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Journal of Virology, January 2009, p. 167-180, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01719-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Control of the Papillomavirus Early-to-Late Switch by Differentially Expressed SRp20{triangledown} ,{dagger}

Rong Jia,1 Xuefeng Liu,1 Mingfang Tao,1 Michael Kruhlak,2 Ming Guo,3 Craig Meyers,4 Carl C. Baker,5 and Zhi-Ming Zheng1*

HIV and AIDS Malignancy Branch,1 Experimental Immunology Branch,2 Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,5 Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030,3 Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 170334

Received 7 August 2008/ Accepted 9 October 2008


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ABSTRACT
 
The viral early-to-late switch of papillomavirus infection is tightly linked to keratinocyte differentiation and is mediated in part by alternative mRNA splicing. Here, we report that SRp20, a cellular splicing factor, controls the early-to-late switch via interactions with A/C-rich RNA elements. An A/C-rich SE4 element regulates the selection of a bovine papillomavirus type 1 (BPV-1) late-specific splice site, and binding of SRp20 to SE4 suppresses this selection. Expression of late BPV-1 L1 or human papillomavirus (HPV) L1, the major capsid protein, inversely correlates with SRp20 levels in the terminally differentiated keratinocytes. In HPV type 16, a similar SRp20-interacting element also controls the viral early-to-late switch. Keratinocytes in raft cultures, which support L1 expression, make considerably less SRp20 than keratinocytes in monolayer cultures, which do not support L1 expression. Conversely, abundant SRp20 in cancer cells or undifferentiated keratinocytes is important for the expression of the viral early E6 and E7 by promoting the expression of cellular transcription factor SP1 for transactivation of viral early promoters.


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INTRODUCTION
 
Papillomaviruses are small DNA tumor viruses that infect cutaneous or mucosal epithelial cells and cause benign tumors and sometimes malignant neoplasms, including cervical cancer in women (36). Papillomavirus infections are transmitted mainly by close skin-to-skin or mucosa-to-mucosa contact. Infecting viral particles reach the keratinocytes in the basal layer of the squamous epithelium via microwounds that expose the basal keratinocytes to incoming virus. After infection of the basal keratinocytes, viral-gene expression and replication proceed in a tightly controlled fashion regulated by keratinocyte differentiation (25, 34).

Although we do not fully understand how keratinocyte differentiation regulates papillomavirus gene expression and virus production, different parts of the viral life cycle occur at different stages of keratinocyte differentiation. The early stage of virus infection takes place in undifferentiated or intermediately differentiated keratinocytes in basal or parabasal layers; at this stage, the viral early genes (E1, E2, E5, E6, and E7) are expressed from the early region of the viral genome and encode all five viral regulatory nonstructural proteins. In contrast, the expression of two structural viral capsid proteins (L1 and L2) from the late region of the virus genome at the late stage of viral infection occurs only in keratinocytes undergoing terminal differentiation in the granular and cornified layers of the epithelium (34, 41). Although the early-to-late switch of viral-gene expression involves a switch in the use of viral promoters during the viral life cycle (21, 48, 49), strict regulation of viral-RNA processing, including alternative RNA splicing and polyadenylation, is absolutely necessary for expression of the viral genes at the appropriate times (42, 57).

Alternative RNA splicing and polyadenylation occur during RNA processing in most eukaryotic and viral genes, usually when the RNA bears weak splice sites or multiple poly(A) signals (38, 56). Because it depends on the local availability of the correct forms of splicing factors, alternative splicing of a particular RNA can be found in different cell types and at different stages of cell differentiation. Although the exact mechanism by which alternative RNA splicing is regulated remains largely unknown, it is the general consensus that one or more cis-acting elements in the regulated exons or introns interact with one or more locally available cellular splicing factors to select an alternative splice site (24, 54). This alternative RNA splicing provides an alternative poly(A) site, which may or may not be used for RNA polyadenylation (7, 57). In papillomaviruses, both viral early and late transcripts are in bicistronic or polycistronic forms that contain two or more open reading frames (ORFs) and multiple introns and exons. These bicistronic or polycistronic transcripts overlap with each other and feature suboptimal splicing signals. Thus, their expression undergoes extensive alternative RNA splicing and polyadenylation at either an early or a late poly(A) site (57). At the early stage of papillomavirus infection, only the early promoters are activated, and viral early transcripts utilize early splice sites and the early poly(A) signal; this occurs in undifferentiated or intermediately differentiated keratinocytes, where the late splice sites and the late poly(A) site are blocked. This strategy allows the virus to avoid any expression of the viral late proteins from the early transcripts and enables the viral early proteins to take control of various cellular pathways before an infectious virus can be produced. In contrast, at the late stage of papillomavirus infection, when the infected cell differentiates and transcription from the late promoter becomes active, the late-specific splice sites and the late poly(A) signal are activated for the expression of viral late genes. However, during this time, the usage of the viral early splice sites and the early poly(A) signal continues, suggesting that some cellular differentiation factors are crucial for this regulation.

To investigate this process in more detail, our laboratories and others have attempted to elucidate the underlying mechanisms that control the viral early-to-late switch. In particular, we have focused on the viral-RNA processing that occurs during late-gene expression of bovine papillomavirus type 1 (BPV-1), which has served as a model system for studies of the molecular biology of papillomaviruses. In the late stage of BPV-1 infection in differentiated cells, the late leader 5' splice site (5' ss) of bicistronic L1L2 transcripts alternatively splices to a proximal 3' splice site (3' ss) (nucleotide [nt] 3225 3' ss) to express L2 or to a distal 3' ss (nt 3605 3' ss) to express L1. This alternative splicing of late transcripts involves the active participation of three exonic splicing enhancers (ESEs), SE1, SE2, and SE4, and two exonic splicing suppressors (ESS), ESS1 and ESS2, in exon 2 (56, 57). SE1, SE2, and ESS1 regulate the selection of the proximal 3' ss, which is also active in the expression of viral early genes. SE4 is an A/C-rich ESE and functions along with ESS2 in vitro to select the distal 3' ss, which is essential for defining a downstream 5' ss (nt 3764 5' ss) to remove intron 2 during splicing of the L1 pre-mRNAs (61); retention of intron 2 results in production of the viral minor capsid protein L2 instead of the viral major capsid protein L1. Notably, selection of the distal 3' ss to remove intron 2 takes place only in the upper layers of the epidermis, whereas selection of the proximal 3' ss, which also occurs during splicing of viral early transcripts, appears in both the basal and upper layers of the epidermis (4). However, how SE4 functions remains unknown.

Here, we verified the role of SE4 in the selection of the 3' ss and further investigated its function by searching for cellular proteins that bind to it. We identified SRp20, a cellular splicing factor (8) and RNA export mediator (27), as a specific SE4-interacting protein. Subsequently, we examined the effect of SRp20 on the splicing of BPV-1 and human papillomavirus type 16 (HPV16) late transcripts. We found that low levels of cellular SRp20 permit the expression of viral late genes and control the early-to-late switch in viral-RNA processing. In contrast, a high level of SRp20 was found to promote the expression of the HPV early genes E6 and E7.


