Transcription Of Human Papillomavirus Type Research Articles

Experimental Support for Human Papillomavirus Genome Amplification Early after Infectious Delivery.

Even though replication and transcription of human papillomavirus type 16 (HPV16) has been intensively studied, little is known about immediate-early events of the viral life cycle due to the lack of an efficient infection model allowing genetic dissection of viral factors. We employed the recently developed infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846) to investigate genome amplification and transcription immediately after infectious delivery of viral genome to nuclei of primary keratinocytes. Using 5-ethynyl-2'-deoxyuridine (EdU) pulse-labeling and highly sensitive fluorescence in situ hybridization, we observed that the HPV16 genome is replicated and amplified in an E1- and E2-dependent manner. Knockout of E1 resulted in failure of the viral genome to replicate and amplify. In contrast, knockout of the E8^E2 repressor led to increased viral genome copy number, confirming previous reports. Genome copy control by E8^E2 was confirmed for differentiation-induced genome amplification. Lack of functional E1 had no effect on transcription from the early promoter, suggesting that viral genome replication is not required for p97 promoter activity. However, infection with an HPV16 mutant virus defective for E2 transcriptional function revealed a requirement of E2 for efficient transcription from the early promoter. In the absence of the E8^E2 protein, early transcript levels are unaltered and even decreased when normalized to genome copy number. Surprisingly, a lack of functional E8^E2 repressor did not affect E8^E2 transcript levels when normalized to genome copy number. These data suggest that the main function of E8^E2 in the viral life cycle is to control genome copy number. IMPORTANCE It is being assumed that human papillomavirus (HPV) utilizes three different modes of replication during its life cycle: initial amplification during the establishment phase, genome maintenance, and differentiation-induced amplification. However, HPV16 initial amplification was never formally proven due to a lack of an infection model. Using our recently established infection model (Bienkowska-Haba M, Luszczek W, Myers JE, Keiffer TR, et al. 2018. PLoS Pathog 14:e1006846), we demonstrate herein that viral genome is indeed amplified in an E1- and E2-dependent manner. Furthermore, we find that the main function of the viral repressor E8^E2 is to control viral genome copy number. We did not find evidence that it regulates its own promoter in a negative feedback loop. Our data also suggest that the E2 transactivator function is required for stimulation of early promoter activity, which has been debated in the literature. Overall, this report confirms the usefulness of the infection model for studying early events of the HPV life cycle using mutational approaches.

Characterization of novel transcripts of human papillomavirus type 16 using cap analysis gene expression technology.

We have performed cap-analysis gene expression (CAGE) sequencing to identify the regulatory networks that orchestrate genome-wide transcription in human papillomavirus type 16 (HPV16)-positive cervical cell lines of different grades: W12E, SiHa, and CaSki. Additionally, a cervical intraepithelial neoplasia grade 1 (CIN1) lesion was assessed for identifying the transcriptome expression profile. Here we have precisely identified a novel antisense noncoding viral transcript in HPV16. In conclusion, CAGE sequencing should pave the way for understanding a diversity of viral transcript expression.

Can a test for E6/E7 transcripts of human papillomavirus type 16 serve as a diagnostic tool for the detection of micrometastasis in cervical cancer?

Tissue from 11 cases of cervical cancer positive for human papillomavirus (HPV) type 16 DNA and 69 pelvic lymph nodes from the same patients were examined for HPV 16 DNA and mRNA from the E6/E7 genes. Five of the tumors were squamous, 3 adeno- and 3 adenosquamous carcinoma. From the primary tumors and the extirpated lymph nodes DNA and RNA or mRNA was subjected to PCR and RT-PCR. Three transcription profiles (only E6*I, E6*I and E6*II or full-length E6-E7 plus both of the spliced transcripts) were found in all of the 11 HPV 16 DNA-positive primary tumors. From the total of 69 lymph nodes analyzed 28 were positive for mRNA. HPV 16 DNA was found in 7 additional samples. Cytokeratin was found in 19 of these lymph nodes, indicating epithelial origin of tumor cells. Only 1 patient had 2 metastases evidenced by histology. These were both positive for HPV DNA and mRNA. The finding of HPV DNA, mRNA and cytokeratin in lymph nodes of patients with cervical cancer should be an indication of lymphogenically driven micrometastases of the tumor. The HPV mRNA assay should offer higher specificity than the DNA test since mRNA can be found in live cells only, while HPV DNA also can originate from dead cell material sequestered in the lymph nodes.

Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers.

