Identification of the E9 ∧ E2C cDNA and functional characterization of the gene product reveal a new repressor of transcription and replication in cottontail rabbit papillomavirus

Cottontail rabbit papillomavirus (CRPV) genomes mutated in the trans-activation domain of the E2 protein, which stimulates both viral DNA replication and transcription, are severely impaired in their ability to induce tumors in New Zealand White rabbits. A number of papillomaviruses encode, in addition to full-length E2, a shortened E2 protein or an E2 protein fused to a short stretch of amino acids derived from the small E8 open reading frame that counteract the activities of E2. We identified and cloned the novel cDNA E9 boolean AND E2C of CRPV from papillomas of New Zealand White and cottontail rabbits and characterized the functions of the encoded gene product. E9 boolean AND E2C was shown to be a bona fide repressor of minimal viral promoters, with the E9 domain being essential for this activity, and to repress E1/E2-dependent replication of a CRPV origin construct. In addition, E9 boolean AND E2C counteracted the transactivation effect of the full-length E2 on minimal promoters containing several E2 binding sites. To investigate the role of E9 boolean AND E2C in tumorigenesis, we constructed two CRPV genomes mutated in E9 boolean AND E2C. One, designated CRPV-E9atgmut-pLAII, contained a mutation in the unique start codon in the E9 open reading frame, and the second E9 boolean AND E2C mutant was constructed by the introduction of a stop codon close to the splice donor site at nucleotide 3714 that additionally prevented the correct splicing of the transcript. When we infected New Zealand White rabbits with these constructs, we surprisingly noted no differences in tumor induction efficiency, viral genome copy number, and viral transcription in comparison to wild-type CRPV.