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MATERIALS AND METHODS
 
Plasmids. Plasmids SE4m1 (pXFL102), SE4m2 (pXFL103), and SE4m3 (pXFL106) were all derived from plasmid p3033 (SE2m) (59) and contain point mutations in both the SE2 and SE4 elements. Plasmid pTMF25-8 was derived from plasmid p3231(59) and has no point mutations in SE2 but has the same point mutations in SE4 elements as SE4m2. Plasmid pJR1, derived from pCBG1(33), has a simian virus 40 promoter-driven BPV-1 late minigene with a mutant (mt) SE2 and wild-type (wt) SE4. Plasmid pXFL101 has a simian virus 40 promoter-driven BPV-1 late minigene similar to that of plasmid p3231(59). Both plasmids pJR1 and pXFL101 contain a blasticidin resistance gene for selection of stable transfection.

Plasmid pJR5 contains a subgenomic HPV16 DNA from nt 686 to 7471 under the control of a cytomegalovirus (CMV) immediate-early (IE) promoter. Plasmid pJR9, derived from plasmid pJR5, contains point mutations in the HPV16 ESE sequence (see Fig. 8B).


Figure 8
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  		FIG. 8. SRp20 suppresses the expression of HPV16 late genes. (A) Diagram of an HPV16 late transcript, the splicing pattern, and the position of an identified ESE. The numbers below the diagram are the nucleotide positions in the HPV16 genome. The arrows indicate a pair of primers used for the RT-PCR assays shown in panel F. (B) Sequences of biotin-labeled wt ESE, mt ESE, and wt2 SE4; underlining indicates mutations. (C) Interactions of HPV16 ESE with cellular splicing factors from U2OS total cell extracts. BPV-1 SE4 was used for comparison in the binding assays. The proteins in the biotin-labeled RNA pull-down assays were examined by Western blotting with anti-SRp20 7B4, anti-YB-1, or MAb104 antibody. The cell extracts (15% of the input in the binding assays) were blotted as positive controls. (D) The CAC motifs in HPV16 ESE are the binding sites for SRp20 and hnRNP L (hnR L). Wt ESE and SE4 were compared with mt ESE for hnRNP L and SRp20 binding in the presence of U2OS total cell extracts or 293 cell nuclear extracts. The cell extracts (10% for U2OS or 40% for 293 of the input in the binding assays) were also blotted as positive controls. (E) Knockdown of SRp20 expression in U2OS cells. U2OS cells transfected once with an SRp20 siRNA or an NS siRNA were cotransfected with a subgenomic (nt 686 to 7471) HPV16 late-gene expression vector, pJR5, along with the second siRNA treatment. Protein samples collected 24 h after the cotransfection were examined by Western blotting with the indicated antibodies. hnRNP K in each sample served as a loading control. (F) Interaction of SRp20 and HPV16 ESE plays a negative role in the expression of HPV16 L1. Knocking down SRp20 in U2OS cells (left) or disruption of HPV16 ESE by point mutation (right) increases the expression of HPV16 late genes. HPV16 vector (pJR5)-transfected U2OS cells with or without knockdown of SRp20 or U2OS cells transfected only with an HPV16 vector containing a wt ESE (pJR5) or mt ESE (pJR9), as shown in panel B, were examined by RT-PCR for L1 expression 24 h after transfection. One representative experiment of three is shown. (G) Northern blot analysis for the expression of HPV16 late genes from HPV16 vector containing a wt ESE (pJR5) in U2OS cells with or without knocking down SRp20. Cellular GAPDH RNA served as a loading control. L1 and L2 expression levels were normalized to GAPDH before the ratio to corresponding NS siRNA controls was calculated. (H) Expression of HPV16 L1 and SRp20 in human vaginal keratinocytes in raft and monolayer cultures. HPV16+ CaSki cells were used as a cancer cell control for Western blotting. Tubulin served as a loading control.

Raft cultures. Raft (organotypic) cultures were derived from HPV16-immortalized primary human vaginal keratinocytes and were grown as previously described (14, 40).

Transient and stable transfections. Both U2OS cells and 293 cells were used for transfection with mammalian expression vectors in the presence of Lipofectamine 2000 (Invitrogen). Total cell RNA was prepared 48 h after transfection and analyzed for viral-late-gene expression by reverse transcription (RT)-PCR or Northern blotting. For stable transfection, U2OS cells transfected with 2 µg of EcoRI-linearized plasmid pJR1 or pXFL101 were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum with the addition of 10 µg/ml of blasticidin (Invitrogen).

Synthetic siRNAs and RNAi. All small interfering RNAs (siRNAs) were synthesized from Dharmacon, Inc. (Lafayette, CO), siRNA 393 (5'-UGACACCAAGGAAGAUGUATT-3'), siRNA 394 (5'-GCAGCCGAUCCACCAGCUGTT-3'), and siRNA oJR1 (5'-GGUCAUCGCAACGAAGGUU-3') for human YB-1. SRp20 siRNA was purchased as a siGenome SMARTpool (SFRS3; catalog no. M-030081-00), and a nonspecific (NS) siRNA with 52% GC content (catalog no. D-001206-08-20) was used as a negative control. For RNA interference (RNAi), U2OS cells transfected with BPV-1 or HPV16 and CaSki and HeLa cells were transfected with each gene-specific siRNA in two separate transfections at an interval of 48 h in the presence of Lipofectamine 2000 (52, 53). The siRNAs used for transfection were SRp20 siRNA (40 nM), YB-1 siRNA (30 nM; 10 nM each of siRNAs 393, 394, and oJR1), and NS siRNA (30 nM for U2OS cells and 40 nM for CaSki or HeLa cells). Protein samples and total cell RNA were prepared 24 or 48 h after the second siRNA transfection.

Western blotting. Protein samples were blotted separately with the following mouse monoclonal antibodies (MAbs): anti-SRp20 (7B4; ATCC, Manassas, VA), anti-pan-SR protein MAb104 (ATCC), anti-hnRNP K (D-6), anti-SP1 (1C6), and anti-HPV16 E7 (ED17), all from Santa Cruz Biotechnology (Santa Cruz, CA); anti-β-tubulin (5H1), anti-SC35 ({alpha}SC35), anti-p21 (6B6), and anti-pRb (G3-245), all from BD Pharmingen (San Diego, CA); anti-ASF/SF2 antibody (clone 96; Invitrogen); anti-HPV L1 (K1H8; Lab Vision Corporation, Fremont, CA); anti-hnRNP L (4D11; Abcam, Cambridge, MA); and anti-p53 (Ab-6; Oncogene, Cambridge, MA). Goat anti-HPV18 E7 (SC-1590) was from Santa Cruz Biotechnology, and rabbit polyclonal anti-YB-1 was from Abcam.

