The E1circumflexE4 protein of human papillomavirus interacts with the serine-arginine-specific protein kinase SRPK1

Abstract

Human papillomavirus (HPV) infections of the squamous epithelium are associated with high-level expression of the E1circumflexE4 protein during the productive phase of infection. However, the precise mechanisms of how E1circumflexE4 contributes to the replication cycle of the virus are poorly understood. Here, we show that the serine-arginine (SR)-specific protein kinase SRPK1 is a novel binding partner of HPV type 1 (HPV1) E1circumflexE4. We map critical residues within an arginine-rich domain of HPV1 E1circumflexE4, and in a region known to facilitate E1circumflexE4 oligomerization, that are requisite for SRPK1 binding. In vitro kinase assays show that SRPK1 binding is associated with phosphorylation of an HPV1 E1circumflexE4 polypeptide and modulates autophosphorylation of the kinase. We show that SRPK1 is sequestered into E4 inclusion bodies in terminally differentiated cells within HPV1 warts and that colocalization between E1circumflexE4 and SRPK1 is not dependent on additional HPV1 factors. Moreover, we also identify SRPK1 binding of E1circumflexE4 proteins of HPV16 and HPV18. Our findings indicate that SRPK1 binding is a conserved function of E1circumflexE4 proteins of diverse virus types. SRPK1 influences important biochemical processes within the cell, including nuclear organization and RNA metabolism. While phosphorylation of HPV1 E4 by SRPK1 may directly influence HPV1 E4 function during the infectious cycle, the modulation and sequestration of SRPK1 by E1circumflexE4 may affect the ability of SRPK1 to phosphorylate its cellular targets, thereby facilitating the productive phase of the HPV replication cycle.

FIG. 1.

GST-HPV1 E1^E4 coprecipitates SRPK1. (A) Silver-stained 5 to 15% SDS-polyacrylamide gel of GST coprecipitations from lysates prepared from SV-JD keratinocytes showing GST, GST-HPV E1^E4 fusion proteins (lower panel), and associated cellular factors (upper panel). +, lysate present; −, lysate absent. Mass spectrometry analysis by LC MS/MS of trypsin-digested peptides prepared from silver-stained bands identified SRPK1 peptides present in a prominent band (indicated by an asterisk) within the GST-HPV1 E1^E4 coprecipitate. (B) The full-length amino acid sequence of SRPK1 (gene identifier 47419936). Underlined in boldface type are the peptides sequenced from the GST-HPV1 E1^E4 coprecipitation.

FIG. 2.

HPV1 E1^E4 associates with Flag-SRPK1 in human keratinocytes. Western analysis of anti-Flag and isotype control immunoprecipitations from lysates prepared from 293T cells coexpressing Flag-SRPK1 and HPV1 E1^E4 or Flag-HPV1 E1 and HPV1 E1^E4. HPV1 E1^E4 was present in immunoprecipitates of Flag-SRPK1 complexes but not in those containing Flag-HPV1 E1, confirming the specificity of the SRPK1-HPV1 E1^E4 association. WB, Western blot; +, present; −, absent.

FIG. 3.

The E1^E4 interaction with SRPK1 is conserved between different HPV types. SRPK1 binding to E1^E4 proteins of various HPV types was investigated by using GST-E1^E4 fusion proteins of HPV1, HPV16, and HPV18 to coprecipitate endogenous SRPK1 derived from SV-JD lysates (upper panel), Flag-SRPK1 expressed in 293T cells (middle upper panel), and His-SRPK1 expressed in bacteria (lower middle panel). A Ponceau-stained nitrocellulose membrane used for the SRPK1 coprecipitation demonstrates that the relative amounts of GST and GST fusion proteins (indicated by asterisks) used in these experiments is approximately equal (lower panel). In all cases, SRPK1 forms complexes specifically with all of the different GST-E1^E4 proteins but not GST alone. In the lower middle panel, the upper band migrating with an apparent molecular mass of over 120 kDa represents the full-length His-SRPK1, while the smaller band is likely to be a breakdown product of His-SRPK1. WB, Western blot.

FIG. 4.

Mapping the domains within HPV1 E1^E4 that mediate the association with SRPK1. Anti-Flag immunoprecipitations (IP) from lysates prepared from 293T cells coexpressing Flag-SRPK1 and mutant HPV1 E1^E4 proteins were performed to identify key residues in HPV1 E1^E4 necessary for SRPK1 binding. (A) A diagrammatic summary of the data from the immunoprecipitation experiments is shown. The white domains correspond to regions within E1^E4 that are not required for the interaction with Flag-SRPK1, and the black domains identify residues found necessary for the association to occur. Alanine-scanning mutagenesis of residues in regions 44 to 48 and 109 to 114 involved in SRPK1 binding identified individual amino acids that are key participants in the association. +, binding; −, no binding. The relationship of the different domains to known E1^E4 functions and sequence characteristics is also shown. (B) Western blot (WB) analysis of immunoprecipitations between Flag-SRPK1 and E1^E4 proteins containing various deletions. (C) Western blot analysis of immunoprecipitations between alanine point substitutions and Flag-SRPK1. All of these mutants aside from F114A are expressed to a similar or greater level than the wild-type E1^E4. Analysis of the immunoprecipitations indicate that the amino acids G44, R45, R47, D110, L111, and D113 are each required to maintain the association of HPV1 E1^E4 with Flag-SRPK1.

