pBEL (29). The 5⬘- (filled circles) and 3⬘-splice sites (open circles) and the early and late poly(A) sites pAE and pAL are indicated. pBEL is driven by the human CMV promoter (29). An enlargement of the region upstream of pAE indicates the deletion of the entire early UTR in plasmid pBELDUTR (18). The nucleotide positions of the borders are indicated. Nucleotide positions refer to the HPV16R sequence. B, RT-PCR on total RNA extracted from the C33A cells transfected with pBEL (29) or pBELDUTR in the absence or presence of plasmids expressing RALY, RALYL, or hnRNP C1. RT-PCR was performed with primers 880S and E4a to detect spliced E4 mRNAs and with 880S and L1A to detect L1 and L1i mRNAs. *, a primer-dimer band. C, RT-PCR on cytoplasmic RNA extracted from the 293T cells transfected with pBEL (29) or pBELDUTR (18) in the absence or presence of plasmids expressing RALYL or hnRNP C1. RT-PCR was performed with primers 880S and E4a to detect spliced E4 mRNAs, with 880S and L1A to detect L1 and L1i mRNAs, and with ACTINS and ACTINA to monitor spliced actin mRNA. RT-PCR was performed in the absence (⫺RT) or presence (⫹RT) of reverse transcriptase. D, RT-qPCR on the HPV16 E4 and L1 mRNAs produced in C33A cells transfected with pBEL (29) or pBELDUTR (18) in the absence or presence of pCRALYL or pChnRNPC1. E, RT-PCR on nuclear (N) and cytoplasmic (C) RNA extracted from C33A cells transfected with pBEL in the absence or presence of plasmid expressing hnRNP C1. RT-PCR was performed with primers 880S and E4a to detect spliced E4 mRNAs, with 880S and L1A to detect L1 and L1i mRNAs, with actins and actina to monitor spliced actin mRNA, and with ACTINS-1 and ACTINA to detect unspliced actin pre-mRNA. F, RT-PCR on nuclear (N) RNA extracted from C33A cells transfected with pBEL (29) or pBELDUTR (18) in the absence or presence of plasmid expressing hnRNP C1. RT-PCR was performed with primers 880S and E4a to detect spliced E4 mRNAs, with 880S and L1A to detect L1 and L1i mRNAs, with actins and actina to monitor spliced actin mRNA, and with ACTINS-1 and ACTINA to detect unspliced actin pre-mRNA. Error bars, S.D.





2

L1 mRNA

28

p4

C

xM

L1 mRNA

3

p4

B

+ E4

M

1

4

-

+

pBELDUTR

pBEL

M

E4

E

pBEL +

ut

+

+ -

M

pBEL -

pCRALYL + - - + -

+ -

inp

D pChnRNPC1:

140 120 100 80 60 40 20

G Ig yc: m α-

2 3 pBEL

pCRALYL + - +

α-myc: IgG:

pB E

-

-

+

pB EL pB EL DU TR pB EL pB EL DU TR

α-myc: + IgG: -

C

B

pCRALYL

Fold increase in E4 mRNA

A

L


pChnRNPC1: + + M

+ +

E4 E4

α-h inp nR ut NP C1

input

-

DU EL

EL

86 L0 - +

+

-

+

E4

α-hnRNPC1

3 Fold increase in E4 mRNA

+ M

pB

pB -

pB

+

pC

DU

TR pChnRNPC1: - + -

EL

EL

86

pB

L0 pC

F

TR

G

E4 mRNA

2,5 2 1,5 1

input pChnRNPC1:

- + - + pBEL pBELDUTR

FIGURE 10. A, RT-PCR on HPV16 E4 mRNAs with primers 880S and E4a on cDNA synthesized from in vivo UV cross-linked, RNA-protein complexes immunoprecipitated with monospecific antibodies to the c-Myc tag on RALYL or mouse IgG. Cells were transfected with pBEL in the absence or presence of pCRALYL. B, RT-qPCR on the HPV16 E4 mRNAs immunoprecipitated with antibodies to the c-Myc tag on RALYL or mouse IgG using primers 880S and E4a. C, RT-PCR on HPV16 E4 mRNAs with primers 880S and E4a on cDNA synthesized from in vivo UV-cross-linked RNA-protein complexes immunoprecipitated with monospecific antibodies to the c-Myc tag on RALYL or mouse IgG. Cells where transfected with pBEL (29) or pBELDUTR (18) in the absence or presence of pCRALYL. D, RT-PCR on HPV16 E4 mRNAs with primers 880S and E4a on cDNA synthesized from in vivo UV-cross-linked RNA-protein complexes immunoprecipitated with IgG or monospecific antibodies to hnRNP C1. Cells were transfected with pBEL (29) in the absence or presence of pChnRNPC1. RT-PCR was also performed on the input cellular extract prior to immunoprecipitation, and 2% of the PCR was loaded on the gel. E, RT-PCR on HPV16 E4 mRNAs with primers 880S and E4a on cDNA synthesized from in vivo UV cross-linked, RNA-protein complexes immunoprecipitated with hnRNP C1 antibody. Cells were transfected with pBEL (29) or pBELDUTR (18) in the presence of pChnRNPC1. RT-PCR was also performed on the input cellular extract prior to immunoprecipitation, and 10% of the PCR-reaction was loaded on the gel. F, RT-PCR on HPV16 E4 mRNAs with primers 880S and E4a on cDNA synthesized from in vivo UV cross-linked, RNA-protein complexes immunoprecipitated with hnRNP C1 antibody. Cells were transfected with empty plasmid (pCL086), pBEL (29), or pBELDUTR (18) in the absence or presence of pChnRNPC1. RT-PCR was also performed on the input cellular extract prior to immunoprecipitation, and 10% was loaded on the gel. G, RT-qPCR on HPV16 E4 mRNAs detected by immunoprecipitation with anti-hnRNP C1 antibody in pBEL- or pBELDUTR-transfected cells in the absence or presence of cotransfected pChnRNPC1 plasmid. Error bars, S.D.

from pBELDUTR-transfected cells were unaffected (Fig. 10G). These results suggested that the HPV16 early UTR is a major hnRNP C1 binding site on HPV16 mRNAs and further strengthened the conclusion that hnRNP C1 can bind to the HPV16 early UTR and control HPV16 late gene expression. hnRNP C1 Binds to the HPV16 Early Untranslated Region in Vitro and Is Present in the Protein Complex on the Splicing Silencer Upstream of SD3632—Our results suggested that hnRNP C1 and RALYL bind to the HPV16 early UTR in living cells. To investigate whether RALYL and hnRNP C1 bind to the HPV16 early UTR in vitro, we performed pull-down experiments with biotinylated oligonucleotides representing wild type and mutant HPV16 early UTR sequences (Fig. 11A). The MAY 22, 2015 • VOLUME 290 • NUMBER 21

results revealed that WT RNA BRUWT2 could pull down hnRNP C1 and RALYL from a nuclear extract, whereas the mutant RNA (BRUMUT2) was less efficient and pulled down very low levels of hnRNP C1 and RALYL (Fig. 11B). We concluded that hnRNP C1 and RALYL bind to the HPV16 early UTR with sequence specificity. If there is cross-talk between hnRNP C1 and/or RALYL in the HPV16 early UTR protein complex and the splicing silencer complex containing primarily hnRNP D proteins, hnRNP D might be pulled down by the BRUWT2 RNA too. Indeed, low levels of hnRNP D were pulled down by the BRUWT2 RNA but not by the BRUMUT2 RNA (Fig. 11B). Based on these results, one should be able to find hnRNP C1 and RALYL in the protein complex that forms on JOURNAL OF BIOLOGICAL CHEMISTRY

13367


α-hnRNPC1

IgG

hnRNP C Family Interacts with HPV16 Early UTR A

BRUWT2: uuguuuguuuguuuguuuuuu BRUMUT2: GuguGuguGuguGuguGuuGu BRAUA: aauaguaauaguaauaguaauaguaa

