Transforming properties of the cottontail rabbit papillomavirus oncoproteins Le6 and SE6 and of the E8 protein

Abstract

Cottontail rabbit papillomavirus induces on cottontail and domestic rabbits papillomas which progress at a high frequency to carcinoma. The virus encodes three transforming proteins; one is translated from open reading frame (ORF) E7 and binds the retinoblastoma protein, and two, LE6 and SE6, are translated from the first and second ATGs of ORF E6, respectively. Here we show that neither of the E6 proteins coprecipitated with p53 in vitro, nor did they bind to a recently identified E6-binding protein (J. J. Chen, C. E. Reid, V. Band, and E. Androphy, Science 269:529-531, 1995). This protein was shown to bind to the E6 proteins of the high-risk human papillomavirus types 16 and 18 but not to the low-risk human papillomavirus types 6 and 11. In-frame deletions cloned into the pZipNeo vector were used to identify structural features of SE6 and LE6 important for transformation of NIH 3T3 cells. Three deletions covering the amino-terminal half of SE6 did not transform cells. In two of the three deletions, two Cys-X-X-Cys motifs were deleted, each deletion preventing the formation of one of the potential small Zn fingers of SE6. Among the LE6 deletions, only one had a reduced transformation efficiency, while seven transformed cells at least as efficiently as wild-type LE6. In each of three of these seven mutants, two Cys-X-X-Cys motifs were deleted. None of the three amino acid deletions which abolished transformation by SE6 reduced transformation by LE6. Furthermore, transformation did not correlate with the level of SE6 or LE6 proteins detectable. ORF E8 colinear with ORF E6, which could generate a 50-amino-acid protein with a hydrophobic segment, did not transform cells when cloned into the pZipNeo vector. However, mutation of the E8 ATG, which did not alter the amino acid sequence of LE6, increased transformation by LE6 without affecting the level of LE6 expression. The data suggest that transformation by the E6 proteins is not mediated by interfering with p53 function or through binding to the E6-binding protein. Furthermore, different structural features are important to maintain transformation functions and protein stability of LE6 and SE6. Finally, E8 seems not to be a transforming protein but rather appears to modulate transformation by LE6.

GM-CSF enhances protective immunity to cottontail rabbit papillomavirus E8 genetic vaccination in rabbits

“…The CRPV E8 gene is co-linear with the E6 gene and encodes a 50 amino acid hydrophobic protein with features similar to the E5 genes of both bovine papillomavirus type 1 (BPV-1) and most HPVs [43,44,53]. However, in the context of the CRPV genome, E8 has impact on papilloma outgrowth and host immune responses when used to vaccinate rabbits.…”

The rabbit papillomavirus model: a valuable tool to study viral–host interactions

“…Strongest homologies were found with HPV1 [15,16]; (vii) viral transcripts have been mapped and the cap sites have been determined [17]. The virus encodes 10 genes, two of which (LE6, the corresponding protein is translated from the first open reading frame starting at 154 bp, with 272aa in length and E7) code for two essential transforming proteins [18,19], two of which are capsid proteins (L1 and L2) [14,20], two of which are involved in the control of replication and gene regulation (E1 and E2) [21 -26] and two others, E9^E2C (renamed E8^E2) which has no effect on growth of the lesions but acts as a transcriptional repressor in mammalian cells [26] 38,39]. Viral infections are initiated in the basal cells and thus must gain access to these cells via abrasions in the skin.…”

“…The SfPV1 (CRPV) genome. Genes found to be essential to produce papillomas include LE6, E7, E1 and E2 [19]. Two late genes L1 and L2 are required for the viral life cycle [14].…”

“…A study in domestic rabbits using tumour suspensions to immunize animals demonstrated that tumour cells contained Genes found to be essential to produce papillomas include LE6, E7, E1 and E2 [19]. Two late genes L1 and L2 are required for the viral life cycle [14].…”

“…The virus encodes 10 genes, two of which (LE6, the corresponding protein is translated from the first open reading frame starting at 154 bp, with 272aa in length and E7) code for two essential transforming proteins [18,19], two of which are capsid proteins (L1 and L2) [14,20], two of which are involved in the control of replication and gene regulation (E1 and E2) [21 -26] and two others, E9^E2C (renamed E8^E2) which has no effect on growth of the lesions but acts as a transcriptional repressor in mammalian cells [26] (https://pave.niaid.nih.gov/) and E4 which plays a role in DNA amplification and L1 expression [27] (figure 2); (viii) unlike LE6 and E7 that are essential for SfPV1 infection, two additional oncogenes SE6, the corresponding protein is translated from the first open reading frame starting at 445 bp, with 175aa in length and E8 (renamed E10) (https://pave.niaid.nih.gov/) both have an effect on tumour growth but not on viral production [31 -33]; (ix) early genes E1 and E2 are also absolutely required for infection [24,28,34] (figure 2); (x) E6 and E7 are expressed in malignant lesions and have been posited to be targets for therapeutic interventions [17,31]; (xi) both episomal and integrated DNAs were found in SfPV1induced carcinomas, and DNA was highly methylated in malignancies in domestic rabbits [32]; (xii) both progressive and regressive variants of the virus exist [33,35,36]; and (xiii) cells infected with SfPV1 were found to co-localize with hair follicle stem cells [37].…”