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Quantitative detection of spliced E6-E7 transcripts of human papillomavirus type 16 in cervical premalignant lesions

The splicing patterns of E6-E7 transcripts of human papillomavirus type 16 (HPV16) in cervical premalignant lesions were quantitatively analyzed by S1 nuclease protection assay. The major E6-E7 transcripts in HPV16-containing cervical lesions (four cervical intraepithelial neoplasias and one invasive carcinoma) were from spliced E6 ∗I/E7 mRNA. The unspliced E6/E7 mRNA, which can encode the full-length zinc finger protein E6, is expressed as 8 to 15% of E6-E7 transcripts. The spliced E6 ∗II/E7 mRNAs were expressed as 14 to 24% of E6-E7 transcripts inmost tissues. However, in HPV16-containing cell lines, the expression levels of spliced and unspliced E6-E7 transcripts were variable.

Oestrogen Stimulates Differential Transcription of Human Papillomavirus Type 16 in SiHa Cervical Carcinoma Cells

Human papillomavirus (HPV) type 16 is highly associated with cervical cancer, but it seems that cofactors such as hormones affect its potential oncogenicity. We have analysed the HPV-16 gene expression in response to sex hormones and glucocorticoids in SiHa cells, a human cervical carcinoma cell line. An eightfold induction of HPV-16 transcripts was obtained in oestrogen-treated SiHa cells. Of the five HPV-16 transcripts detected in these cells only the two major ones, the 4.6 kb and the 4.1 kb mRNA species, were affected by oestrogen. Since the five transcripts span the E6 and E7 open reading frames of the HPV-16 genome, these results suggest that the expression of the various transcripts is differentially controlled, as oestrogen regulates only two of them. We have identified in the HPV-16 genome seven different regions with a high degree of similarity to the oestrogen-responsive element consensus sequence (GGTCANNNTGACC). These sequences are located throughout the entire HPV-16 genome. Progesterone or dexamethasone had no detectable stimulatory effect on the various transcripts of HPV-16 in SiHa cells, up to 24 h after treatment of the cells. Since the E6 and E7 open reading frames have been associated with the oncogenic potential of HPV-16, the effect of oestrogen on the transcription of these viral genes may be of biological relevance in the malignant transformation of HPV-16-infected cervical cells.

Selective suppression of human papillomavirus transcription in non-tumorigenic cells by 5-azacytidine.

The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non-tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5-azacytidine. In contrast, viral gene expression is not influenced by 5-azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run-on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down-regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non-tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5-azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5-azacytidine and appears to correlate with the presence of specific chromosomes in non-tumorigenic cells.

Integration and transcription of human papillomavirus type 6 recombinant DNA in mouse cells

Human papillomavirus (HPV) DNA is found in nature mostly in an episomal form. However, in permanent cell lines established from cervical carcinomas in which HPV sequences are present, they are usually integrated in the host genome. In vitro studies of HPV have been hampered because of the difficulty of stably maintaining HPV sequences in transfected cells. We have cloned the entire HPV6 genome into three vectors: pML2, a derivative of pBR322 lacking the “poison sequences”; p142-6, a plasmid containing the complete bovine papillomavirus type 1 (BPV-1) genome which usually remains extrachromosomal in transfected mouse cells; and p302-3, a plasmid containing the bacterial gene conferring resistance to neomycin. Each of the three constructs has been transfected onto C127 mouse cells. In the case of the recombinant molecules cloned into pML2 and into p42-6, the transfection had been done together with the p302-3 plasmid. G418 resistant colonies were selected and analyzed for their DNA content. In all cases ( 5 5 ) the transfected molecules were present and integrated in the cellular genome. No free episomal DNA was detected, even in the cells transfected with the BPV-1 containing plasmid. One junction with the flanking cellular sequences is located in the late open reading frames region of the HPV6 genome and there is a disruption of the early region, as has been described for the HPV18 genome integrated into the cervical carcinoma cell line HeLa. Transcription analysis revealed transcripts of heterogeneous sizes, spanning the distal early region (E2E4E5) of the HPV6 genome, as happens in vivo in Condylomata acuminata, although in these genital lesions the HPV6 genome is maintained as a non-integrated episome.

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein.

Human papillomavirus type 16 DNA and RNA were characterized in the cervical cancer-derived CaSki cell line, which contains only integrated DNA, and in a cervical cancer, which contains predominantly plasmid DNA. In both, a major RNA can code for the early open reading frame E7 and a minor one can code for E6. The cervical cancer, but not the CaSki cell line, contains a minor RNA that can code for an intact E2 protein, and this may relate to the continued presence of plasmid DNA. The RNA mapping data suggest that the poly(A)+ RNA is transcribed from a minor fraction of the several hundred gene copies present, and in the cervical cancer these genomes appear to be integrated. The E7 protein has been identified in CaSki cells and the prevalence of its mRNA suggests a possible function in progression to, or long-term maintenance of, the malignant state.