RNA

CU C BR

AU A

s ad Be

t pu In

BR

UM UT 2

C

BR

s ad

BR

In

Be

pu

t

B

UW

T2

BRCUC: aCACUGCCACUGCCACUGCCACUGCa

hnRNP C1 hnRNP C1

RALYL

RALYL

hnRNP D

D UTR probe

HIShnRNP C1:

HIShnRNP C1:

-

-

R

R

UT

BP

DU

C1

Comp:

FIGURE 11. A, sequences of the RNA oligonucleotides used for protein pull-down experiments. BRUWT2 and BRUMUT2 represent the wild type and mutant U-rich region of the HPV16 early untranslated region, respectively, whereas RNA oligonucleotides BRAUA and BRCUC contain five copies of the wild type (AUAGUA) or mutant RNA (CACUGC) motif in the splicing silencer upstream of HPV16 SD3632, respectively. B, Western blot analysis of proteins pulled down by RNA oligonucleotides BRUWT2 and BRUMUT2 using antibodies to hnRNP C1, RALYL, or hnRNP D. C, Western blot analysis of proteins pulled down by RNA oligonucleotides BRAUA and BRCUC using antibodies to hnRNP C1, RALYL, or hnRNP D. D, UV-cross-linking of radiolabeled HPV16 early UTR RNA to recombinant His-tagged hnRNP C1 protein in the absence or presence of competitor RNA. C1BP, RNA consisting four copies of hnRNP C1 binding site AUUUUUA; DUTR, HPV16 early UTR with a deletion that removes the U-rich region of the UTR; UTR, full-length HPV16 early UTR.

the splicing silencer sequence upstream of SD3632. Proteins pulled down by the HPV16 wild type splicing silencer RNA (BRAUA) were also subjected to Western blotting with hnRNP C1 and RALYL antibody (Fig. 11C). Both hnRNP C1 and RALYL were pulled down by the splicing silencer RNA BRAUA (Fig. 11C) and less efficiently by the mutant splicing silencer RNA BRCUC (Fig. 11C). As expected, hnRNP D bound to the WT splicing silencer and not to the mutant (Fig. 11C). Finally, we wished to investigate whether hnRNP C1 binds directly to the HPV16 early UTR RNA. We performed an RNA-protein UV cross-linking experiment with in vitro synthesized, radiolabeled RNA representing the entire early UTR and recombinant His-tagged hnRNP C1. The results demonstrated that hnRNP C1 binds directly to the HPV16 UTR probe (Fig. 11D) and demonstrated that this interaction was specific for the U-rich region of the HPV16 early UTR because preincubation with a cold RNA competitor encompassing the entire UTR competed well with the probe for hnRNP C1, whereas a shorter HPV16 UTR RNA lacking the U-rich 3⬘-end did not (Fig. 11D). The full UTR competed to the same extent as an artificial RNA containing five copies of and hnRNP C1 binding site AUUUUUA (C1BP) (Fig. 11D). Taken together, these results strongly suggested that


hnRNP C1 derepresses HPV16 late 5⬘-splice site SD3632 by interfering with the splicing silencer complex upstream of SD3632.

Discussion We have previously reported that the HPV16 late 5⬘-splice site SD3632 is strongly suppressed in cervical cancer cells (22, 28). These results were further substantiated here. When we selected cervical cancer cells that were permissive for HPV16 late gene expression, many of these cells bypassed SD3632 by activating novel, perhaps cryptic, splice sites to circumvent SD3632. Whether these splicing events are part of the HPV16 gene expression program and take place in vivo remains to be seen. However, utilization of alternative splice sites as well as direct splicing from SD880 to SA5639 to circumvent SD3632 certainly supports the idea that SD3632 is strongly suppressed in cancer cells. The results also suggested that suppression is mediated by factors that are essential for dividing cells or essential to the tumor cell phenotype, because very rarely was SD3632 used in the permissive HPV16 clones. Instead, other splice sites that obviated the need for SD3632 were used. Indeed, the hnRNP D proteins and hnRNP A2/B1 that supVOLUME 290 • NUMBER 21 • MAY 22, 2015


