Phenotypic analysis of bovine papillomavirus type 1 E2 repressor mutants

Abstract

The bovine papillomavirus type 1 (BPV-1) E2 open reading frame encodes three proteins: the E2 transcriptional transactivator, the E2 transcriptional repressor (E2-TR), and the E8/E2 fusion peptide. In this study, we describe the phenotypes of BPV-1 mutants which are disrupted in their capacity to encode either the E2 transcriptional repressor or the E8/E2 fusion peptide. We also describe experiments which demonstrate that the E8/E2 gene product functions similarly to E2-TR. In the context of the entire viral genome, disruption of E8/E2 expression had little effect on viral processes, whereas disruption of E2-TR expression resulted in a complex phenotype marked by a 10to 20-fold increase in viral DNA plasmid copy number as well as increased transformation potential. A double mutant, defective in the expression of both E8/E2 and E2-TR proteins, had high levels of E2 transactivation activity yet had reduced plasmid replication capacity and a delayed capacity to transform rodent cells.

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

“…This effect is not due to increased viral DNA copy number, because our expression assays were performed in CV1 cells in which BPV-1 DNA does not replicate (29). The biological activity of the repressor mutant in C127 cells implies that viral gene expression is increased in these cells also, a fact documented by the RNA mapping and immunoprecipitation experiments of Lambert et al (18). In C127 a.…”

“…Lambert et al (18,19) and Choe et al (6) have described subgenomic BPV-1 plasmids that express repressor activity under the control of heterologous promoters. On the basis of these studies, they have suggested that E2 repressor proteins can be synthesized by translation initiation at an internal methionine codon at nucleotide (nt) 3091 or by translation of a spliced ORF E8/E2 mRNA.…”

The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA

“…To generate the pHAGE lentiviral expression vectors used in the proteomics studies, BPV1 E2 and E2TR (P. M. Howley laboratory plasmid numbers p7211 and p7212, respectively) were PCR amplified from a BPV1 genomic plasmid and E8/E2C was PCR amplified from the pE8E2 plasmid and cloned into the lentiviral vector using Gateway cloning (p7214) (Invitrogen) (62,63). Human papillomavirus E2 ORFs with XhoI and NotI restriction sites at their 5= and 3= ends, respectively, were amplified from cloned HPV genomes (22,64,65) and subcloned into the pOZ-N-HA-PURO vector (where PURO stands for puromycin resistance) using the XhoI and NotI sites, and the sequence was verified.…”

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

“…It is unclear what the role of the P3 promoter might be, however, since P3 is the weakest promoter under any circumstance and appears to express both the E2 activator (49) and the E8/E2 repressor (4). Perhaps this promoter supplies some sort of back-up system, as suggested by the finding that an E8/E2 mutant is phenotypically normal, whereas mutations that inactivate E8/E2 and E2C have a more severe effect than a mutation that affects E2C only (20). The E2 titration, while informative, clearly represents only part of the BPV gene expression program.…”

“…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).…”