Serine/Arginine-rich Splicing Factor 2 Modulates Herpes Simplex Virus Type 1 Replication via Regulating Viral Gene Transcriptional Activity and Pre-mRNA Splicing

Abstract

Once it enters the host cell, herpes simplex virus type 1 (HSV-1) recruits a series of host cell factors to facilitate its life cycle. Here, we demonstrate that serine/arginine-rich splicing factor 2 (SRSF2), which is an important component of the splicing speckle, mediates HSV-1 replication by regulating viral gene expression at the transcriptional and posttranscriptional levels. Our results indicate that SRSF2 functions as a transcriptional activator by directly binding to infected cell polypeptide 0 (ICP0), infected cell polypeptide 27 (ICP27), and thymidine kinase promoters. Moreover, SRSF2 participates in ICP0 pre-mRNA splicing by recognizing binding sites in ICP0 exon 3. These findings provide insight into the functions of SRSF2 in HSV-1 replication and gene expression.

Keywords: RNA splicing; gene expression; herpesvirus; host-pathogen interaction; transcription factor.

FIGURE 1.

SRSF2 promoted HSV-1 replication. A, HeLa cells were transfected with the SRSF2 siRNA or negative control for 36 h. The SRSF2 expression levels were measured with Western blotting. B, HeLa cells were transfected with SRSF2-FLAG, FLAG, or BLANK for 36 h. The SRSF2 expression levels were measured with Western blotting. C, after 36 h of transfection with SRSF2 siRNA or the negative control, the HeLa cells were infected with HSV-1 at an m.o.i. of 1 for 12 h, fixed, and stained with an anti-HSV-1 glycoprotein antibody (green) and subjected to confocal microscopy analysis. DAPI (blue) was used to stain the nuclei. The right histogram shows the relative fluorescence intensity of the HSV-1 glycoproteins. The fluorescence signal value was measured using ImageJ software and normalized to a single cell. The data are normalized to the control (siCTRL) level and are presented as the mean ± S.D. (*, p < 0.01, Student's t test). D, after 36 h of transfection with SRSF2-FLAG, FLAG, or BLANK, the HeLa cells were infected with HSV-1 at an m.o.i. of 1 for 12 h, fixed, stained with an anti-HSV-1 glycoprotein antibody (green), and subjected to confocal microscopy analysis. DAPI (blue) was used to stain the nuclei. The right histogram shows the relative fluorescence intensity of the HSV-1 glycoproteins. The fluorescence signal value was measured using ImageJ software and normalized to a single cell. The data were normalized to the control level (FLAG) and are presented as the mean ± S.D. (*, p < 0.01, Student's t test). E, after 36 h of transfection with the SRSF2 siRNA or negative control siRNA, HeLa cells were infected with HSV-1 at an m.o.i. of 1. The culture media were collected at the indicated time points after infection and subjected to the plaque assay. The data points represent the mean values determined from three independent experiments. The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). F, after 36 h of transfection with SRSF2-FLAG, FLAG, or BLANK, the HeLa cells were infected with HSV-1 at an m.o.i. of 1. The culture media were collected at the indicated time points after infection and subjected to the plaque assay. The data points represent mean values determined from three independent experiments. The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). The cell viability and proliferative ability were determined in HeLa cells transfected with SRSF2 siRNA (G) or SRSF2-FLAG (H) with a CCK8 assay. The data points represent mean values determined from three independent experiments. The data are presented as the mean ± S.D. (no significance, Student's t test).

FIGURE 2.

SRSF2 regulated viral gene expression. A, after 36 h of transfection with the SRSF2 siRNA or negative control siRNA, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. The ICP0, ICP27, and TK expression levels relative to β-actin expression were determined with qRT-PCR in three independent experiments. An unrelated RNA (NEAT1) was used as the control. The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). B, after 36 h of transfection with the SRSF2 siRNA or negative control siRNA, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 8 h. The ICP0, ICP27, and TK expression levels were measured with Western blotting. Protein ratios of the indicated proteins/β-actin were analyzed with ImageJ software, and statistical analysis was conducted with data from three independent experiments (right panel). The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). C, after 36 h of transfection with SRSF2-FLAG, FLAG, or BLANK, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. The ICP0, ICP27, and TK expression levels relative to β-actin expression were determined by qRT-PCR for three independent experiments. An unrelated RNA (NEAT1) was used as the control. The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). D, after 36 h of transfection with SRSF2-FLAG, FLAG, or BLANK, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 8 h. The ICP0, ICP27, and TK expression levels were measured with Western blotting. Protein ratios of the indicated proteins/β-actin were analyzed with ImageJ software, and statistical analysis was conducted with data from three independent experiments (right panel). The data are presented as the mean ± S.D. (*p < 0.01, Student's t test).

FIGURE 3.