FIG. 5.

SRPK1 distribution is altered in the presence of HPV1 E1^E4. (A and B) Confocal analysis of 4-μm sections of an HPV1-induced wart costained for E4 (red) and SRPK1 (green); nuclei were identified using DAPI (blue). (A) SRPK1 expression in regions of the wart tissue that are E4 negative and do not show evidence of productive HPV1 infection. Arrowheads indicate the basal cell layer. (B) In regions of the wart positive for E4 expression, SRPK1 is contained within E4 inclusions present in cells of the granular layers (upper panel, examples of costained inclusions are indicated by arrows) but not in those formed in cells of the lower (spinous) layers (bottom panel). (C) Confocal analysis of distribution of E4 (red) and SRPK1 (green) in SV-JD cells cotransfected with plasmids that express HPV1 E1^E4 and Flag-SRPK1 or Flag-SRPK1 alone. Nuclei were identified using DAPI (blue).

FIG. 6.

SRPK1 phosphorylates HPV1 E1^E4 in vitro. (A) SDS-polyacrylamide gel of in vitro kinase reaction mixtures containing His-SRPK1 and GST-E1^E4 proteins of HPV1, HPV16, and HPV18. The amounts of GST-E1^E4 fusion proteins (indicated by asterisks) and His-SRPK1 in the kinase reactions are shown in the Coomassie-stained gel. The autoradiograph demonstrates the presence of phosphorylated protein species. The arrows adjacent to lane 5, which contained only His-SRPK1, indicate the presence of phosphorylated full-length His-SRPK1 (SRPK1-P) as well as smaller phosphopeptide species, which may be breakdown products of SRPK1 or phosphorylated bacterial contaminants. Lanes 1, 3, and 4 show similar patterns of protein phosphorylation, indicating that neither GST, GST-HPV16 E1^E4, nor GST-HPV18 E1^E4 is phosphorylated by SRPK1 under these conditions. In contrast, the GST-HPV1 E1^E4 substrate (lane 2), produces a distinct phosphorylated species (E4-P) which is not present in the reaction mixture containing His-SRPK1 alone (lane 5). (B) An in vitro kinase reaction mixture containing His-SRPK1 and GST-HPV1 E1^E4 proteins was analyzed by Western blotting (WB) with an HPV1 E1^E4-specific monoclonal antibody prior to autoradiography (³²P). Major E4 phosphospecies (E4-P) corresponds to one of a series of peptides (identified with arrowheads) smaller than the full-length GST fusion (FL). The faint band (marked with an asterisk) appearing in the autoradiograph is derived from the strong chemiluminescence signal of the full-length E1^E4 fusion protein.

FIG. 7.

In vitro phosphorylation of HPV1 E1^E4 is dependent on SRPK1 binding. In vitro kinase reactions containing His-SRPK1 and GST-HPV1 E1^E4 fusion proteins that bind SRPK1 (E1^E4 and Δ49-53) or are defective for binding (Δ44-48) titrated at 25, 5, and 1 μg. The relative amounts of His-SRPK1 and the different GST proteins (indicated by asterisks) present in the kinase reactions are shown in the Coomassie-stained gel. A major phosphospecies (E4-P) is present in in vitro kinase reaction mixtures containing the wild-type E1^E4 protein (lanes 4 to 6) and the Δ49-53 deletion mutant (lanes 10 to 12) but is not detected in reactions containing the Δ44-48 deletion mutant (lanes 7 to 9) or the GST control protein (lanes 1 to 3). In the titrations of E1^E4 substrates that interact with SRPK1, we observed a dose-dependent reduction in autophosphorylation of the full-length His-SRPK1 polypeptide (SRPK1-P).

FIG. 8.

The sequence of regions of HPV E4 proteins that are rich in basic amino acids. The amino acid sequences of E4 regions of HPV1, -16, -18, and -63 that are particularly rich in basic amino acids are shown with arginine residues identified in boldface type. Note that bipartite arginine-rich motifs are present in HPV1, -18, and -63 but not HPV16. Amino acids within the HPV1 E4 region involved in the interaction with SRPK1 are underlined.