hnRNP D



the splicing silencer complex upstream of SD3632 (28). Indeed, we found that hnRNP C1 was present in this splicing silencer complex, but at low levels. The hnRNP C family of hnRNPs consists of hnRNP C1/C2, hnRNP CL1, RALY, and RALYL (40), and it has been shown that hnRNP A2/B1 and hnRNP D are part of the interactome of RALY (54), suggesting that the hnRNP C1, hnRNP D, and hnRNP A2/B1 proteins are in contact in the cells. hnRNP C1/C2 is one of the more common RNA-binding proteins in the cell and is known as a strictly nuclear protein (55). Despite its nuclear localization, it is involved in many aspects of RNA processing, ranging from mRNA polyadenylation to splicing, nuclear export, and translation. The subcellular localization of hnRNP C1 can be altered, depending on the conditions of the cell. For example, cells that are stressed or simply mitotic exhibit elevated levels of hnRNP C1 protein in the cytoplasm (56, 57). Thus, hnRNP C1 could also control translation of the cellular amyloid precursor protein mRNA (58). Effects on translation are also associated with a perturbed nucleocytoplasmic ratio of the hnRNP C1 protein brought about by virus infections. hnRNP C1 has been shown to bind specifically to the 5⬘- and/or 3⬘-untranslated regions of various RNA viruses that replicate in the cytoplasm (59 – 62) and to control viral replication (60). Although the subcellular distribution of hnRNP C1 in uninfected or HPV16-infected human keratinocytes has not been studied, levels of expression of hnRNP C1 have been investigated by immunohistochemistry in low or high grade cervical lesions as well as in cervical cancer biopsies obtained for the purpose of cervical cancer screening (44). The results revealed that hnRNP C1 was overexpressed in the HPV-infected cells and that the levels of hnRNP C1 correlated with the severity of the HPV16-infected lesion (44). These results strongly suggested that the levels of hnRNP C1 increased as a result of the HPV16 infection. In contrast, in low grade HPV16-positive lesions, as in uninfected cervical epithelium, the levels of hnRNP C1 were highest in suprabasal layers and decreased again at the very top of the epithelium (44). Thus, high hnRNP C1 expression correlates with high levels of HPV16 replication, suggesting that hnRNP C1 may contribute to HPV16 late gene expression as viral DNA replication peaks. In conclusion, it appears that many viruses depend on the presence of hnRNP C1 in the infected cell and that many also alter the levels or subcellular distribution of hnRNP C1. Acknowledgments—We are grateful to all members of Stefan Schwartz’s group for reagents and discussion. We thank students on the M.Sc. course BIMM31 in molecular virology at Lund University, summer student Sara Nolbrant, and M.Sc. project student Jon Eriksson for contributing to this project.

References 1. Walboomers, J. M., Jacobs, M. V., Manos, M. M., Bosch, F. X., Kummer, J. A., Shah, K. V., Snijders, P. J., Peto, J., Meijer, C. J., and Munõz, N. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189, 12–19 2. Bouvard, V., Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Benbrahim-Tallaa, L., Guha, N., Freeman, C., Galichet, L., Cogliano, V., and WHO International Agency for Research on Cancer Monograph