RNA preparation and RT-PCR. Following DNase I treatment, 1 µg of RNA was reverse transcribed at 42°C using random hexamers and then amplified for 35 cycles using the primer pair Pr7345 (5'-CAATGGGACGCGTGCAAAGC-3') and Pr3738 (5'-CAGTATTTGTGCTTGTCCTT-3') or Pr3715 (5'-TTTCAGCACCGTTGTCAGCAACTGTG-3') for BPV-1 L1 and L2 detection. BPV-1 L2 and L1 cDNAs spliced using the nt 3225 3' ss and nt 3605 3' ss, respectively, were used as positive controls. The primer pair oSB23 (nt 3385 to 3402; 5'-TATTAGGCAGCACTTGGC-3') and oXHW41 (nt 5723 to 5699; 5'-CAACATATTCATCCGTGCTTACAAC-3') was also used for HPV16 L1 detection.

RNA pull-down assays. Biotin-labeled BPV-1 RNA oligonucleotides oJR4 (5'-biotin-CUGCACCACCACCUGGUUCTT-3'; wt1 SE4), oJR5 (5'-biotin-CUGUGUCACUGUCUGGUUCTT-3' [underlining indicates mutations]; mt1 SE4), oJR6 (5'-biotin-GGCAGGAAGAAGAGGAGCATT-3'; wt SE2), oJR7 (5'-biotin-CUGCACCACCACCUAUCUATT-3'; wt2 SE4), and oJR8 (5'-biotin-CUGUGUCACUGUCUAUCUATT-3'; mt2 SE4), and biotin-labeled HPV16 RNA oligonucleotides oJR9 (5'-biotin-CCAGACACCGGAAACCCCUGCCACACCAC-3'; wt ESE) and oJR10 (5'-biotin-CCAGAUGUCGGAAACCUCUGCUGUGCUGU-3'; mt ESE) were synthesized by Integrated DNA Technologies (Coralville, IA). We obtained the 293 nuclear extract (7.5 mg/ml) from ProteinOne (Bethesda, MD). The U2OS cell extract was prepared from actively growing cells in exponential phase. After washes, the cell pellets were resuspended in radioimmunoprecipitation assay (RIPA) buffer (Boston Bio-Products, Ashland, MA) with the addition of Complete Mini EDTA-free Protease Inhibitor Mixture (Roche, Indianapolis, IN), repeat pipetted, and sonicated (10 s) on ice. After centrifugation, the supernatant was collected as U2OS total cell extract. Each biotin-labeled RNA oligonucleotide (10 µl at 40 µM) was first immobilized onto 100 µl of NeutrAvidin beads (50% slurry; Pierce, Rockford, IL) in a final volume of 300 µl of 1x binding buffer (20 mM Tris, 200 mM NaCl, 6 mM EDTA, 5 mM potassium fluoride, 5 mM β-glycerophosphate, 2 µg/ml aprotinin, pH 7.5) at 4°C for 2 h, followed by incubation with 10 µl of 293 nuclear extract or 100 µl of U2OS total cell extract in 1x binding buffer in a final volume of 400 µl at 4°C for 2 h. The beads were washed three times with binding buffer, resuspended in 40 µl of 2x sodium dodecyl sulfate sample buffer, and boiled for 5 min. Proteins in the pull-down assays were analyzed by Western blotting.

In vitro splicing assay and spliceosome assembly. In vitro splicing of 4 ng of 32P-labeled BPV-1 late pre-mRNAs was carried out using 40% of the HeLa nuclear extract (ProteinOne) in a volume of 25 µl in splicing buffer by incubation at 30°C for 2 h. The spliced products were analyzed by electrophoresis in a denaturing 8% polyacrylamide-8 M urea gel. Spliceosomal complex formation was performed under the conditions described previously (60).

Northern blotting. Equal amounts of the poly(A)-selected RNAs from U2OS cells transiently transfected with a papillomavirus late-minigene vector were run in 1% agarose-formaldehyde gels and blotted onto a GeneScreen Plus membrane (Perkin Elmer, Waltham, MA). To detect CaSki and HeLa E6E7 RNAs, approximately 10 µg of the cytoplasmic and nuclear fractions of total RNA with (CaSki) or without (HeLa) poly(A) selection was prepared 24 h after the second siRNA transfection. Northern blot analyses were performed with a 32P-labeled antisense HPV16 E6E7 probe (nt 442 to 816) transcribed in vitro or with an oligonucleotide probe end labeled with 32P. The following oligonucleotide probes were used: oXHW41 for HPV16 L1, oZMZ433 (5'-CACTGAGGTAC/CTGCTGGGATGCACACCAC-3') for HPV18 E6E7, Pr3738 for BPV-1 late transcripts, and oZMZ270 (5'-TGAGTCCTTCCACGATACCAAA-3') for cellular GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

Immunohistochemical and immunofluorescence staining. Bovine fibropapilloma tissues and cervical tissues with cervical intraepithelial neoplasia I (CIN I) lesions were from previous studies (4, 5, 22). Normal cervical sections were purchased from US Biomax (Rockville, MD). Immunohistochemistry was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA). Sections were incubated with MAb 7B4 (anti-SRp20; ATCC), anti-BPV-1 L1 (Chemicon) (60), or anti-HPV L1 K1H8 overnight at 4°C, followed by secondary antibody for 30 min and ABC reagent for 30 min. The specific signal was developed with a DAB substrate kit (Vector). Immunofluorescence double staining was performed with the Vector M.O.M. Immunodetection Kit. Briefly, tissue sections were processed as described above without endogenous peroxidase quenching and incubated with anti-SRp20 7B4 overnight at 4°C, followed by biotin-labeled secondary antibody for 30 min. The specific signal was developed by using Alexa Fluor 488 streptavidin for 5 min. The same section was then treated with the reagents in the Streptavidin/Biotin Blocking Kit (Vector), followed by Image-iT FX Signal Enhancer (Invitrogen) for 30 min and M.O.M Mouse Ig Blocking Reagent for 1 h. Subsequently, the sections were incubated with anti-BPV-1 L1 or anti-HPV L1 K1H8 antibody overnight at 4°C and with biotin-labeled secondary antibody for 30 min. The specific signal was developed using Alexa Fluor 546 streptavidin for 5 min and was imaged by epifluorescence or confocal microscopy. The background signals captured from control slides stained with no primary antibody, anti-SRp20 only, anti-BPV-1 L1 only, or anti-HPV L1 only were used to subtract the background signal (if any) from double-stained images.


