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

Abstract

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.Human papillomavirus type 16 (HPV-16) is the major cause of cervical cancer (41, 60) and is one of the most common cancer-causing infections in the world. Cervical cancer accounts for 6% of all malignancies in women in developed countries but 26% in developing countries (37). While the majority of HPV-16 infections are cleared in less than a year, in rare cases, despite immunosurveillance, persistence is established. Persistence of HPV-16 infection is itself the highest risk factor for the development of cervical cancer (28) and requires successful inhibition of late gene expression early in the life cycle to avoid the immune response. As cervical cancer cells never express the late viral mRNAs or proteins, one can speculate that inhibition of late gene expression is a prerequisite for cancer progression.Expression of HPV-16 genes is complex and is tightly regulated by differentiation-dependent transcription and RNA processing events (2, 40). Production of the late mRNAs encoding the antigenic capsid proteins L1 and L2 is restricted to terminally differentiated keratinocytes (41). The inhibition of the late genes may be partly attributed to RNA elements in the late region, suggested to regulate various RNA processing events such as mRNA stability, splicing, and translation (2, 40). Regulatory RNA elements have been identified in HPV-16 coding regions (42,45) and also in the late untranslated region of HPV-1 (46), HPV-16 (31), HPV-31 (13), and bovine papillomavirus type 1 (BPV-1) (23), while differentiation-dependent splicing of t...

Human Papillomavirus E2 Regulates SRSF3 (SRp20) To Promote Capsid Protein Expression in Infected Differentiated Keratinocytes

“…Further, wild-type HPV genomes are present in NIKS16 and W12E and CIN612-9E cells. In contrast, in the previous studies, subgenomic constructs with deletions in important cis-acting elements (e.g., the late negative regulatory region [51]) controlling gene expression were used (20,21,50). In our HPV-infected lines, viral transcription is driven from the natural viral promoters (10,38) which are relatively weakly active.…”

Role of Calpain in the Formation of Human Papillomavirus Type 16 E1^E4 Amyloid Fibers and Reorganization of the Keratin Network

“…Either the DNA sequence present at these sites or the corresponding sequence of the mRNA message may be sensitive to mutation; perhaps changing the secondary structure and hence the access of binding proteins. In fact sequences in this region have already been shown to affect HPV16 splicing (32), demonstrating the care that must be taken when making and interpreting mutant HPV genomes. We experienced additional problems while trying to characterize our noncleavable mutant 16E1 E4 proteins expressed from transient transfection cDNAs.…”

Serine- and Arginine-rich Proteins 55 and 75 (SRp55 and SRp75) Induce Production of HIV-1 vpr mRNA by Inhibiting the 5′-Splice Site of Exon 3

“…7C). If the HIV-1 GAR enhancer or the core HPV-16 enhancer (E) was added back into the internal exon of pT1sd to generate pT1-5/GAR and pT1sde (29), respectively (Fig. 7A), splicing shifted to L1 mRNA (Fig.…”

“…Plasmid pNL13a7di3 was constructed by first PCR-amplifying nucleotides 245-289 and 4936 -5055 using oligonucleotides BSS and di3sd3as (Table 1) followed by cloning into pNL13a7 (7) using BssHII and EcoRI. The plasmids pT1sd and pT1sde have been described previously (29). Plasmid pT1-5/GAR was generated by first PCRamplifying HIV-1 sequences between nucleotides 5523 and 5591 using oligonucleotides AnnealGARBss/XbaS and AnnealGARBss/XbaAS (Table 1).…”