13369


press SD3632 are both overexpressed in cervical cancer cells (44), suggesting an important role for these proteins in carcinogenesis. A number of alternatively spliced HPV16 mRNAs encoding L1 have been found in HPV16-infected cells (15, 16). However, the major L1 mRNA candidate mRNAs expressed from the late promoter p670 are either spliced SD880-SA3358-SD3632SA5639 (L1 mRNA) or SD880-SA5639 (L1i mRNA), and both contain the E1/E4 ATG upstream of the L1 ATG. Because the E1/E4 ATG matches the Kozak sequence for efficient translation initiation very well (45), it is likely to constitute an obstacle to L1 translation. Indeed, it has been shown to have a negative impact on translation of L1 (46). The novel XL1 mRNA detected here solves that problem by fusing the E1/E4 ATG to the L1 ORF with the E1/E4 ORF, thereby using the strong E1/E4 ATG for L1 production. However, the E1/E4 ORF and the little exon in L2 add 70 amino acids to the N terminus of L1. It remains to be seen whether this protein is produced in vivo and whether it can function as a capsid protein. A similarly spliced mRNA found in HPV31 that does not fuse the E1/E4 ATG to L1 (39), would seem to argue that these splicing events occur to bypass suppressed splice sites rather than translation start codons. Human cells contain a large number of RNA binding hnRNPs, many of which have been shown previously to interact with HPV mRNAs and/or regulate HPV gene expression, including hnRNP A1 (29 –31, 47, 48), hnRNP A2/B1 (28), hnRNP C1/C2 (18, 41, 42), hnRNP D (28), hnRNP E (49), hnRNP H (21), hnRNP I (37), and hnRNP K (49). We have shown that hnRNP C1 binds specifically to the HPV16 early UTR. Because the HPV16 early UTR stimulates polyadenylation at pAE (18) and, as such, prevents HPV16 late gene expression, it was surprising to find that hnRNP C1 and, to a lesser extent, RALYL enhanced HPV16 late gene expression by activating splicing of HPV16 late 5⬘-splice site SD3632. Binding of hnRNP C1 to the HPV16 early UTR appeared to have less impact on HPV16 early polyadenylation, because the effect of hnRNP C1 on the levels of L2 mRNAs in general was lower than on the spliced L1 mRNAs. A recent publication of individual nucleotide resolution UV cross-linking and immunoprecipitation data on hnRNP C1 revealed that hnRNP C1 can bind to the polypyrimidine tract upstream of 3⬘- splice sites and to the polyU tract downstream of 5⬘-splice sites (50). The U-rich HPV16 early UTR may therefore function as a landing pad for hnRNP C1 proteins that control the activity of HPV16 late 5⬘-splice site SD3632. The role of hnRNP C1 in the regulation of splicing of cellular mRNAs has often been as a repressor of splicing (50) or exon exclusion (51), but hnRNP C1-mediated enhancement of splicing has been suggested as well (52). This is consistent with the activation of HPV16 late 5⬘-splice site SD3632 reported here. We speculate that this effect of hnRNP C1 RNA binding is dependent on the exact context of the RNA binding site, as recently suggested for hnRNP L (53). Our results indicate that hnRNP C1 and RALYL can activate splicing from the suppressed HPV16 late 5⬘-splice site SD3632 by interacting with the HPV16 early UTR. One may therefore speculate that hnRNP C1 and/or RALYL control SD3632 by interacting with the hnRNP D and hnRNP A2/B1 proteins in


3.

4. 5. 6. 7.

8. 9.

10.

12. 13. 14. 15. 16.

17.

18.

19.

20.

21.

22.

23.

24.

25.