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RESULTS
 
Selection of a viral late-specific splice site is facilitated by an exonic A/C-rich element. SE4, an A/C-rich element reported to be an ESE in the BPV-1 late transcript (61), has three consecutive tandem repeats of CAC sequences followed by a suppressor (ESS2) core, UGGU. We analyzed the structure and function of SE4 by introducing point mutations. As shown in Fig. 1A and B, point mutations in the third CAC (from 5' to 3') reduced the ability of SE4 to promote RNA splicing in vitro (compare pre-mRNAs 2, 5, and 6 to pre-mRNA 1). In contrast, introduction of point mutations into the first CAC had no effect on the function of SE4 (compare pre-mRNA 4 to pre-mRNA 1); however, mutation of both the first and third CAC together (pre-mRNA 5) was more suppressive than mutation of the third CAC alone (pre-mRNA 2). Mutations in the second CAC had little effect (pre-mRNA 3), and changes of sequences outside of the CAC repeats (pre-mRNA 8) alone or in combination with mt CACs (pre-mRNAs 6 and 7) did not have any additional effect. We confirmed this observation in a heterologous pre-mRNA by using a Drosophila double-sex (dsx) pre-mRNA (58). The Drosophila dsx pre-mRNA contains a suboptimal intron and cannot be spliced in vitro in the absence of an ESE. When the dsx exon 4 was attached to a wt SE4 or a purine-rich enhancer (AAG)8, the dsx pre-mRNAs were spliced efficiently (Fig. 1C and D, pre-mRNAs 1 and 5). As predicted, an mt SE4 (SE4m1 or SE4m2) or a negative control sequence, Py3, lacked this activity when placed in the same position (Fig. 1C and D, pre-mRNAs 2, 3, and 4). Mechanistically, SE4, but not its mutant, more efficiently enhances the formation of spliceosomal complexes A and B (Fig. 1E). Together, these data suggest that the A/C-rich SE4 functions as an ESE mainly through its A/C-rich motif.


Figure 1
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  		FIG. 1. Mapping of SE4 motifs that function in splicing of BPV-1 late and Drosophila dsx pre-mRNAs in vitro. (A) Schematic diagram of pre-mRNAs containing a wt or mt SE4. Unchanged nucleotides are indicated by dots. The numbers above the pre-mRNA exons (boxes) and an intron (line) are the nucleotide positions in the BPV-1 genome. The splicing efficiency for each pre-mRNA was calculated from the splicing gel in panel B as described previously (61). (B) Splicing gel, showing the corresponding splicing products on the left (from top to bottom, splicing intermediates, pre-mRNAs, fully spliced products, and 5' exons). The numbers at the top of the gel indicate the corresponding pre-mRNAs in panel A used for splicing. (C) Structures of the Drosophila dsx chimeric pre-mRNAs used to test wt and mt SE4. The lengths (nt) of the exons (boxes) and an intron (line) are indicated below the respective regions of the pre-mRNA. The synthetic oligonucleotides Py3 and (AAG)8 served as negative and positive controls, respectively, for splicing enhancement. The hatched box indicates the location where the tested sequences were cloned. (D) BPV-1 wt SE4, but not mt SE4, functions in vitro as a splicing enhancer in Drosophila dsx pre-mRNA. The numbers at the top of the gel indicate the corresponding pre-mRNAs in panel C used for splicing. (E) Formation of spliceosome complexes on pre-mRNAs 1 (wt SE4) and 2 (SE4m1). Spliceosome complex formation was performed under standard splicing conditions at 30°C, stopped after the indicated incubation times by the addition of heparin, and loaded and run immediately on a native 4% polyacrylamide gel. Spliceosome complexes A and B and the NS complex H are indicated on the left of the gel. One representative experiment of two is shown in panels B, D, and E.

To understand how the A/C-rich SE4 functions in vivo in alternative RNA splicing, we utilized a BPV-1 late-minigene expression vector driven by a CMV IE promoter for in vivo assays. This minigene has a large deletion in its intron 1 to remove all viral early promoters and contains two alternative 3' ss in its exon 2 (Fig. 2a). A proximal 3' ss at nt 3225 in the virus genome is a major 3' ss for all early transcripts, but the same splice site is also used for splicing of viral late transcripts to express the viral minor capsid protein L2. Thus, selection of this 3' ss takes place in both undifferentiated and highly differentiated keratinocytes and in both the early and late stages of virus infection (4). It is controlled by three downstream RNA cis elements, SE1, SE2, and ESS1(57). In contrast, a distal 3' ss at nt 3605 in the virus genome is a late-specific 3' ss and is controlled by two other downstream cis elements, SE4 and ESS2 (61). However, selection of this distal 3' ss is active only in highly differentiated keratinocytes at the late stage of virus infection, and its selection is essential for activating a downstream 5' ss at nt 3764 for splicing to remove intron 2, where the L2 ORF resides (4), to produce the viral major capsid protein L1.


Figure 2
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  		FIG. 2. SE4 functions as an ESE in vivo. (a) Diagrams of a BPV-1 late-minigene expression vector (long open box) and the splicing pattern (line below the box) of pre-mRNAs expressed from the vector. At the ends of the late minigene are the CMV IE1 promoter (solid box) and pUC18 (shaded box). The vector expresses a late pre-mRNA that has a large deletion (from nt 7563 to 3073) in intron 1, indicated by a vertical line. The numbers below the lines are the nucleotide positions in the BPV-1 genome. Early and late poly(A) sites are indicated as AE and AL, respectively, above the lines. Shown at the top are the relative positions of the L2 and L1 ORFs (open boxes), as indicated by the first nucleotide of the start codon and the last nucleotide of the stop codon from each ORF. Shown below is the partial structure of the minigene transcript delineating the relative positions of SE1, ESS1, SE2, SE4, and ESS2; the nucleotide positions of the 5' ss and 3' ss; and splicing directions. A pair of primers used for RT-PCR in panel C are indicated by arrows under the transcript and are named for the locations of their 5' ends. The drawings are not to scale. (b) Sequences of wt and mt SE2 and wt and mt SE4 in the pre-mRNAs expressed from plasmids used for transfection of 293 cells. Unchanged nucleotides (dots) and nucleotides changed by point mutation (letters) are indicated. The boxed sequences are functional motifs in each splicing enhancer. (c) RT-PCR analysis of BPV-1 late transcripts prepared from 293 cells transfected with 1 µg of individual expression vectors as indicated above the gel. Total cell RNA was extracted and digested with (lanes 2 to 6) or without (lane 7) RNase-free DNase I before RT-PCR analysis. BPV-1 L1 and L2 cDNAs were included as positive controls, and water was used as a negative control. One representative experiment of two is shown.

The introduction of point mutations into SE2 (Fig. 2b) led to a switch of 3' ss usage from nt 3225 (proximal) to nt 3605 (distal) (Fig. 2c, lane 3), showing the importance of SE2 in the selection of the proximal 3' ss. As predicted, the introduction of additional point mutations into SE4 to disrupt its CAC motif partially restored the usage of the proximal 3' ss (Fig. 2c, lanes 4 and 5), indicating that SE4 plays a key role in selection of the distal 3' ss in vivo. Consistent with the dominant function of SE4, introduction of point mutations into the UGGU sequence immediately downstream of the CAC motif in SE4 did not affect the choice of splice sites (Fig. 2c, lane 6). This was further confirmed by RT-PCR analysis when SE4 was mutated on its own in the context of wt SE2. The choice of the nt 3605 3' ss was not detectable in vivo when the late transcript contained an mt SE4 (SE4m2) alone (data not shown).

