Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression

HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16⁺ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of ⁹¹QYNK⁹⁴ to ⁹¹PSFW⁹⁴ displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.     

DNA Replication of Human Papillomavirus Type 31 Is Modulated by Elements of the Upstream Regulatory Region That Lie 5′ of the Minimal Origin

The viral replication factors E1 and E2 of papillomaviruses are necessary and sufficient to replicate plasmids containing the minimal origin of DNA replication in transient assays. Under physiological conditions, the upstream regulatory region (URR) governs expression of the early viral genes. To determine the effect of URR elements on E1 and E2 expression specifically, and on the regulation of DNA replication during the various phases of the viral life cycle, we carried out a systematic replication study with entire genomes of human papillomavirus type 31 (HPV31), a high-risk oncogenic type. We constructed a series of URR deletions, spacer replacements, and point mutations to analyze the role of the keratinocyte enhancer (KE) element, the auxiliary enhancer (AE) domain, and the L1-proximal end of the URR (5′-URR domain) in DNA replication during establishment, maintenance, and vegetative viral DNA amplification. Using transient and stable replication assays, we demonstrate that the KE and AE are necessary for efficient E1 and E2 gene expression and that the KE can also directly modulate viral replication. KE-mediated activation of replication is dependent on the position and orientation of the element. Mutation of either one of the four Ap1 sites, the single Sp1 site, or the binding site for the uncharacterized footprint factor 1 reduced replication efficiency through decreased expression of E1 and E2. Furthermore, the 5′-URR domain and the Oct1 DNA binding site are dispensable for viral replication, since such HPV31 mutants are able to replicate efficiently in a transient assay, maintain a stable copy number over several cell generations, and amplify viral DNA under vegetative conditions. Interestingly, deletion of the 5′-URR domain leads to increased transient and stable replication levels. These findings suggest that elements in the HPV31 URR outside the minimal origin modulate viral replication through both direct and indirect mechanisms.     

Engagement of the ATR-Dependent DNA Damage Response at the Human Papillomavirus 18 Replication Centers during the Initial Amplification

We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high- and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G₂ phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of γH2AX, ATR-interacting protein (ATRIP), and topoisomerase IIβ-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification.     

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

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.     

Genetic analysis of high-risk e6 in episomal maintenance of human papillomavirus genomes in primary human keratinocytes

Papillomaviruses possess small DNA genomes that encode five early (E) proteins. Transient DNA replication requires activities of the E1 and E2 proteins and a DNA segment containing their binding sites. The E6 and E7 proteins of cancer-associated human papillomavirus (HPV) transform cells in culture. Recent reports have shown that E6 and E7 are necessary for episomal maintenance of HPV in primary keratinocytes. The functions of E6 necessary for viral replication have not been determined, and to address this question we used a recently developed transfection system based on HPV31. To utilize a series of HPV16 E6 mutations, HPV31 E6 was replaced by its HPV16 counterpart. This chimeric genome was competent for both transient and stable replication in keratinocytes. Four HPV16 E6 mutations that do not stimulate p53 degradation were unable to support stable viral replication, suggesting this activity may be necessary for episomal maintenance. E7 has also been shown to be essential for episomal maintenance of the HPV31 genome. A point mutation in the Rb binding motif of HPV E7 has been reported to render HPV31 unable to stably replicate. Interestingly, HPV31 genomes harboring two of the three p53 degradation-defective E6 mutations combined with this E7 mutation were maintained as replicating episomes. These findings imply that the balance between E6 and E7 functions in infected cells is critical for episomal maintenance of high-risk HPV genomes. This model will be useful to dissect the activities of E6 and E7 necessary for viral DNA replication.     

Papillomavirus Genomes Associate with BRD4 to Replicate at Fragile Sites in the Host Genome

It has long been recognized that oncogenic viruses often integrate close to common fragile sites. The papillomavirus E2 protein, in complex with BRD4, tethers the viral genome to host chromatin to ensure persistent replication. Here, we map these targets to a number of large regions of the human genome and name them Persistent E2 and BRD4-Broad Localized Enrichments of Chromatin or PEB-BLOCs. PEB-BLOCs frequently contain deletions, have increased rates of asynchronous DNA replication, and are associated with many known common fragile sites. Cell specific fragile sites were mapped in human C-33 cervical cells by FANCD2 ChIP-chip, confirming the association with PEB-BLOCs. HPV-infected cells amplify viral DNA in nuclear replication foci and we show that these form adjacent to PEB-BLOCs. We propose that HPV replication, which hijacks host DNA damage responses, occurs adjacent to highly susceptible fragile sites, greatly increasing the chances of integration here, as is found in HPV-associated cancers.     

Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication

We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.     

The fidelity of HPV16 E1/E2-mediated DNA replication

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 eta (poleta)), XP30eta (expressing a restored wild-type poleta), 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. For example, restoring poleta to the XP30 cell line results in a 3-fold drop in the number of mutants obtained following replication of a UVC-damaged template. A relatively high percentage of the mutant-replicated molecules arise as a result of genetic rearrangement. This is the first time such studies have been carried out with an HPV replication system, and the results are discussed in the context of the HPV life cycle and what is known about HPV genomes in human cancers.