keratinocytes. PLoS One 8, e72776 26. Jia, R., Li, C., McCoy, J. P., Deng, C. X., and Zheng, Z. M. (2010) SRp20 is a proto-oncogene critical for cell proliferation and tumor induction and maintenance. Int. J. Biol. Sci. 6, 806 – 826 27. Jia, R., Liu, X., Tao, M., Kruhlak, M., Guo, M., Meyers, C., Baker, C. C., and Zheng, Z. M. (2009) Control of the papillomavirus early-to-late switch by differentially expressed SRp20. J. Virol. 83, 167–180 28. Li, X., Johansson, C., Glahder, J., Mossberg, A. K., and Schwartz, S. (2013) Suppression of HPV-16 late L1 5⬘-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs. Nucleic Acids Res. 22, 10488 –10508 29. Zhao, X., Rush, M., and Schwartz, S. (2004) Identification of an hnRNP A1 dependent splicing silencer in the HPV-16 L1 coding region that prevents premature expression of the late L1 gene. J. Virol. 78, 10888 –10905 30. Zhao, X., and Schwartz, S. (2008) Inhibition of HPV-16 L1 expression from L1 cDNAs correlates with the presence of hnRNP A1 binding sites in the L1 coding region. Virus Genes 36, 45–53 31. Zhao, X., Fay, J., Lambkin, H., and Schwartz, S. (2007) Identification of a 17-nucleotide splicing enhancer in HPV-16 L1 that counteracts the effect of multiple hnRNP A1-binding splicing silencers. Virology 369, 351–363 32. Collier, B., Oberg, D., Zhao, X., and Schwartz, S. (2002) Specific inactivation of inhibitory sequences in the 5⬘ end of the human papillomavirus type 16 L1 open reading frame results in production of high levels of L1 protein in human epithelial cells. J. Virol. 76, 2739 –2752 33. Nagy, A. (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99 –109 34. Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U., and Speed, T. P. (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249 –264 35. Tusher, V. G., Tibshirani, R., and Chu, G. (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. U.S.A. 98, 5116 –5121 36. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 37. Somberg, M., Zhao, X., Fröhlich, M., Evander, M., and Schwartz, S. (2008) PTB induces HPV-16 late gene expression by interfering with splicing inhibitory elements at the major late 5⬘-splice site SD3632. J. Virol. 82, 3665–3678 38. Orru`, B., Cunniffe, C., Ryan, F., and Schwartz, S. (2012) Development and validation of a novel reporter assay for human papillomavirus type 16 late gene expression. J. Virol. Methods 183, 106 –116 39. Ozbun, M. A., and Meyers, C. (1997) Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b. J. Virol. 71, 5161–5172 40. Busch, A., and Hertel, K. J. (2012) Evolution of SR protein and hnRNP splicing regulatory factors. Wiley Interdiscip. Rev. RNA 3, 1–12 41. Sokolowski, M., Zhao, C., Tan, W., and Schwartz, S. (1997) AU-rich mRNA instability elements on human papillomavirus type 1 late mRNAs and c-fos mRNAs interact with the same cellular factors. Oncogene 15, 2303–2319 42. Sokolowski, M., and Schwartz, S. (2001) Heterogeneous nuclear ribonucleoprotein C binds exclusively to the functionally important UUUUUmotifs in the human papillomavirus type-1 AU-rich inhibitory element. Virus Res. 73, 163–175 43. McCloskey, A., Taniguchi, I., Shinmyozu, K., and Ohno, M. (2012) hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts for export. Science 335, 1643–1646 44. Fay, J., Kelehan, P., Lambkin, H., and Schwartz, S. (2009) Increased expression of cellular RNA-binding proteins in HPV-induced neoplasia and cervical cancer. J. Med. Virol. 81, 897–907 45. Kozak, M. (1992) Regulation of translation inititaion in eucaryotic systems. Annu. Rev. Cell Biol. 8, 197–225 46. Tomita, Y., and Simizu, B. (1993) Translational properties of the human papillomavirus type-6 L1-coding mRNA. Gene 133, 223–225 47. Rosenberger, S., De-Castro Arce, J., Langbein, L., Steenbergen, R. D. M., and Rösl, F. (2010) Alternative splicing of human papillomavirus type-16



11.