Interaction of SRp20 with the CAC motif in SE4 blocks selection of the viral late-specific splice site. To understand which cellular factors are involved in the SE4-mediated selection of splice sites, we carried out RNA pull-down assays using biotin-labeled SE4 RNA with or without the presence of mutations. The UGGU sequence immediately downstream of the CAC motif was also mutated (Fig. 3A, wt2 and mt2) to avoid possible base pairing with the overlapped ACCACCAs in SE4 that might affect the binding of cellular proteins to SE4. In pull-down assays using nuclear extracts from 293 cells, as shown in Fig. 3B, the CAC motif in SE4 selectively interacted with SRp20, the smallest member of the SR protein family (55), with high affinity (lanes 1 and 2). When point mutations were introduced into the first and third CAC in the SE4 CAC motif, the mt SE4 bound weakly to SRp30 and SRp75 (lane 3). Interestingly, wt2 SE4 bound more strongly to SRp20 than the wt1 SE4, suggesting that the UGGU motif does affect protein binding to the CAC motif in SE4. In contrast to SE4, SE2, which contains AG-rich sequences, bound all other classical SR proteins (SRp30, SRp40, SRp55, and SRp75), as described previously (59), but did not bind SRp20 (lane 4). We confirmed these observations by using two antibodies against individual SR proteins, anti-ASF/SF2 and anti-SC35 (Fig. 3C). Strikingly, SE2 was found to bind hnRNP L as well as SE4 did (Fig. 3C). hnRNP L has been reported to bind only CA repetitive sequences in the regulation of RNA splicing, stability, and poly(A) site selection (28, 29). In addition, we found that SE4 bound YB-1 protein (Fig. 3C, lane 2) when the UGGU motif in the RNA oligonucleotide was mutated, suggesting that the UGGU motif also interferes with YB-1 binding to SE4. To exclude the possibility that the point mutations in the UGGU motif might create a binding site for SRp20 orYB-1, we examined one additional RNA oligonucleotide (mt2 SE4 in Fig. 3A) in which the mutations in mt1 and wt2 SE4 were combined. There was no binding of SRp20 (Fig. 3D) or YB-1 (data not shown) to this oligonucleotide in the pull-down assays. Thus, we conclude that the CAC motif in SE4 is the binding site for SRp20, but also for the hnRNP L and YB-1, which bind an AG-rich SE2, as well.


Figure 3
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  		FIG. 3. SRp20 controls the selection of the splice sites via interactions with SE4. (A) Sequences of various biotin (B)-labeled RNA oligonucleotides, wt1 SE4 (wt1), mt1 SE4 (mt1), wt2 SE4 (wt2), mt2 SE4 (mt2), and wt SE2, used in the studies. (B) SE4 selectively interacts with the cellular splicing factor SRp20. Proteins from 293 nuclear extracts were used in RNA pull-down assays; blotted with the MAb104 antibody, which recognizes a phosphoepitope of all classical SR proteins in the SR protein family; and reprobed with an anti-SRp20 MAb, 7B4. (C) Comparison of SE4 with SE2 for protein binding of ASF/SF2, SC35, YB-1, and hnRNP L. Nuclear extracts of 293 cells or total cell extracts of U2OS cells were incubated with each biotin-labeled RNA oligonucleotide immobilized on avidin-conjugated beads. After washes, the proteins in the RNA pull-down assays were detected by Western blotting with each indicated antibody. (D) SRp20 binding to SE4 in 293 cell nuclear extracts or U2OS total cell extracts in RNA pull-down assays blotted with anti-SRp20 7B4 antibody. (E) SRp20 affects the selection of splice sites. The structure of the BPV-1 late-minigene transcript expressed from plasmid pXFL101 and its splicing pattern are the same as those described in Fig. 2a. After SRp20 and YB-1 expression in U2OS cells stably expressing BPV-1 late transcripts was knocked down, total cell RNA from each group was digested with RNase-free DNase I and analyzed by RT-PCR. A mixture of BPV-1 L2 and L1 cDNAs was used as a size control. Water was used as a negative control. The ratio (percent; shown at the bottom) of the nt 3605 3' ss usage was calculated as follows: (splicing at the nt 3605 3' ss/splicing at the nt 3225 3' ss) x 100. One representative experiment of three is shown. (F) Knockdown by siRNA of SRp20 and YB-1 expression in U2OS cells stably transfected with plasmid pXFL101, as determined by Western blotting with an anti-SRp20 (7B4) or anti-YB-1 antibody. The same membrane was also blotted with anti-tubulin to control for sample loading. (G) SRp20-SE4 interaction blocks selection of the 3605 3' ss. U2OS cells with or without knockdown of SRp20 by an SRp20-specific siRNA or an NS siRNA were transiently transfected with p3231 vector (wt SE4) or pTMF25-8 (SE4m2) and examined by Northern blotting for splicing selection. One representative experiment of two is shown. W.B., Western blot. GAPDH in Northern blotting and hnRNP K in Western blotting served as sample loading controls in each assay. The ratio (percent) of the nt 3605 3' ss usage was calculated as follows, after being normalized to GAPDH: (splicing at nt 3605 3' ss/splicing at nt 3225 3' ss) x 100.

Next, we wished to determine how the interactions of SRp20 and YB-1 with SE4 affect the alternative RNA splicing of BPV-1 late transcripts. YB-1 was chosen for the comparison because of its role in stimulating the splicing of a CD44 alternative exon via interaction with an A/C-rich element (50). We examined the effect of knocking down SRp20 and YB-1 by RNAi on the switching between alternative 3' ss in U2OS cells stably transfected with a BPV-1 late-minigene expression vector. Stably transfected cells lacking SRp20 showed about threefold more viral-RNA splicing at the late-specific nt 3605 3' ss than stable cells with no siRNA treatment or with NS siRNA treatment (Fig. 3E and F), indicating that the interaction of SRp20 with the CAC motif in SE4 blocks the selection of the late-specific 3' ss. In contrast, siRNA knockdown of YB-1 in stably transfected cells had no effect on the splicing selection (Fig. 3E and F).

When U2OS cells were transiently transfected with a BPV-1 late-minigene expression vector containing a wt SE4 or an mt SE4 (SE4m2) and were examined by Northern blotting for splicing selection in the presence or absence of SRp20 expression, we found that the negative role of SRp20 in the selection of the viral late-specific 3' ss was SE4 dependent. As shown in Fig. 3G, knocking down SRp20 expression was found to promote the usage of the nt 3605 3' ss from the wt SE4 vector but had only a minimal effect on the splicing selection from the mt SE4 vector. We also created a separate U2OS cell line stably transfected with a BPV-1 late-minigene transcript containing an mt SE2 (SE2m in Fig. 2b). Consistent with the previous observation, only a small amount of splicing (~37%) occurred at the nt 3225 3' ss in this cell line (see Fig. S1 in the supplemental material). However, cells lacking SRp20 had a further decrease (>4-fold) in the usage of the nt 3225 3' ss relative to cells not treated with siRNA or treated with an NS siRNA (see Fig. S1 in the supplemental material). Cells lacking both SRp20 and YB-1 had a decrease of only about twofold in usage of the nt 3225 3' ss, suggesting that YB-1 might act to slightly promote selection of the viral late-specific nt 3605 3' ss despite the previous finding that lack of YB-1 alone had no effect on splicing. Overall, we conclude that cellular SRp20 overwhelms YB-1, not only in the interaction with SE4 (Fig. 3), but in selection of the late-specific 3' ss, and therefore plays a dominant-negative role in the SE4-mediated selection of the late-specific splice site.

