Abstract
The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. We show here for the first time that the interaction between HPV1 E1^E4 and SRPK1 leads to potent inhibition of SRPK1 phosphorylation of host serine-arginine (SR) proteins that have critical roles in mRNA metabolism, including pre-mRNA processing, mRNA export, and translation. Furthermore, we show that SRPK1 phosphorylates serine residues of SR/RS dipeptides in the hinge region of the HPV1 E2 protein in in vitro kinase assays and that HPV1 E1^E4 inhibits this phosphorylation. After mutation of the putative phosphoacceptor serine residues, the localization of the E2 protein was altered in primary human keratinocytes; with a significant increase in the cell population showing intense E2 staining of the nucleolus. A similar effect was observed following coexpression of E2 and E1^E4 that is competent for inhibition of SRPK1 activity, suggesting that the nuclear localization of E2 is sensitive to E1^E4-mediated SRPK1 inhibition. Collectively, these data suggest that E1^E4-mediated inhibition of SRPK1 could affect the functions of host SR proteins and those of the virus transcription/replication regulator E2. We speculate that the novel E4 function identified here is involved in the regulation of E2 and SR protein function in posttranscriptional processing of viral transcripts. The HPV life cycle is tightly linked to the epithelial terminal differentiation program, with the virion-producing phase restricted to differentiating cells. While the most abundant HPV protein expressed in this phase is the E4 protein, we do not fully understand the role of this protein. Few E4 interaction partners have been identified, but we had previously shown that E4 proteins from diverse papillomaviruses interact with the serine-arginine-specific protein kinase SRPK1, a kinase important in the replication cycles of a diverse range of DNA and RNA viruses. We show that HPV1 E4 is a potent inhibitor of this host cell kinase. We show that E4 inhibits SRPK1 phosphorylation, not only of cellular SR proteins involved in regulating alternative splicing of RNA but also the viral transcription/replication regulator E2. Our findings reveal a potential E4 function in regulation of viral late gene expression through the inhibition of a host cell kinase.
Highlights
The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types
An in vitro kinase assay system was used to establish whether SRPK1 binding to HPV1 E1^E4 alters the activity of the kinase
glutathione S-transferase (GST) or GST-HPV1 E1^E4 proteins were titrated into the assay and, after 30 min, half of the reaction mixture was resolved by SDS-PAGE, and the dried gel was exposed to X-ray film
Summary
The serine-arginine-specific protein kinase SRPK1 is a common binding partner of the E1^E4 protein of diverse human papillomavirus types. Genetic knockdown of E4 expression in papillomavirus life cycle models can result in aberrations in the productive cycle, including reduced viral DNA amplification and decreased viral late gene expression [2,3,4,5] These studies indicated that E4 function(s) are important at multiple stages of virion production, there may be differences in the role of E4 between genotypes [6]. Overexpression of E4 proteins leads to a G2/M arrest of the cell cycle, a function common to HPV types with different epithelial tropisms [7,8,9] Abrogation of this function in an HPV18 replication model did not affect viral genome amplification or expression of viral late genes [10]. Journal of Virology p. 12599 –12611 jvi.asm.org 12599 newly synthesized virus, confirmation of this role has yet to obtained [11,12,13]
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