Importance of the bovine papillomavirus P2443 promoter in the regulation of E2 and E5 expression

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded transacting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes

Pol

Howley

1995

“…To facilitate these experiments, we made use of constructs which have the CAT gene fused in frame with either E2 or E5 (46). Both of these proteins are translated from mRNAs made predominantly from the P4 promoter (3,57). Figure 9 shows that each of these E2 mutant proteins provided by the CMV expression vector activated the P4 promoter.…”

Section: Fig 2 Identification Of Phosphorylated Tryptic Fragments Amentioning

confidence: 99%

“…E2 acts as a transcriptional activator by interacting with E2 binding sites, thereby enhancing transcription of all early viral transcripts (60). For example, E2 expression is autocatalytic, as the protein activates expression from several promoters that can produce an effective E2 mRNA (3,57,63). The transcription of viral promoter P4 (also known by the coordinate of a major start site P 2443 ) is stimulated by E2, producing a predominant unspliced mRNA encoding E2 and a spliced transcript which is the major message for E5 production (3,57).…”

mentioning

confidence: 99%

A papillomavirus E2 phosphorylation mutant exhibits normal transient replication and transcription but is defective in transformation and plasmid retention

Lehman

King

Botchan

1997

Papillomavirus DNA persists in infected cells as a nuclear plasmid, causing epithelial lesions in many hosts, including humans. The viral protein E2 is required for both replication and transcription to facilitate this persistence. Bovine papillomavirus E2 protein is phosphorylated at two predominant sites. Phosphorylation of one of these sites (serine 301) inhibits replication of the genome. Using mass spectrometry and Edman sequencing, we have mapped additional phosphorylation sites in tryptic peptides to positions which lie primarily in the putatively unstructured hinge region of E2. Mutation of the major sites facilitates transformation in the absence of viral repressors and only has a minor effect on transformation when the repressors are present. Mutation of the major phosphorylation sites combined with one additional change at a newly discovered site (serine 235) blocks transformation. Transformation can be restored by mutating this residue to aspartic acid, mimicking a phosphorylated amino acid, suggesting that phosphorylation is key to the regulation. Transformation by the mutant genome can also be rescued by ectopic expression of the E2 enhancer protein, demonstrating a loss of function by the mutant protein and not a toxic defect. In transient assays, phosphorylation site mutants of E2 protein were normal for all viral functions tested, including replication, transcriptional activation and repression (by the overlapping mutant repressors), protein accumulation, and surprisingly, viral oncogene E5 promoter activation. While the mutant genome transiently replicated to high levels, stable replication was defective, suggesting that a function of E2 required for plasmid retention is regulated by phosphorylation.

Human Papillomavirus Type 31 Replication Modes during the Early Phases of the Viral Life Cycle Depend on Transcriptional and Posttranscriptional Regulation of E1 and E2 Expression

Hubert

Laimins

2002

The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient assays with cotransfected E1 or E2 expression vectors. Both reporters were moderately activated by E1 in a dose-dependent manner. In addition, E1Luc was activated by low doses of E2 but was repressed at high doses. In contrast, E2 had little effect on E2Luc activity. These data indicate that E1 expression and that of E2 are interdependent and regulated differentially. When the E6 splicing mutations were analyzed in both reporter backgrounds, only E1Luc activities correlated with splicing competence in the E6 ORF. These findings support the hypothesis that the E6 intron primarily regulates expression of E1. Finally, in long-term replication assays, none of the E6 mutant genomes could be stably maintained. However, cotransfection of the E6 splicing mutant genomes with pHPV31-E7NS, which contains a nonsense mutation in the E7 coding sequence, restored stable replication of some mutants. Our observations indicate that E1 expression and that of E2 are differentially regulated at multiple levels and that efficient expression of E1 is required for transient and st...