These conclusions were further supported by transient cotransfection of 293 cells with a BPV-1 late-minigene expression vector containing a wt SE4 or mt SE4 (Fig. 4A) and an SRp20, ASF/SF2, or YB-1 expression vector. Although the transcripts with an mt SE4 alone reduced selection of the nt 3605 3' ss for RNA splicing compared to the wt SE4 transcripts (Fig. 4B, compare lane 2 to lane 6), the overexpression of SRp20 did not have any effect on switching of the nt 3605 3' ss (data not shown). As predicted, the overexpression of ASF/SF2, which interacts with SE2 (Fig. 3C) and SE1 (59), although it showed no effect on splicing of the mt SE4 transcripts (Fig. 4B, lanes 6 to 9), slightly increased the choice of the nt 3225 3' ss for the splicing of wt SE4 transcripts, along with decreased usage of the nt 3605 3' ss (Fig. 4B, lanes 2 to 5). In contrast, the overexpression of YB-1 appeared to increase splicing at the nt 3605 3' ss of wt SE4 transcripts but without much effect on the mt SE4 transcripts (Fig. 4B, lanes 11 to 16).


Figure 4
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  		FIG. 4. Effects of ASF/SF2 and YB-1 on SE4-mediated selection of the BPV-1 late-specific 3605 3' ss. (A) Schematic diagram of a BPV-1 late-minigene expression vector with a mutant SE4 alone and its pre-mRNA derived from plasmid pTMF25-8. See other descriptions in Fig. 2a. The crossed-out SE4 indicates that SE4 contains point mutations, as seen in SE4m2 in Fig. 2b. (B) Selection of alternative splice sites by overexpression of ASF/SF2 or YB-1. Total RNA extracted from 293 cells 48 h after cotransfection was digested with RNase-free DNase I and analyzed for RNA splicing by RT-PCR. The cotransfection was carried out with 0.5 µg of a BPV-1 late-minigene expression vector with wt SE4 (p3231) or mt SE4 plus 0, 0.1, 0.5, or 2.5 µg of an ASF/SF2 or a YB-1 expression vector. BPV-1 L1 and L2 cDNAs were included as positive controls, and water was used as a negative control. One representative experiment of two is shown.

Expression of viral L1 in terminally differentiated keratinocytes with low levels of SRp20. Selection of the 3225 3' ss for RNA splicing of both viral early transcripts and L2 pre-mRNAs appears in both the basal and upper layers of the epidermis, whereas selection of the L1-specific 3605 3' ss and activation of the 3764 5' ss downstream (Fig. 2a) take place only in the upper layers of the epidermis (4). To investigate whether SRp20 is a limiting factor in the selection of the L1-specific 3605 3' ss, we used immunohistochemistry to examine bovine fibroma tissues for the expression of SRp20 and BPV-1 L1. In these wart tissues, SRp20 was abundantly expressed in both the basal cells and undifferentiated keratinocytes but was expressed at substantially reduced levels in highly differentiated keratinocytes or the cornified upper layers of the wart tissues, where viral L1 is produced (Fig. 5A and B; see Fig. S2A in the supplemental material). Double staining of the wart tissues for L1 and SRp20 expression demonstrated that viral L1 is produced only in terminally differentiated keratinocytes with substantially reduced SRp20 expression (Fig. 5C and D; see Fig. S2B in the supplemental material). These data indicate that the expression of cellular SRp20 is inversely correlated with BPV-1 L1 expression in keratinocytes.


Figure 5
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  		FIG. 5. Inverse expression of SRp20 and L1 in bovine warts caused by BPV-1 infection. (A and B) Immunohistochemical staining of wart tissues with anti-SRp20 antibody 7B4 (A) or an anti-BPV-1 L1 antibody (B), showing that BPV-1 L1 is expressed in superficial layers of the wart tissues, where SRp20 expression is low. (C) Expression of BPV-1 L1 in keratinocytes with terminal differentiation and substantially reduced SRp20. The confocal images were taken separately from the superficial layers of wart tissues double-stained for BPV-1 L1 (red) and SRp20 (green) and then merged to show the colocalization of the two proteins. DAPI (4',6'-diamidino-2-phenylindole) shows nuclear staining of the cells. Scale bar, 50 µm. (D) (Left) Enlarged, merged image from panel C showing the colocalization and expression of BPV-1 L1 (red) and SRp20 (green) in individual cells. (Right) L1 and SRp20 staining in individual cells.

This inverse correlation of BPV-1 L1 and SRp20 expression was also verified in the HPV-infected cervix (Fig. 6). When cervical tissue infected with HPV18, -52, and -66 with active viral-DNA replication (22) was used for double staining with viral L1 and SRp20 antibodies, we demonstrated that viral-L1 expression took place only in the upper layers of the infected cervix, where the infected keratinocytes exhibited remarkable deficiency of SRp20 (Fig. 6A). In contrast, SRp20 was found to be expressed abundantly in the undifferentiated or intermediately differentiated keratinocytes in the basal and parabasal layers of the cervix. This was further confirmed by immunohistochemical staining of the cervical tissues with the two antibodies (data not shown). In double-staining and confocal-imaging analyses, a few cells showed high levels of SRp20 but low levels of L1 (Fig. 6A and B) or vice versa, indicating an inverse correlation of the expression of SRp20 and viral L1 in these cervical cells, as shown in bovine wart tissues.


Figure 6
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  		FIG. 6. Inverse expression of SRp20 and L1 in HPV-infected cervical tissues. The cervical tissue had mixed infections with HPV18, HPV52, and HPV56 and displayed CIN I lesions with active viral-DNA replication (22). (A) Expression of HPV L1 is inversely correlated with the SRp20 level in cervical keratinocytes. Confocal images of cervical tissues double stained with anti-SRp20 7B4 antibody (green) and anti-HPV L1 K1H8 antibody (red) were taken separately and merged to show the colocalization of the two proteins. The arrows and arrowheads indicate, respectively, the cells inversely coexpressing SRp20 and L1. (B) Colocalization and expression of HPV L1 (red) and SRp20 (green) in individual cells enlarged from a merged image in panel A (magnification, x20).

