Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35)

Abstract

High-risk human papillomaviruses (HR-HPV) cause anogenital cancers, including cervical cancer, and head and neck cancers. Human papillomavirus 16 (HPV16) is the most prevalent HR-HPV. HPV oncogenesis is driven by two viral oncoproteins, E6 and E7, which are expressed through alternative splicing of a polycistronic RNA to yield four major splice isoforms (E6 full length, E6*I, E6*II, E6*X). The production of multiple mRNA isoforms from a single gene is controlled by serine/arginine-rich splicing factors (SRSFs), and HPV16 infection induces overexpression of a subset of these, SRSFs 1, 2, and 3. In this study, we examined whether these proteins could control HPV16 oncoprotein expression. Small interfering RNA (siRNA) depletion experiments revealed that SRSF1 did not affect oncoprotein RNA levels. While SRSF3 knockdown caused some reduction in E6E7 expression, depletion of SRSF2 resulted in a significant loss of E6E7 RNAs, resulting in reduced levels of the E6-regulated p53 proteins and E7 oncoprotein itself. SRSF2 contributed to the tumor phenotype of HPV16-positive cervical cancer cells, as its depletion resulted in decreased cell proliferation, reduced colony formation, and increased apoptosis. SRSF2 did not affect transcription from the P97 promoter that controls viral oncoprotein expression. Rather, RNA decay experiments showed that SRSF2 is required to maintain stability of E6E7 mRNAs. These data show that SRSF2 is a key regulator of HPV16 oncoprotein expression and cervical tumor maintenance.

Importance: Expression of the HPV16 oncoproteins E7 and E6 drives HPV-associated tumor formation. Although increased transcription may yield increased levels of E6E7 mRNAs, it is known that the RNAs can have increased stability upon integration into the host genome. SR splicing factors (SRSFs) control splicing but can also control other events in the RNA life cycle, including RNA stability. Previously, we demonstrated increased levels of SRSFs 1, 2, and 3 during cervical tumor progression. Now we show that SRSF2 is required for expression of E6E7 mRNAs in cervical tumor but not nontumor cells and may act by inhibiting their decay. SRSF2 depletion in W12 tumor cells resulted in increased apoptosis, decreased proliferation, and decreased colony formation, suggesting that SRSF2 has oncogenic functions in cervical tumor progression. SRSF function can be targeted by known drugs that inhibit SRSF phosphorylation, suggesting a possible new avenue in abrogating HPV oncoprotein activity.

FIG 1

E6 splice isoform expression in epithelial cell transformation. (A) Diagram of the E6E7 region of the HPV16 genome. Shaded gray boxes, open reading frames; arrow, P₉₇, HPV16 early promoter. The numbers (nucleotides) refer to the end of the E6 (560) and the beginning of the E7 (562) open reading frames. The four E6E7 mRNAs are shown below the genomic map. Numbers mark the nucleotide positions of one splice donor (226) and three possible splice acceptor sites (409, 526, 742). Gray boxes, exons; black lines, introns. (B) RT-qPCR quantification of the expression levels of total E6E7 RNA relative to levels of GAPDH in the four W12 cervical epithelial cell lines (W12E, W12G, W12t, W12ti). The graph shows the means and standard errors of the means from three separate experiments. (C) Ethidium bromide-stained polyacrylamide gel electrophoresis of RT-PCR products amplified from polyadenylated RNA isolated from the above-described W12 cell lines using primers that amplify all E6 isoforms. The various isoforms are indicated to the right of the gel (E6fl, E6*I, E6*II, E6*X). A second gel identical to that for fractionating GAPDH control PCR products from the same RNA/cDNA preparations was electrophoresed at the same time and is shown below. M, marker; RT, reverse transcriptase; −, RT-PCR in the absence of reverse transcriptase; +, RT-PCR in the presence of reverse transcriptase.

