Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.     

The genomes of the animal papillomaviruses European elk papillomavirus, deer papillomavirus, and reindeer papillomavirus contain a novel transforming gene (E9) near the early polyadenylation site.

We report that the genomes of reindeer papillomavirus (RPV), European elk papillomavirus (EEPV), and deer papillomavirus (DPV) contain a short conserved translational open reading frame (ORF), E9, which is located between the E5 ORF and the early polyadenylation site. In RPV, DPV, and EEPV, E9 ORFs have the potential to encode extremely hydrophobic polypeptides of approximately 40 amino acids. In mouse C127 cells transformed by EEPV and RPV, there exists a unique, abundant mRNA species of approximately 700 nucleotides which has the capacity to encode an E9 polypeptide. This mRNA is transcribed from a previously unrecognized promoter at position 4030 in the EEPV genome. The EEPV E9 ORF exhibits weak transforming activity in C127 cells and primary rat embryo fibroblasts. We also show that EEPV E5 is the major oncogene in the EEPV genome when assayed in C127 cells, although it is less efficient in transformation than the E5 genes of bovine papillomavirus type 1, DPV, and RPV.     

Accumulation of RNA homologous to human papillomavirus type 16 open reading frames in genital precancers.

The accumulation of human papillomavirus type 16 (HPV-16)-specific RNAs in tissue sections from biopsies of patients with genital precancers was studied by in situ hybridization with single-stranded 35S-labeled RNA. These analyses revealed that the most abundant early-region RNAs were derived from the E4 and E5 open reading frames (ORFs). RNAs homologous to the E6/E7 ORFs were also detected, whereas RNAs homologous to the intervening E1 ORF were not. This suggests that the E4 and E5 mRNAs are derived by splicing to the upstream E6/E7 ORFs, consistent with studies of HPV-11 in condylomata (L. T. Chow et al., Cancer Cells (Cold Spring Harbor) 5:55-72, 1987). Abundant RNAs homologous to the 5' portion of L1 were also detected. These RNAs were localized to the apical strata of the epithelium. HPV-16 RNAs accumulated in discrete regions of these lesions, and when present were most abundant in the upper cell layers of the precancerous epithelium. RNAs homologous to early ORFs were also detected in some germinal cells within the basal layer of the epithelium.     

Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells.

A series of mutations in open reading frames (ORFs) E6 and E7 of bovine papillomavirus type 1 (BPV1) was constructed to analyze the roles of these ORFs in transformation of mouse C127 cells. The mutations were designed to prevent synthesis of specific proteins encoded by these genes. None of the mutations caused a decrease in the focus-forming activity of the full-length viral genome or in the ability of the viral DNA to replicate as a high-copy-number plasmid. Analysis of these mutants in the absence of a functional BPV1 E5 gene revealed a weak focus-forming activity encoded by ORF E6. Mutations preventing synthesis of the E6 protein did cause defects in anchorage-independent growth and tumorigenicity of transfected and transformed cells. However, a frameshift mutation between the first and second ATG codons of ORF E6 did not inhibit induction of colony formation, suggesting that translation from the first methionine codon is not required. Mutations that inactivated ORF E7 or E6/E7 individually did not inhibit induction of colony formation in agarose. However, a defect in this activity was caused by simultaneous disruption of both ORF E7 and ORF E6/E7 when they were expressed from the full-length viral genome but not when they were expressed under the control of a retrovirus long terminal repeat. These results suggest that translation of both ORF E6 and the 3' end of ORF E7 is required for efficient induction of anchorage-independent growth by the intact BPV1 genome.