In raft cultures derived from human vaginal keratinocytes immortalized by HPV16, we also demonstrated that SRp20 appeared mostly in basal cells but only weakly in spinous cells, with none in granular or cornified cells (Fig. 7), indicating its differentiation-dependent expression.


Figure 7
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  		FIG. 7. SRp20 expression in 10-day-old raft cultures derived from HPV16-immortalized vaginal keratinocytes. Immunohistochemical staining of the raft tissue section was performed with an anti-SRp20 antibody (7B4), showing strong nuclear SRp20 staining mainly in the basal-layer cells and weak nuclear staining in the spinous layer cells, but no staining in granular and cornified cells.

SRp20 suppresses the expression of HPV late genes but promotes the expression of the HPV early genes E6 and E7. Recent studies have shown that HPV16 late transcripts also contain an A/C-rich, SE4-like element (45) in a corresponding exon, as illustrated in Fig. 8A. Interestingly, this ESE element also interacts with SRp20, YB-1, and hnRNP L (Fig. 8B to D) through its CAC motifs. However, unlike BPV-1 SE4, which does not interact at all with SRp30, SRp55, and SRp75 (Fig. 8C and 3B), HPV16 ESE binds to all three (Fig. 8C). Since YB-1-SE4 interaction had only a minimal effect on BPV-1 alternative splicing and hnRNP L also bound to AG-rich SE2 (Fig. 3C) and a UAUCUA motif (data not shown), we focused on SRp20 binding in the regulation of the expression of HPV16 late genes. U2OS cells were cotransfected with SRp20 siRNA or an NS siRNA (Fig. 8E), along with a subgenomic HPV16 construct, pJR5 (wt ESE), or were transfected only with pJR5 or pJR9 (an HPV16 construct with an mt ESE) (Fig. 8F). Because the viral late promoter has no activity in cell cultures, transcription of viral late transcripts from pJR5 and pJR9 was driven by a CMV IE promoter. We found an increase in HPV16 late transcripts in the cells either with reduced SRp20 but transfected with pJR5 (wt ESE) (Fig. 8F, left) or with a normal level of SRp20 but transfected with pJR9 (mt ESE) (Fig. 8F, right), by RT-PCR analysis. The former was further verified by Northern blot analysis (Fig. 8G), showing a substantial increase in both L1 and L2 in the cells with knocking down of SRp20 expression. These data suggest that SRp20 suppresses the expression of HPV16 late genes through an SRp20-ESE interaction. To test this hypothesis, we compared the expression levels of SRp20 in human vaginal keratinocytes in raft cultures, which support L1 production, and in monolayer cultures, which do not support L1 production (Fig. 8H) but do support the expression of viral early genes. We found that keratinocytes in raft cultures make considerably less SRp20 than the corresponding cells in monolayer cultures or HPV16+ CaSki cells (Fig. 8H), indicating that a natural reduction of SRp20 in raft cultures is at least partially responsible for the early-to-late switch and the resulting expression of viral late genes.

Subsequently, we asked whether the presence of SRp20 at a high level is required for expression of viral early genes. We used an siRNA to knock down the production of SRp20 in HPV-positive cervical cancer cells, which produce large amounts of the E6 and E7 oncoproteins but do not express viral late genes. In sharp contrast to the effect of SRp20 on the expression of viral late genes, the expression of viral early E6 and E7 depended on a high level of SRp20 in the cells (Fig. 9). Reduction of cellular SRp20 by siRNA led to an approximately twofold reduction of bicistronic viral E6E7 transcripts (Fig. 9A to C) and a substantial reduction of the viral E7 oncoprotein in both HPV16+ CaSki cells (Fig. 9D) and HPV18+ HeLa cells (Fig. 9E). The accumulation of p53 and activation of a p53 downstream target, p21, in these cells were indications that viral E6 was downregulated, because viral E6 targets cellular p53 for degradation (47). Downregulation of E7 expression in both cell lines also resulted in the accumulation of hypophosphorylated p105Rb, an E7 target (20). Strikingly, SP1, an essential transcription factor for E6 and E7 expression (2, 13, 15, 51), was also greatly reduced in both cells with SRp20 knockdown (Fig. 9D and E). Altogether, these data indicate that cellular SRp20 promotes the expression of viral early genes by maintaining SP1 at a physiological level in the cells.


Figure 9
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  		FIG. 9. SRp20 promotes the expression of the HPV16 and HPV18 early genes E6 and E7. (A) Diagrams of the HPV16 and HPV18 E6 and E7 ORFs, the splicing directions of the HPV16 and HPV18 E6E7 bicistronic pre-mRNAs, and the positions of antisense probes used for Northern blot analysis. The numbers above the E6 and E7 ORFs are start and stop codons for each ORF, and above the pre-mRNA are splice sites. Exons (box 1 and box 2) and introns (lines) of the E6E7 pre-mRNA are shown. The drawings are not to scale. (B to E) Knocking down SRp20 in CaSki and HeLa cells with siRNA decreases the expression of HPV16 and HPV18 E6 and E7. HPV16+ CaSki and HPV18+ HeLa cells were treated twice with SRp20-specific siRNA or an NS siRNA (B). HPV16 and HPV18 E6E7 RNAs were detected by Northern blotting (C), and E7 protein and E6 and E7 downstream targets, as well as SP1, were detected by Western blotting (D and E). For the Northern blotting (C), total cytoplasmic or nuclear RNA was isolated with (CaSki) or without (HeLa) poly(A) selection, and the E6E7 RNA in each fraction was detected. After normalization to GAPDH, the relative level of E6E7 RNA in each fraction of SRp20 siRNA-treated cells was expressed as a ratio to the level obtained from a corresponding fraction of cells treated with an NS siRNA. C, cytoplasmic; N, nuclear. One representative experiment of two is shown. For the Western blots in panels B, D, and E, hnRNP K or tubulin in each sample served as a sample loading control. (F) Relationship of SRp20 expression in the cervix to viral E6E7 and L1L2 production. Normal cervical tissues were examined for SRp20 expression by immunohistochemistry with an anti-SRp20 7B4 antibody. Abundant SRp20 expression in the basal and parabasal layers of the cervix is depicted as paralleling the level of E6E7 expression, but the reduction of SRp20 expression in the upper layers of the cervix is inversely related to L1L2 expression (arrows). Infection of the cervical basal cells by viruses (circles, top left) is initiated through a microtrauma.


