SPLICE SITES OF HUMAN PAPILLOMAVIRUS TYPE-16 E6 GENE OR HETEROLOGOUS GENE REQUIRED FOR TRANSFORMATION BY E7 AND ACCUMULATION OF E7 RNA

Transformation of primary baby rat kidney cells by the human papilloma virus type 16 (HPV 16) E7 gene and efficient accumulation of E7 RNA ha ve been shown by this laboratory to depend on the integrity of the nuc leotide position (nt) 880 splice donor site. Here, the splice sites wi thin the HPV 16 E6 open reading frame (ORF) and the sites of the SV40 splicing unit were examined for an ability to provide this requirement . Constructs containing the HPV 16 E6 sites and the SV40 splice site s equences were used for transformation and RNase protection assays. E6 splice sites supported a low level of transformation, in assays for co mplete HPV 16 early region constructs containing loss-of-function muta tions of the nt 880 site. Using constructs with wild-type E6 or SV40 s plice sites showed that both splice sites could substitute similarly f or the requirement in cis of the nt 880 site for transformation. HPV 1 6 E6 mutated splice site and SV40 splice site in reverse, nonfunctiona l orientation relative to the promoter, were not transformation compet ent. The HPV 16 E7 RNA levels for the E6 splice site constructs correl ated closely with the transformation frequency. The SV40 splice sites were required for E7 transcript accumulation. The results showed E6 sp lice site function and evidence for enhanced exon skipping from E6 spl ice donor site to acceptor sites 3' of the E7 ORF. This was shown with constructs containing loss-of-function mutations of the nt 880 site. These results confirmed the function of the splice sites by the transf ormation competent constructs and suggested lower transformation frequ ency than for wild type was due to skipping of the E7 exon. These patt erns of transcripts may have a role in the regulation of gene expressi on during progression to malignancy. The combined results revealed tha t the general presence of a functional splice donor site was absolutel y required for transformation by HPV 16 E7 and accumulation of E7 RNA. (C) 1995 Wiley-Liss, Inc.