Bovine papillomavirus E2 repressor mutant displays a high-copy-number phenotype and enhanced transforming activity

Abstract

The methionine codon at bovine papillomavirus type 1 nucleotide 3091 was mutated to determine whether it may serve as an initiation codon for an E2 transcriptional repressor protein and to determine the role of the repressor in the biological activities of the virus. A series of transient expression experiments with CV1 cells documented that the mutation reduced expression of repressor activity from the viral genome and resulted in increased expression of the E5 transforming gene. Viral genomes containing the mutation displayed enhanced transforming activity in several assays in mouse C127 cells, including focus formation, colony formation in agarose, and tumorigenicity. In transformed cells, the mutant viral DNA was maintained as a plasmid with approximately 500 genomes per cell, whereas the wild-type copy number was approximately 75. These results indicate that the wild-type bovine papillomavirus type 1 genome encodes an E2 repressor protein that moderates the viral transforming activity and allows maintenance of the viral DNA at a relatively low copy number.

Phenotypic analysis of bovine papillomavirus type 1 E2 repressor mutants

“…repress E2 transactivation (data not shown), in agreement with the finding that this ATG codon is utilized for translation initiation of E2-TR (21). The capacity for this mutation to disrupt E2-TR expression is further described in the accompanying paper by Riese et al (35).…”

“…This change in relative abundance was significant, despite an increased level of E8/E2 expression by this mutant (Table 1). A similar study was performed on an identical ATG missense mutant at nt 3092 and arrived at a similar conclusion that disruption of E2-TR resulted in an increase in the potential of the virus to transactivate, replicate, and transform (35). It will be of interest to understand the consequences of disrupting E2-TR expression in dermal fibroblasts and in basal epithelial cells of a bovine fibropapilloma.…”

“…This corresponded both to the highly transformed phenotype of these cells (Fig. 2 and 3) and to elevated expression of an E5-CAT fusion gene (35). Cells harboring the double mutant p1474-1 had E5 levels which were up to threefold higher than those of the wild type.…”

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells

“…Therefore, it seems probable that maintenance of BPV as a multicopy episome over numerous generations requires a finely tuned regulatory system. It has been shown that mutation of one form of E2 affects the level of the other forms and that E2 repressor mutants display a high-copy-number phenotype (19,20,34). These findings, together with our results that all promoters respond to the E2 activator through the URR and that no other BPV gene products are involved, suggest that viral homeostasis could be maintained by a delicate balance between the E2 activator and repressor proteins.…”

“…Since at least the P2 and P4 promoters have been shown to be regulated by the E2 activator and repressor proteins in a URR-dependent manner (8,9,12,33,40,41), it is possible that an autoregulatory circuit based on the E2 proteins is crucial for control of BPV gene expression. Indeed, it appears that a precise regulation of gene expression is necessary for control of BPV copy number, since mutations in the E2C repressor result in a high-copy-number phenotype in stably transformed cell lines (20,34). In addition, repressor mutants replicate to higher levels than the wild type in transient replication assays (47).…”