Working Group (2009) A review of human carcinogens: part B: biological agents. Lancet Oncol. 10, 321–322 Bosch, F. X., Lorincz, A., Munõz, N., Meijer, C. J., and Shah, K. V. (2002) The causal relation between human papillomavirus and cervical cancer. J. Clin. Pathol. 55, 244 –265 zur Hausen, H. (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat. Rev. Cancer 2, 342–350 Howley, P. M., and Lowy, D. R. (2006) in Virology, 5th Ed. (Knipe, D. M., and Howley, P. M., eds) pp 2299 –2354, Lippincott, Philadelphia Doorbar, J. (2005) The papillomavirus life cycle. J. Clin. Virol. 32, S7–S15 Chow, L. T., Broker, T. R., and Steinberg, B. M. (2010) The natural history of human papillomavirus infections of the mucosal epithelia. APMIS 118, 422– 449 Bodily, J., and Laimins, L. A. (2011) Persistence of human papillomavirus infection: keys to malignant progression. Trends Microbiol. 19, 33–39 Johansson, C., and Schwartz, S. (2013) Regulation of human papillomavirus gene expression by splicing and polyadenylation. Nat. Rev. Microbiol. 11, 239 –251 Thierry, F. (2009) Transcriptional regulation of the papillomavirus oncogenes by cellular and viral transcription factors in cervical carcinoma. Virology 384, 375–379 McBride, A. A. (2013) The papillomavirus E2 proteins. Virology 445, 57–79 Bernard, H. U. (2013) Regulatory elements in the viral genome. Virology 445, 197–204 Graham, S. V. (2008) Papillomavirus 3⬘ UTR regulatory elements. Front. Biosci. 13, 5646 –5663 Jia, R., and Zheng, Z. M. (2009) Regulation of bovine papillomavirus type 1 gene expression by RNA processing. Front. Biosci. 14, 1270 –1282 Schwartz, S. (2013) Papillomavirus transcripts and posttranscriptional regulation. Virology 445, 187–196 Baker, C., and Calef, C. (1997) in Human Papillomaviruses: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences (Billakanti, S. R., Calef, C. E., Farmer, A. D., Halpern, A. L., and Myers, G. L., eds) Los Alamos National Laboratory, Los Almos, NM Jeon, S., and Lambert, P. F. (1995) Integration of human papillomavirus type 16 DNA into the human genome leads to increased stability of E6 and E7 mRNAs: implications for cervical carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 92, 1654 –1658 Zhao, X., Oberg, D., Rush, M., Fay, J., Lambkin, H., and Schwartz, S. (2005) A 57 nucleotide upstream early polyadenylation element in human papillomavirus type 16 interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB. J. Virol. 79, 4270 – 4288 Terhune, S. S., Hubert, W. G., Thomas, J. T., and Laimins, L. A. (2001) Early polyadenylation signals of human papillomavirus type 31 negatively regulate capsid gene expression. J. Virol. 75, 8147– 8157 Johansson, C., Somberg, M., Li, X., Backström Winquist, E., Fay, J., Ryan, F., Pim, D., Banks, L., and Schwartz, S. (2012) HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation. EMBO J. 31, 3212–3227 Oberg, D., Fay, J., Lambkin, H., and Schwartz, S. (2005) A downstream polyadenylation element in human papillomavirus type 16 encodes multiple GGG-motifs and interacts with hnRNP H. J. Virol. 79, 9254 –9269 Rush, M., Zhao, X., and Schwartz, S. (2005) 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. J. Virol. 79, 12002–12015 Somberg, M., Li, X., Johansson, C., Orru, B., Chang, R., Rush, M., Fay, J., Ryan, F., and Schwartz, S. (2011) SRp30c activates human papillomavirus type 16 L1 mRNA expression via a bimodal mechanism. J. Gen. Virol. 92, 2411–2421 Somberg, M., and Schwartz, S. (2010) Multiple ASF/SF2 sites in the HPV-16 E4-coding region promote splicing to the most commonly used 3⬘-splice site on the HPV-16 genome. J. Virol. 84, 8219 – 8230 Li, X., Johansson, C., Cardoso Palacios, C., Mossberg, A., Dhanjal, S., Bergvall, M., and Schwartz, S. (2013) Eight nucleotide substitutions inhibit splicing to HPV-16 3⬘-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human


48.

49.

50.

51.

52.

54.


55. 56.

57.

58.

59.

60.

61.