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DISCUSSION
 
SRp20 is the smallest member of the SR protein family (55), with a single N-terminal RNA recognition motif (RRM) immediately followed by an RS-rich domain. In addition to its regulation of RNA splicing via interactions with ESEs (17, 18), SRp20 has been found to play an important role in alternative RNA polyadenylation (35, 37), RNA export (26, 27), and translation (6). Furthermore, blastocyst formation is blocked in mouse embryos lacking SRp20 (31). Although SRp20 was initially shown to bind a pyrimidine-rich sequence with a consensus (A/U)C(A/U)(A/U)C sequence (8), a recent study demonstrated that the SRp20 RRM binds the RNA sequence 5'-CAUC-3' in a semi-sequence-specific manner, with only the 5' cytosine recognized specifically by the RRM (23). In this report, we have described SRp20 as an important factor controlling the papillomaviral early-to-late switch through its interactions with an A/C-rich element in viral transcripts, SE4, that controls the selection of a BPV-1 late-specific splice site. We found that binding of SRp20 to the three consecutive CAC motifs in SE4 suppresses this selection, whereas knocking down SRp20 expression in U2OS cells promotes selection of the L1-specific splice site. We also found that the terminally differentiating keratinocytes in bovine wart tissues that express BPV-1 L1 or in human cervical tissues with HPV infection-induced CIN I lesions that express HPV L1 have much reduced expression of SRp20, suggesting an inverse correlation between SRp20 expression and L1 production during differentiation. This observation is consistent with the concept that viral early genes are expressed in the basal and undifferentiated cells of the cervix but that the expression of papillomavirus late genes and production of the viral major capsid protein L1 take place only in the most terminally differentiated keratinocytes in the most superficial layers of the cervix (25). Finding of a suppressive, rather than enhancing, activity of SRp20 on the selection of a viral late-specific splice site is consistent with the report that SRp20 inhibits fibronectin EDI (extra domain I) splicing (12).

In HPV16, a similar SRp20-interacting element, ESE, controls the viral early-to-late switch of gene expression. An early study of this ESE by deletion mutations showed that it promotes the use of a common 3' ss at nt 3358, an important 3' ss in E6, E7, E4, and L1 pre-mRNAs, and the use of polyadenylation at an early poly(A) site. Thus, ESE indirectly blocks the expression of HPV16 late genes (45). In this report, we found that this ESE interacts with SRp20, YB-1, and hnRNP L through its CAC motifs. It also interacts with SRp30s, SRp55, and SRp75, deviating somewhat from BPV-1 SE4, which does not bind any of the SR proteins other than SRp20. Knocking down SRp20 expression in U2OS cells with an siRNA or disruption of the SRp20 binding sites in the ESE by point mutation promoted the expression of viral late genes from a subgenomic HPV16 expression vector, confirming that the ESE suppresses the expression of viral late genes (45), presumably by interacting with SRp20. The lack of conventional cell cultures for HPV multiplication has largely restrained our understanding of the HPV life cycle. However, raft cultures derived from human keratinocytes have been successfully utilized to produce infectious HPVs (14, 40). We found that human keratinocytes in raft cultures, which support viral-L1 expression, make much less SRp20 than the corresponding monolayer cultures, which do not support viral-L1 expression. Conversely, cervical cancer cell lines produce a large amount of SRp20 but do not express any late genes. Instead, the abundant SRp20 in cervical cancer cell lines is important for the substantial expression of the viral early genes E6 and E7. The finding that SRp20 is produced primarily in basal and parabasal cells of the cervix, where only viral early genes (including viral E6 and E7) are expressed (41), further supports an important role for SRp20 in the regulation of the viral life cycle. Altogether, along with the observation of viral-L1 expression inversely related to reduced SRp20 in terminally differentiated keratinocytes, we propose a model to illustrate our current understanding of SRp20 expression in the cervical epithelium over the viral life cycle (Fig. 9F). This model differs from the ASF/SF2 model proposed in the study of a negative regulatory element in the 3' end of HPV16 late pre-mRNAs (39), in which upregulation of ASF/SF2 upon differentiation of W12E cells was ascribed to the expression of E2 from episomal HPV16 genomes rather than epithelial differentiation.

Despite the fact that the HPV16 ESE regulates the selection of the splice site and poly(A) site, the integration of an HPV genome into a host chromosome in cervical cancer cells disrupts the integrity of viral E1 and E2 ORFs (3, 30) and positions the identified SRp20-interacting ESE downstream of the integration site, interrupting the ability of the HPV16 ESE to access and regulate the use of the upstream 3' ss. Because the viral E6 and E7 ORFs are positioned upstream of E1 and E2 in the virus genome, regulation by SRp20 of viral E6 and E7 expression in cervical cancer cells must be attributed to this ESE-independent pathway. In the case of HPV18, no ESE element has been identified in any of its transcripts. Notably, the downregulation of viral E6 and E7 expression by SRp20 knockdown appeared in association with a reduction in transcription factor SP1 and viral E6E7 transcription, but not with a reduction in viral RNA export, because the cells with reduced SRp20 expression showed no accumulation of bicistronic E6E7 RNAs in the nuclear fraction. Instead, the number of bicistronic E6E7 transcripts was reduced two- to fourfold in both the cytoplasmic and nuclear fractions (Fig. 9C). Thus, reduced viral E6 and E7 transcription might be a result of the reduced expression of SP1 in the SRp20 knockdown cells, since SP1 is essential for transcription activation of both HPV16 and HPV18 E6E7 promoters (2, 13, 15, 51).

Initially, we thought that YB-1 could be a major player in SE4-mediated regulation of BPV-1 L1 expression. YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein that is involved in regulation of DNA repair (19), mRNA transcription (46), splicing (1, 50), translation (10, 43), and stability (9, 16). YB-1 has been identified as a spliceosome-associated factor (11) and interacts with splicing factors SRp30c (44), SRrp86 (32), and phosphatase PP2C{gamma} (1) to regulate alternative RNA splicing. In human CD44 alternative RNA splicing, YB-1 binding to an A/C-rich exon enhancer controls the inclusion of variable exon 4 (50). However, in our study, YB-1 bound the A/C-rich SE4 only with low affinity compared to its binding of the purine-rich SE2 and had little positive effect on the selection of a viral-L1-specific 3' ss. Although YB-1 is an abundant cellular protein, the observed minimal effect of YB-1 on the function of A/C-rich SE4 in selection of the L1-specific 3' ss could be a consequence of the overwhelming suppressive effect of the SRp20-SE4 interaction on splice site selection. Altogether, our data support a compelling model of the papillomavirus early-to-late switch that is closely tied to SRp20 function and its differential expression in infected keratinocytes.


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ACKNOWLEDGMENTS
 
We thank Tom Cooper (Baylor College of Medicine) for YB-1 plasmids, James Manley (Columbia University) for the ASF/SF2 plasmid pCGNF1, and Javier Caceres (MRC Human Genetics Unit, Edinburgh, United Kingdom) for the SRp20 plasmid. We also thank Jennice Gullett for preparation of the raft culture tissues.

This study was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, NIH, and NIH grant R01 AI057988-01 to C.M.


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FOOTNOTES
 
* Corresponding author. Mailing address: HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, 10 Center Dr. 6N106, Bethesda, MD 20892-1868. Phone: (301) 594-1382. Fax: (301) 480-8250. E-mail: zhengt{at}exchange.nih.gov Back

{triangledown} Published ahead of print on 22 October 2008. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back


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Journal of Virology, January 2009, p. 167-180, Vol. 83, No. 1
0022-538X/09/$08.00+0     doi:10.1128/JVI.01719-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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