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Human papillomaviruses (HPV) are epitheliotropic DNA tumor viruses that are prevalent in the human population. A subset of the HPVs termed high-risk HPVs (HR-HPVs) are causative agents of anogenital cancers and head-and-neck cancers. Cancer is the result of persistent high-risk HPV infections that have not been cleared by the immune system of the host. These infections are characterized by dysregulated HPV gene expression, in particular constitutive high expression of the HPV E6 and E7 oncogenes and absence of the highly immunogenic viral L1 and L2 capsid proteins. HPVs make extensive use of alternative mRNA splicing to express its genes and are therefore highly dependent on cellular RNA-binding proteins for proper gene expression. Levels of RNA-binding proteins are altered in HPV-containing premalignant cervical lesions and in cervical cancer. Here we review our current knowledge of RNA-binding proteins that control HPV gene expression. We focus on RNA-binding proteins that control expression of the E6 and E7 oncogenes since they initiate and drive development of cancer and on the immunogenic L1 and L2 proteins as there silencing may contribute to immune evasion during carcinogenesis. Furthermore, cellular RNA-binding proteins are essential for HPV gene expression and as such may be targets for therapy to HPV infections and HPV-driven cancers.

Human papillomavirus type 16 (HPV16) E2 is an essential HPV16 protein. We have investigated how HPV16 E2 expression is regulated and have identifed a splicing enhancer that is required for production of HPV16 E2 mRNAs. This uridine-less splicing enhancer sequence (ACGAGGACGAGGACAAGGA) contains 84% adenosine and guanosine and 16% cytosine and consists of three ‘AC(A/G)AGG’-repeats. Mutational inactivation of the splicing enhancer reduced splicing to E2-mRNA specific splice site SA2709 and resulted in increased levels of unspliced E1-encoding mRNAs. The splicing enhancer sequence interacted with cellular RNA binding protein hnRNP G that promoted splicing to SA2709 and enhanced E2 mRNA production. The splicing-enhancing function of hnRNP G mapped to amino acids 236–286 of hnRNP G that were also shown to interact with splicing factor U2AF65. The interactions between hnRNP G and HPV16 E2 mRNAs and U2AF65 increased in response to keratinocyte differentiation as well as by the induction of the DNA damage response (DDR). The DDR reduced sumoylation of hnRNP G and pharmacological inhibition of sumoylation enhanced HPV16 E2 mRNA splicing and interactions between hnRNP G and E2 mRNAs and U2AF65. Intriguingly, hnRNP G also promoted intron retention of the HPV16 E6 coding region thereby inhibiting production of spliced E7 oncogene mRNAs.

Human papillomavirus type 16 (HPV16) E1 and E6 proteins are produced from mRNAs with retained introns, but it has been unclear how these mRNAs are generated. Here, we report that hnRNP D act as a splicing inhibitor of HPV16 E1/E2- and E6/E7-mRNAs thereby generating intron-containing E1- and E6-mRNAs, respectively. N- and C-termini of hnRNP D contributed to HPV16 mRNA splicing control differently. HnRNP D interacted with the components of splicing machinery and with HPV16 RNA to exert its inhibitory function. As a result, the cytoplasmic levels of intron-retained HPV16 mRNAs were increased in the presence of hnRNP D. Association of hnRNP D with HPV16 mRNAs in the cytoplasm was observed, and this may correlate with unexpected inhibition of HPV16 E1- and E6-mRNA translation. Notably, hnRNP D40 interacted with HPV16 mRNAs in an HPV16-driven tonsillar cancer cell line and in HPV16-immortalized human keratinocytes. Furthermore, knockdown of hnRNP D in HPV16-driven cervical cancer cells enhanced production of the HPV16 E7 oncoprotein. Our results suggest that hnRNP D plays significant roles in the regulation of HPV gene expression and HPV-associated cancer development.

We have determined the effect of seven serine- and arginine-rich (SR) proteins and 15 heterogenous nuclear ribonucleoproteins (hnRNPs) on human papillomavirus type 16 (HPV16) late gene expression. Of the seven SR proteins analyzed here, SRSF1, SRSF3, and SRSF9 induced HPV16 late gene expression, and five of the SR proteins affected HPV16 L1 mRNA splicing. Of the 15 hnRNP proteins analyzed here, hnRNP A2, hnRNP F, and hnRNP H efficiently induced HPV16 late gene expression, and all of the hnRNPs affected HPV16 L1 mRNA levels or mRNA splicing. Thus, the majority of SR proteins and hnRNPs have the potential to regulate HPV16 L1 mRNA splicing. Strict control of the expression of the immunogenic L1 and L2 capsid proteins may contribute to the ability of HPV16 to cause persistence.