FIG 2

Levels of SRSF proteins 1, 2, and 3 are higher in the W12 tumor lines (W12t and W12ti) than in the nontumor lines (W12E and W12G). (A) Western blot showing SRSF1 levels in the four W12 lines; (B) Western blot showing SRSF2 levels in the four W12 lines; (C) Western blot showing SRSF3 levels in the four W12 lines. GAPDH was used as a loading control. Lane 1, W12E (nontumor cell line) protein extracts; lane 2, W12G (nontumor cell line) protein extracts; lane 3, W12t (tumor cell line) protein extracts; lane 4, W12ti (tumor cell line) protein extracts.

FIG 3

The effect of SRSF depletion on E6E7 RNA expression in cervical tumor and nontumor cells. (A) SRSF protein levels before and after depletion by specific siRNA pools in the W12ti cells; (B) ethidium bromide-stained polyacrylamide gel electrophoresis of RT-PCR products amplified from polyadenylated RNA isolated from W12ti cells using primers that amplify all E6 isoforms. The effect of depletion of SRSF1 (lanes 5 and 6), SRSF3 (lanes 9 and 10), and SRSF2 (lanes 11 and 12) for 48 h on E6 isoform expression in W12ti cervical tumor cells is shown. The E6 RNA isoforms are indicated to the right of the gels. A second gel identical to that for fractionating GAPDH control PCR products from the same RNA/cDNA preparations was electrophoresed at the same time and is shown below each gel. RT, reverse transcriptase; −, RT-PCR in the absence of reverse transcriptase; +, RT-PCR in the presence of reverse transcriptase; no siRNA, cells transfected without siRNA; cntrl, cells transfected with a control siRNA, siGLO; SRSF, cells treated with SRSF siRNA for 48 h. The experiments were repeated at least three times, and similar data were obtained in each experiment. (C) qPCR quantification of total E6 RNA levels following SRSF depletion. The graph shows the means and standard deviations from the means from three separate experiments.

FIG 4

The effect of SRSF depletion on E6E7 RNA expression in CaSki cervical tumor and W12G nontumor cells. (A) SRSF protein levels before and after depletion by specific siRNA pools. The antibody used (MAb104) also detects other SR proteins, and an adjacent band corresponding to SFSR5 is shown as an internal control for protein levels and to show specificity of SRSF2 depletion. As expected, the starting levels of SRSF2 in W12G cells are much lower than in CaSki cells or W12ti cells than in the internal SRSF5 control band. (B and D) Ethidium bromide-stained polyacrylamide gel electrophoresis of RT-PCR products amplified from polyadenylated RNA isolated from cell lines using primers that amplify all E6 isoforms. The E6 RNA isoforms are indicated to the right of the gels. A second gel identical to that for fractionating GAPDH control PCR products from the same RNA/cDNA preparations was electrophoresed at the same time and is shown below each gel. RT, reverse transcriptase; −, RT-PCR in the absence of reverse transcriptase; +, RT-PCR in the presence of reverse transcriptase; no siRNA, cells transfected without siRNA; cntrl, cells transfected with a control siRNA, siGLO; SRSF, cells treated with SRSF siRNA for 48 h. The experiments were repeated at least three times, and similar data were obtained in each experiment. (B) The effect of SRSF2 depletion of E6 isoform expression in CaSki cervical tumor cells. (C) qPCR quantification of total E6 RNA levels in CaSki cells before and after SRSF2 depletion. The graph shows the means and standard deviations from the means from three separate experiments. (D) The effect of SRSF2 depletion on E6 isoform expression in W12G nontumor cervical cells. (E) qPCR quantification of total E6 levels in W12G cells before and after SRSF2 depletion. The graph shows the means and standard deviations from the means from three separate experiments.