62.

binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach. J. Proteome Res. 12, 2869 –2884 Dreyfuss, G., Kim, V. N., and Kataoka, N. (2002) Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell. Biol. 3, 195–205 Lee, H.-H., Chien, C.-L., Liao, H.-K., Chen, Y.-J., and Chang, Z.-F. (2004) Nuclear efflux of heterogeneous nuclear ribonucleoprotein C1/C2 in apoptotic cells: a novel nuclear export dependent on Rho-associated kinase activation. J. Cell Sci. 117, 5579 –5589 Pinõl-Roma, S., and Dreyfuss, G. (1993) Cell cycle-regulated phosphorylation of the pre-mRNA binding (heterogeneous nuclear ribonucleoprotein) C proteins. Mol. Cell. Biol. 13, 5762–5770 Lee, E. K., Kim, H. H., Kuwano, Y., Abdelmohsen, K., Srikantan, S., Subaran, S. S., Gleichmann, M., Mughal, M. R., Martindale, J. L., Yang, X., Worley, P. F., Mattson, M. P., and Gorospe, M. (2010) hnRNP C promotes APP translation by competing with FMRP for APP mRNA recruitment to P bodies. Nat. Struct. Mol. Biol. 17, 732–739 Spångberg, K., Wiklund, L., and Schwartz, S. (2000) HuR, a protein implicated in oncogene and growth factor mRNA decay, binds to the 3⬘ ends of hepatitis C virus RNA of both polarities. Virology 274, 378 –390 Brunner, J. E., Ertel, K. J., Rozovics, J. M., and Semler, B. L. (2010) Delayed kinetics of poliovirus RNA synthesis in a human cell line with reduced levels of hnRNP C proteins. Virology 400, 240 –247 Shabman, R. S., Gulcicek, E. E., Stone, K. L., and Basler, C. F. (2011) The Ebola virus VP24 protein prevents hnRNP C1/C2 binding to karyopherin ␣1 and partially alters its nuclear import. J. Infect. Dis. 204, S904 –S9910 Casaca, A., Fardilha, M., da Cruz e Silva, E., and Cunha, C. (2011) The heterogeneous ribonuclear protein C interacts with the hepatitis ␦ virus small antigen. Virol. J. 8, 358


13371


53.

E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation. Proc. Natl. Acad. Sci. U.S.A. 107, 7006 –7011 Cheunim, T., Zhang, J., Milligan, S. G., McPhillips, M. G., and Graham, S. V. (2008) The alternative splicing factor hnRNP A1 is up-regulated during virus-infected epithelial cell differentiation and binds the human papillomavirus type 16 late regulatory element. Virus Res. 131, 189 –198 Collier, B., Goobar-Larsson, L., Sokolowski, M., and Schwartz, S. (1998) Translational inhibition in vitro of human papillomavirus type 16 L2 mRNA mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2. J. Biol. Chem. 273, 22648 –22656 Zarnack, K., König, J., Tajnik, M., Martincorena, I., Eustermann, S., Stévant, I., Reyes, A., Anders, S., Luscombe, N. M., and Ule, J. (2013) Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements. Cell 152, 453– 466 Izquierdo, J. M. (2010) Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R. Nucleic Acids Res. 38, 8001– 8014 Irimura, S., Kitamura, K., Kato, N., Saiki, K., Takeuchi, A., Gunadi, Matsuo, M., Nishio, H., and Lee, M. J. (2009) HnRNP C1/C2 may regulate exon 7 splicing in the spinal muscular atrophy gene SMN1. Kobe J. Med. Sci. 54, E227–E236 Motta-Mena, L. B., Heyd, F., and Lynch, K. W. (2010) Context-dependent regulatory mechanism of the splicing factor hnRNP L. Mol Cell. 37, 223–234 Tenzer, S., Moro, A., Kuharev, J., Francis, A. C., Vidalino, L., Provenzani, A., and Macchi, P. (2013) Proteome-wide characterization of the RNA-

Microbiology: Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3′ -Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing

J. Biol. Chem. 2015, 290:13354-13371. doi: 10.1074/jbc.M115.638098 originally published online April 15, 2015

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