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

The activation of transcription and of DNA replication are, in some cases, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN6GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, E1. Much of the information on these activities is based on transient-transfection assays as well as biochemical analyses; however, their importance in the productive life cycle of oncogenic HPVs remains unclear. To determine the contributions of these E2 functions to the HPV life cycle, a genetic analysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replication activities but were transactivation defective. Following transfection of normal human keratinocytes, these mutant genomes were established as stable episomes and expressed early viral transcripts at levels similar to those of wild-type HPV31. Upon differentiation in organotypic raft cultures, the induction of late gene expression and amplification of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was observed in the mutant lines. We conclude that the transactivation function of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Transactivation by the E2 Protein of Oncogenic Human Papillomavirus Type 31 Is Not Essential for Early and Late Viral Functions

Stubenrauch

Colbert

Laimins

1998

“…Second, the regulatory region of BPV1 has 12 E2BS with relative affinities varying 100-fold (30), whereas a large group of HPVs (including types 11,16,18, and 31) contain four high-affinity sites whose locations in the upstream regulatory region are highly conserved (2,20,44,50). The role of E2BS has been examined in the genomic context of BPV1 by deletion analysis, which has revealed a high degree of redundancy in terms of transcriptional regulation (53). Also, in studies that addressed the importance of E2BS during the vegetative life cycle of BPV1, mutations in E2BS surrounding the origin showed no effect on transient or stable replication or the copy number of episomes (49).…”

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confidence: 99%

Differential Requirements for Conserved E2 Binding Sites in the Life Cycle of Oncogenic Human Papillomavirus Type 31

Stubenrauch

Lim

Laimins

1998

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062–3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.

“…(In both cases, the reactions represented in lanes marked 142-6 contained one-half the amount of RNA used in the other reactions.) These promoters have been reported to be transactivated by E2 (23,41); however, reduction of E2 consistently did not result in loss of P3080 or P890 activity. This result suggests that under the conditions of this study, the activity seen from P890 and P3080 did not result from E2 transactivation.…”

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confidence: 90%

“…As many of the BPV-1 promoters can be transactivated by E2 (15,17,23,36,41), the effects that the P2.3 mutations have on viral transcription were examined. It should be noted that no substantial difference in total viral DNA copy number was observed between C127 cells transformed by wild-type and mutated genomes.…”

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confidence: 99%

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

Spalholz

1993

The full-length bovine papillomavirus E2 gene product (E2TA), which has a direct role in DNA replication and functions as a transcriptional activator, can be expressed from an unspliced mRNA transcribed from the P2443 promoter or from spliced mRNAs transcribed from other upstream promoters. The regulation of E2 expression from these promoters is still in question. In the background of wild-type protein coding sequences, this study identified the P2443 promoter as the major source of E2TA as well as E5 expression in C127 cells. The bovine papillomavirus type 1 (BPV-1) E2 proteins are important regulators of viral transcription (for a review, see reference 24). In BPV-1, a 48-kDa protein (E2TA) is produced from translation of the full-length E2 open reading frame (ORF). This protein acts as a transcriptional activator when bound to tandem repeats of its cognate binding site, ACCN6GGT (2). Two smaller E2 proteins, each containing the DNA binding domain of the protein, act as repressors of E2-transactivated transcription (10, 20, 21). E2-dependent enhancers located in the long control region of the viral genome can activate transcription from the viral promoters P89 and P2443 and possibly also from P8s9 and P3080 (15, 17, 23, 27, 36, 37, 41). Controlling elements in the regulation of E2 transactivator expression are still under investigation. Three species of mRNA from transformed cells which could encode the full-length E2 ORF have been described: an unspliced spe