Heterogeneous Nuclear Ribonucleoprotein A1 (hnRNP A1) and hnRNP A2 Inhibit Splicing to Human Papillomavirus 16 Splice Site SA409 through a UAG-Containing Sequence in the E7 Coding Region

Human papillomavirus 16 (HPV16) 5′-splice site SD226 and 3′-splice site SA409 are required for production of the HPV16 E7 mRNAs, whereas unspliced mRNAs produce E6 mRNAs. The E6 and E7 proteins are essential in the HPV16 replication cycle but are also the major HPV16 proteins required for induction and maintenance of malignancy caused by HPV16 infection. Thus, a balanced expression of unspliced and spliced mRNAs is required for production of sufficient quantities of E6 and E7 proteins under physiological and pathophysiological conditions. If splicing becomes too efficient, the levels of unspliced E6 mRNAs will decrease below a threshold level that is no longer able to produce E6 protein quantities high enough to significantly reduce p53 protein levels. Similarly, if splicing becomes too inefficient, the levels of spliced E7 mRNAs will decrease below a threshold level that is no longer able to produce E7 protein quantities high enough to significantly reduce pRb protein levels. To determine how splicing between SD226 and SA409 is regulated, we have investigated how SA409 is controlled by the cellular proteins hnRNP A1 and hnRNP A2, two proteins that have been shown previously to control HPV16 gene expression. We found that hnRNP A1 and A2 interacted directly and specifically with a C-less RNA element located between HPV16 nucleotide positions 594 and 604 downstream of SA409. Overexpression of hnRNP A1 inhibited SA409 and promoted production of unspliced E6 mRNAs at the expense of the E7 mRNAs, whereas overexpression of hnRNP A2 inhibited SA409 to redirect splicing to SA742, a downstream 3′-splice site that is used for generation of HPV16 E6̂E7, E1, and E4 mRNAs. Thus, high levels of either hnRNP A1 or hnRNP A2 inhibited production of the promitotic HPV16 E7 protein. We show that the hnRNP A1 and A2 proteins control the relative levels of the HPV16 unspliced and spliced HPV16 E6 and E7 mRNAs and function as inhibitors of HPV16 E7 expression. IMPORTANCE Human papillomavirus type 16 (HPV16) belongs to the high-risk-group of HPVs and is causing a variety of anogenital cancers and head and neck cancer. The two HPV16 oncoproteins E6 and E7 prevent apoptosis and promote mitosis and are essential for completion of the HPV16 life cycle and for transformation of the infected cell and maintenance of malignancy. E6 and E7 are produced from two mRNAs that are generated in a mutually exclusive manner by alternative splicing. While E6 protein is made from the unspliced mRNA, E7 is made from the spliced version of the same pre-mRNA. Since sufficient quantities of both E6 and E7 are required for malignant transformation, this intricate arrangement of gene expression renders E6 and E7 expression vulnerable to external interference. Since antiviral drugs to HPV16 are not available, a detailed knowledge of the regulation of HPV16 E6 and E7 mRNA splicing may uncover novel targets for therapy.