FIG 5

W12ti tumor cells exhibit decreased viral oncoprotein expression upon SRSF2 knockdown. (A) Western blot analysis of p53 protein levels with or without SRSF2 knockdown in W12ti cells. GAPDH is used as a loading control. SFSR2 depletion is shown with SRSF5 as the internal loading control. no siRNA, cells transfected without siRNA; cntrl, cells transfected with a control siRNA; SRSF2, cells transfected with SRSF2 siRNA for 48 h. Below the blots is a quantification of levels of p53 in untreated cells (no siRNA) or treated with the indicated siRNA. The data show the means and standard deviations from the means from three separate experiments. The asterisk indicates a significant change in protein expression (P ≤ 0.05) using a Student t test. (B) Western blot analysis of E7 and pRb protein levels with or without SRSF2 knockdown in W12ti cells. GAPDH is shown as the loading control. The same protein extracts as in panel A were used in this experiment, so the same level of SRSF2 depletion was achieved. no siRNA, cells transfected without siRNA; cntrl, cells transfected with a control siRNA; SRSF2, cells transfected with SRSF2 siRNA for 48 h. Below the blots is a quantification of levels of E7 in cells not treated (no siRNA) or treated with the indicated siRNAs. The data show the means and standard deviations from the means from three separate experiments. The asterisk indicates a significant change in protein expression (P ≤ 0.05) using a Student t test.

FIG 6

W12ti tumor cells undergo apoptosis upon SRSF2 knockdown. (A) FACS plots of annexin V-propidium iodide staining of W12ti cells transfected with the indicated siRNAs above the plots or irradiated with UVB as a positive control for cell death. The percentages of cells occupying each quadrant in one experiment are shown. The lower right-hand quadrant contains early apoptotic cells, while the upper right-hand quadrant contains late apoptotic/dead cells. W12ti cells were treated with control siRNA (siCntrl) or siRNA pool against SRSF2 (siSRSF2) or siRNA against HPV16 E6 (siE6) or treated with UVB irradiation (UVB) for 24 h as a positive indicator of apoptosis. Very similar results were obtained in three separate experiments (see Table 1). (B) Graph of the percentage of W12ti cells from three independent experiments occupying the live and early apoptotic quadrants of the annexin V and propidium iodide staining plots shown in panel A. (C) Western blot showing the levels of SRSF2 in mock-transfected cells (no siRNA) or cells transfected with control siRNA (cntrl) or transfected with siRNA against SRSF2 (SRSF2). SRSF5 is shown as an internal loading control.

FIG 7

The effect of SRSF2 depletion on W12 cell proliferation and colony formation. (A) Proliferation rates over 72 h of W12ti tumor cells treated with control siRNA (cntrl) or treated with an siRNA pool against SRSF2 (siSRSF2). (B) W12G nontumor cells treated with control siRNA (cntrl) or treated with an siRNA pool against SRSF2 (siSRSF2). The inset Western blots in panels A and B show levels of SRSF2 depletion and SRSF5 levels as an internal loading control. (C) Colony formation assay of W12ti cells with (SRSF2 siRNA) or without (cntrl siRNA) SRSF2 knockdown. The graph shows means and standard deviations from the means of numbers of colonies from three independent experiments. Asterisk indicates a significant change in colony formation (P ≤ 0.05) using a Student t test.

FIG 8

SRSF2 does not regulate HPV16 P₉₇ promoter activity but may stabilize E6E7 RNAs. (A) Graph of luciferase activity in 293T cells transiently transfected (48 h) with either a luciferase reporter plasmid containing the control SV40 promoter (pGL3-promoter; Promega) or the HPV16 long control region encompassing the P₉₇ promoter (HPV16 LCR) (39). Cells transfected with the HPV16 LCR construct were also transfected with either control siRNA (cntrl siRNA) or siRNA against SRSF2 (siSRSF2). The means and standard deviations from the means from three separate experiments are shown. RT-PCR analysis of E6E7 RNAs in actinomycin D-treated W12ti cells 24 h following transfection with siRNA against SRSF2 (B) or control siRNA (cntrl siRNA) (C). The number of hours of actinomycin D treatment is shown above the gels. GAPDH RNA levels are stable over this time period and are shown as a loading control.