SRSF2 enhanced ICP0, ICP27, and TK transcriptional activity by binding to their promoters. A, after co-transfection with the SRSF2 siRNA or negative control siRNA and the pGL3 enhancer plasmid containing the ICP0 promoter, ICP27 promoter, or a pRL-TK reporter for 36 h, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. The NEAT1 promoter reporter was used as a negative control. The relative transcriptional activities of the ICP0, ICP27, TK, and NEAT1 promoters were determined with a luciferase assay in three independent experiments. The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). B, after co-transfection with SRSF2-FLAG, FLAG, or BLANK and the pGL3 enhancer plasmid containing the ICP0 promoter, ICP27 promoter, or a pRL-TK reporter vector with the same sequence as the HSV-1 TK gene for 36 h, HeLa cells were infected with HSV-1 for 4 h. The NEAT1 promoter reporter was used as a negative control. The relative transcriptional activity of the ICP0, ICP27, TK, and NEAT1 promoters was analyzed with a luciferase assay in three independent experiments. The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). C, HeLa cells were infected with HSV-1 for 4 h. ChIP assays were performed with an anti-SRSF2 antibody or an anti-IgG antibody (Ab). -Fold enrichment of the ICP0, ICP27, and TK promoters by anti-SRSF2 relative to the input level was examined with qRT-PCR. The NEAT1 promoter was used as a negative control. D, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h, fixed, incubated with a biotin-ICP0-P4 fragment (green), a biotin-ICP27-P2 fragment (green), and a biotin-TK-P3 fragment (green) and SRSF2 (red) and subjected to confocal microscopy analysis. The mock-infected nuclei were used as a negative control. The intensity plots for the red and green channels were analyzed with ImageJ software. DAPI (blue) was used to stain the nuclei. Scale bars, 10 μm.

FIGURE 4.

SRSF2 associated with RNAP II, ICP27, and ICP8. A, after 4 h of infection with HSV-1, HeLa cells were fixed, stained with antibodies against SRSF2 (green), RNA polymerase II (red), ICP27 (red), and ICP8 (red), and subjected to confocal microscopy analysis. The intensity plots for the red and green channels were analyzed with ImageJ software. DAPI (blue) was used to stain the nuclei. Scale bars, 10 μm. B, after 36 h of transfection with the SRSF2 siRNA or negative control siRNA, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. Cell lysates were harvested and subjected to an immunoprecipitation (IP) assay with the anti-SRSF2 antibody or the anti-IgG antibody (Ab). The retrieval of RNA polymerase II, ICP27, SRSF2, and ICP8 by endogenous SRSF2 and IgG was measured by Western blotting. Protein ratios for the indicated proteins/β-actin were analyzed with ImageJ software, and statistical analysis was conducted with data from three independent experiments (right panel). The data are presented as the mean ± S.D. (*, p < 0.01, Student's t test). C, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. Cell lysates were harvested with and without DNase and RNase treatment and then subjected to an immunoprecipitation assay with the anti-SRSF2 antibody. The retrieval of RNA polymerase II, ICP27, SRSF2, and ICP8 by endogenous SRSF2 was measured by Western blotting.

FIGURE 5.

SRSF2 regulated ICP0 pre-mRNA splicing. A, schematic of SRSF2 potential binding sites in the ICP0 pre-mRNA sequence. The black box shows the potential binding site, and red characters indicate matching sequences. B, after 36 h of transfection with the SRSF2 siRNAs or negative control, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. Cell lysates were harvested and subjected to an RNA immunoprecipitation assay. QRT-PCR was performed to detect the retrieval of ICP0 exon 1, ICP0 exon 2, ICP0 exon 3, and NEAT1 by the anti-SRSF2 antibody or anti-IgG antibody over the input level. The unrelated RNA NEAT1 was used as a negative control. The data are represented as the mean ± S.D. (*, p < 0.01, Student's t test). C, HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h, fixed, incubated with DIG-labeled ICP0 exon 3 fragment (green) or ICP0 exon 3 fragment with a deletion in SRSF2 binding motifs (green) and SRSF2 (red) and subjected to confocal microscopy analysis. The intensity plots for the red and green channels were analyzed with the Image J software. DAPI (blue) was used to stain the nuclei. Scale bars, 10 μm. D, after 36 h of transfection with the SRSF2 siRNAs or negative control, the HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. The expression ratio of the ICP0 mRNA to the pre-mRNA was determined by qRT-PCR in three independent experiments. The data are represented as the mean ± S.D. (*, p < 0.01, Student's t test). E, after 36 h of transfection with SRSF2-FLAG, FLAG, or BLANK, the HeLa cells were infected with HSV-1 at a m.o.i. of 1 for 4 h. The expression ratio of the ICP0 mRNA to pre-mRNA was determined by qRT-PCR in three independent experiments. The data are represented as the mean ± S.D. (*, p < 0.01, Student's t test).