Human papillomaviruses (HPVs) depend on the cellular RNA-processing machineries including alternative RNA splicing and polyadenylation to coordinate HPV gene expression. HPV RNA processing is controlled by cis-regulatory RNA elements and trans-regulatory factors since the HPV splice sites are suboptimal. The definition of HPV exons and introns may differ between individual HPV mRNA species and is complicated by the fact that many HPV protein-coding sequences overlap. The formation of HPV ribonucleoproteins consisting of HPV pre-mRNAs and multiple cellular RNA-binding proteins may result in the different outcomes of HPV gene expression, which contributes to the HPV life cycle progression and HPV-associated cancer development. In this review, we summarize the regulation of HPV16 gene expression at the level of RNA processing with focus on the interactions between HPV16 pre-mRNAs and cellular RNA-binding factors.

Inhibition of the Akt kinase activates HPV16 late gene expression by reducing HPV16 early polyadenylation and by activating HPV16 late L1 mRNA splicing. We identified ‘hot spots’ for RNA binding proteins at the early polyA signal and at splice sites on HPV16 late mRNAs. We observed that hnRNP L was associated with sequences at all HPV16 late splice sites and at the early polyA signal. Akt kinase inhibition resulted in hnRNP L dephosphorylation and reduced association of hnRNP L with HPV16 mRNAs. This was accompanied by an increased binding of U2AF65 and Sam68 to HPV16 mRNAs. Furthermore, siRNA knock-down of hnRNP L or Akt induced HPV16 gene expression. Treatment of HPV16 immortalized keratinocytes with Akt kinase inhibitor reduced hnRNP L binding to HPV16 mRNAs and induced HPV16 L1 mRNA production. Finally, deletion of the hnRNP L binding sites in HPV16 subgenomic expression plasmids resulted in activation of HPV16 late gene expression. In conclusion, the Akt kinase inhibits HPV16 late gene expression at the level of RNA processing by controlling the RNA-binding protein hnRNP L. We speculate that Akt kinase activity upholds an intracellular milieu that favours HPV16 early gene expression and suppresses HPV16 late gene expression.

Adenosine plays an important role in cell death and differentiation as well as in tumorigenesis and the intra- and extra-cellular levels range from nanomolar to millimolar levels under various physiological or pathophysiological conditions. Here we report that adenosine can activate HPV16 late gene expression in a dose- and time-dependent manner, but only in the presence of guanosine. This activation occurred within hours after addition of the nucleosides and was primarily dependent on the ENT1 nucleoside transporter protein. Induction of HPV16 late gene expression was mainly the result of increased read-through at the early HPV16 polyadenylation signal into the late region of the HPV16 genome, thereby producing HPV16 late L2 mRNAs. The effect of guanosine and adenosine on HPV16 late gene expression was mediated by the increased binding to HPV16 mRNAs and nuclear export of the cellular HuR protein. Our results demonstrate that nucleosides can affect HPV16 gene expression.

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5′-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5′-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression. Background: HPV16 late gene expression is suppressed in mitotic cells. Results: hnRNP C proteins bind to the HPV16 early, untranslated region and activate HPV16 late mRNA splicing. Conclusion: hnRNP C proteins control HPV16 late gene expression. Significance: hnRNP C proteins may contribute to the ability of HPV16 to avoid the immune system and establish persistent infections that progress to cancer.

The human papillomavirus type 16 (HPV16) life cycle can be divided into an early stage in which the HPV16 genomic DNA is replicated, and a late stage in which the HPV16 structural proteins are synthesized and virions are produced. A strong coupling between the viral life cycle and the differentiation state of the infected cell is highly characteristic of all HPVs. The switch from the HPV16 early gene expression program to the late requires a promoter switch, a polyadenylation signal switch and a shift in alternative splicing. A number of cis-acting RNA elements on the HPV16 mRNAs and cellular and viral factors interacting with these elements are involved in the control of HPV16 gene expression. This review summarizes our knowledge of HPV16 cis-acting RNA elements and cellular and viral trans-acting factors that regulate HPV16 gene expression at the level of splicing and polyadenylation.