The Fidelity of HPV16 E1/E2-mediated DNA Replication

Abstract

Human papillomaviruses (HPV) are causative agents in a variety of human diseases; for example over 99% of cervical carcinomas contain HPV DNA sequences. Often in cervical carcinoma the HPV genome is integrated into the host genome resulting in unregulated expression of the viral transforming proteins E6 and E7. Therefore viral integration is a step toward HPV-induced carcinogenesis. Integration of the HPV genome could occur following double-strand DNA breaks that could arise during viral DNA replication. We investigated the fidelity of HPV 16 E1-and E2-mediated DNA replication of non-damaged and UVC-damaged templates in a variety of cell lines with different genetic backgrounds; C33a (derived from an HPV-negative cervical carcinoma), XP30RO (deficient in the by-pass polymerase (pol )), XP30 (expressing a restored wild-type pol ), XP12RO (nucleotide excision repair defective), and MRC5 (derived from a 14-week-old human fetus). The results demonstrate that the fidelity of E1-and E2-mediated DNA replication is reflective of the genetic background in which the assays are carried out. Human papilloma viruses (HPVs) 1 are small doublestranded DNA tumor viruses that are causative agents in a variety of human diseases. Over 99% of cervical carcinomas contain HPV sequences; these are of the high-risk type with HPV16 being the most common. The low risk HPVs are causative agents of genital warts, and HPV6/11 are the most commonly found in this disease (1). HPVs are maintained as episomal circular DNA molecules in infected cells and are replicated by the viral proteins E1 and E2 (2, 3). The E2 protein forms homodimers and recognizes and binds to 12bp palindromic DNA sequences in the transcriptional control region of HPV (4). E2 can then regulate transcription from the adjacent promoter that controls the expression of the HPV oncogenes E6 and E7; E2 can either activate or repress transcription depending upon E2 protein levels and the cell type (5, 6). As well as regulating transcription E2 is also essential for replication of the viral genome; there are three E2 DNA binding sequences surrounding the viral origin (ori) of replication and a physical interaction between E2 and E1 results in recruitment of E1 to the ori sequence (7,8,9). Following recruitment by E2 the E1 protein interacts with the ori DNA sequence and then forms a hexameric complex required for replication of the viral genome by virtue of its helicase activity (10). E1 is known to interact with subunits of DNA polymerase-␣ and with components of the Swi/Snf complex in order to activate DNA replication (11,12).In many HPV-induced cervical cancers the HPV genome is found integrated into the host genome. This integration usually deletes the coding sequence for the E2 protein and results in elevated expression of the viral oncoproteins E6 and E7 (13). It is proposed that increased E6 and E7 levels are involved in progression toward carcinoma. Therefore the loss of episomal status of the HPV genome is an important step on the way to carcinogenesis. The integration i...

Human Papillomavirus 16 Oncoprotein Expression Is Controlled by the Cellular Splicing Factor SRSF2 (SC35)

“…293T cells were transfected as described above with a control vector, pGL3-promoter (Promega), or with an HPV16 long control region (LCR) expression vector (39) and either siGLO or SRSF2 siRNA. Cells were harvested after 48 h, and luciferase reporter activity (Promega luciferase assay kit) was measured using a Thermolabs Luminoskan Ascent plate reader.…”

“…Therefore, we tested the hypothesis that the depletion of E6E7 RNAs upon SRSF2 knockdown in W12ti cells was a result of SRSF2-mediated downregulation of E6E7 transcription from the viral P 97 promoter. 293T cells were transfected with either control siRNA or siRNA against SRSF2 together with a luciferase reporter construct under the control of the HPV16 long control region (LCR) (genome nt 7101 [Ϫ803] to ϩ137; this region contains the P 97 promoter and a transcription initiation site) (39). The HPV16 LCR was less transcriptionally active than the control SV40 promoter in the pGL3 promoter plasmid.…”

Molecular Methods for a Correct Diagnosis of Multiple HPV Infections and Clinical Implications for Vaccine

“…This latter pathophysiological HPV aspect generally causes a high relative genetic instability characterized by specific genotypic or phenotypic rearrangements that, again, increases the risk of tumor progression. 11 Such assertion was then validated by Giannoudis et al 12 in 2001, who stated that another factor emerging in the cervical cancer development is the role played by the different HPV variants. Based on the sequence variations of the viral regions L1, L2, and LCR, the author has identified 5 phylogenetic variants of HR-HPV 16: European (E), Asian (As), Asian-American (AA), African 1 (Af1), and African 2 (Af 2).…”

“…However, the non-European variants were associated with a higher prevalence and incidence of high-grade lesions, also in their countries of origin. 11,12 The objective of converting the information present in the nucleotide sequences in a more complete genomic and phylogenetic analysis should reconstruct the evolutionary history of those from a common ancestor and could lead to the possiblility of identifying, with accuracy, the broadcast of a new HPV infection in high epidemic proportions. The starting point for the construction of a phylogenetic tree of an HPV is the multiple alignment of genomic sequences to analyze, and, subsequently, through mathematical algorithms, it can generate a detailed tree where nodes represent the different molecular species of viruses; the topology represents the relationships between multiple HPVs.…”

Human papilloma virus and breast cancer

“…The virus genome integration results in unregulated expression of the viral transforming proteins E6 and E7, which are responsible for the malignant phenotype, mainly through inactivation of tumor suppressor proteins p53 and RB1, respectively. Therefore viral integration is a step toward HPVinduced carcinogenesis (Boner and Morgan, 2002;Taylor et al, 2003). Braakhuis et al (2004) also report that head and neck squamous-cell carcinomas with active HPV type 16 DNA (HPV DNA expressing the viral E6 and E7 genes) had a distinct biological activity and have a favorable outcome.…”