A splicing enhancer in the E4 coding region of human papillomavirus type 16 is required for early mRNA splicing and polyadenylation as well as inhibition of premature late gene expression

Successful inhibition of human papillomavirus type 16 (HPV-16) late gene expression early in the life cycle is essential for persistence of infection, the highest risk factor for cervical cancer. Our study aimed to locate regulatory RNA elements in the early region of HPV-16 that influence late gene expression. For this purpose, subgenomic HPV-16 expression plasmids under control of the strong human cytomegalovirus immediate early promoter were used. An exonic splicing enhancer that firmly supported the use of the E4 3' splice site at position 3358 in the early region of the HPV-16 genome was identified. The enhancer was mapped to a 65-nucleotide AC-rich sequence located approximately 100 nucleotides downstream of the position 3358 3' splice site. Deletion of the enhancer caused loss of both splicing at the upstream position 3358 3' splice site and polyadenylation at the early polyadenylation signal, pAE. Direct splicing occurred at the competing L1 3' splice site at position 5639 in the late region. Optimization of the position 3358 3' splice site restored splicing to that site and polyadenylation at pAE. Additionally, a sequence of 40 nucleotides with a negative effect on late mRNA production was located immediately downstream of the enhancer. As the E4 3' splice site is employed by both early and late mRNAs, the enhancer constitutes a key regulator of temporal HPV-16 gene expression, which is required for early mRNA production as well as for the inhibition of premature late gene expression.