HPV Genome Research Articles

Long-distance communication: Looping of human papillomavirus genomes regulates expression of viral oncogenes.

High-risk human papillomaviruses (HPVs) are a major cause of cancers. HPVs infect epithelial cells, and viral oncogenes disrupt several cellular processes, including cell division, differentiation, and apoptosis. Expression of these oncogenes is relatively low in undifferentiated epithelial cells but increases in differentiating cells by unknown mechanisms. In a new study, Parish and colleagues unveil how two cellular proteins, CCCTC-binding factor (CTCF) and Yin Yang 1 (YY1), mediate looping of the HPV18 genome, which regulates expression of viral oncogenes in both dividing and differentiating epithelial cells.

SETD2-dependent H3K36me3 plays a critical role in epigenetic regulation of the HPV31 life cycle

The life cycle of HPV is tied to the differentiation status of its host cell, with productive replication, late gene expression and virion production restricted to the uppermost layers of the stratified epithelium. HPV DNA is histone-associated, exhibiting a chromatin structure similar to that of the host chromosome. Although HPV chromatin is subject to histone post-translational modifications, how the viral life cycle is epigenetically regulated is not well understood. SETD2 is a histone methyltransferase that places the trimethyl mark on H3K36 (H3K36me3), a mark of active transcription. Here, we define a role for SETD2 and H3K36me3 in the viral life cycle. We have found that HPV positive cells exhibit increased levels of SETD2, with SETD2 depletion leading to defects in productive viral replication and splicing of late viral RNAs. Reducing H3K36me3 by overexpression of KDM4A, an H3K36me3 demethylase, or an H3.3K36M transgene also blocks productive viral replication, indicating a significant role for this histone modification in facilitating viral processes. H3K36me3 is enriched on the 3’ end of the early region of the high-risk HPV31 genome in a SETD2-dependent manner, suggesting that SETD2 may regulate the viral life cycle through the recruitment of H3K36me3 readers to viral DNA. Intriguingly, we have found that activation of the ATM DNA damage kinase, which is required for productive viral replication, is necessary for the maintenance of H3K36me3 on viral chromatin and for processing of late viral RNAs. Additionally, we have found that the HPV31 E7 protein maintains the increased SETD2 levels in infected cells through an extension of protein half-life. Collectively, our findings highlight the importance of epigenetic modifications in driving the viral life cycle and identify a novel role for E7 as well as the DNA damage response in the regulation of viral processes through epigenetic modifications.

Human papillomavirus type 16 genomic variation in women with subsequent in situ or invasive cervical cancer: prospective population-based study

BackgroundHPV genomic variation may be involved in viral carcinogenesis.MethodsIn a national register-based nested case–control study, we retrieved archival smears from baseline cytologically normal women who later developed cancer in situ (CIS), squamous cervical cancer (SCC) or remained free of disease. These smears were previously HPV tested by PCR and HPV16 was the strongest risk factor. We now used the Illumina NextSeq platform to sequence HPV16 genomes in cervical smears from 242 women who later developed CIS/CIN3 (n = 134), SCC (n = 92) or remained healthy (n = 16).ResultsThe median sequence depth per sample was high (11,288×). For 218/242 samples (>90%), we covered ≥80% of the complete HPV16 genome with sequencing median depths of >200×. We identified a wide range of unique isolates and 147 novel SNPs across the 218 samples. Most women (97%) had HPV16 lineage A infection, with the sublineages being A1 (66.1%), A2 (28.9%) and A4 (1.8%), respectively. The least variable gene was the E7 (3.4% variability), where 170/204 case women (83%) displayed a fully conserved sequence. There were no obvious differences by disease outcome (CIS or SCC).ConclusionsWe found a high number of novel SNPs. The E7 gene was hypovariable both among women later developing CIN3/CIS, and SCC, respectively.

Abstract 3326: Upregulation of epigenetic changes acquired by HPV16 E6/E7 oncoproteins in mouse keratinocytes: Targeting ATM/PI3K

Abstract Human Papillomavirus (HPV), a non-enveloped epitheliotropic double stranded DNA virus, is an etiological agent of oral cancer. Oral cancer is related to the persistent infection by high-risk HPV type 16, E6/E7 oncoproteins. The E6/E7 oncoproteins significantly contribute to the carcinogenic genetic instability effect of high-risk HPV through the degradation of two gatekeeper proteins, p53 and pRB, respectively. The ATM gene, also known as a gatekeeper protein plays a central role in the complex processes that repair DNA strand breaks. The ATM gene is located at 11q22.3 and belongs to a protein family known as the PI3K. Many studies have tried to clarify the epigenetic instability role of ATM in cancer function and susceptibility. However, there are no studies addressing the relationship between HPV infection-oral cancer, ATM/PI3K expression, and genomic instability. The aim of this study is to analyze the influence of HPV16 E6/E7 oncoproteins in oral cancer and the epigenetic profile. In our experimental procedures, we proposed a novel approach to study HPV-induced epigenetic changes with reconstructed human oral epithelium in SCID mouse. We used HPV-16, which is the most common HPV type found in oral cancer. Two plasmids were used to produce HPV-16 E6/E7. The first plasmid p16sheLL expresses the two viral capsid proteins, L1 and L2. The second plasmid pBR322HPV16 contains the full length HPV-16 genome. This approach closely mimics the architecture of normal human oral epithelium. In our study, we first focused on analyzing selected cellular gene loci where DNA methylation profiles are known to be altered, either by HPV infection or in oral cancer. We examined the following gene promoters DcR1/DcR2 (apoptosis regulation), p16 (cell cycle control), DAPK (cancer metastasis), and MGMT (DNA repair). We also analyzed progressive morphological and cytopathic changes in the HPV-infected oral epithelia. These data indicate that HPV E6/E7 oncoproteins can promote increased upregulation of apoptosis, disrupt cell cycle regulation, and decrease DNA double strand break repair. Epigenetics and chromosomal instability events were noted, specifically, the deletion of the distal region of chromosome 11q22-23 in the E6/E7 transfected keratinocytes. These results indicate that HPV-16, E6/E7 oncoproteins induced epigenetic changes in mouse keratinocytes, which may further promote oral carcinogenesis. Citation Format: Eva McGhee, Mengtao Li, Yi-Ling Lin, Liliana Zarate, Naomi Long, Mai Do, Chinelo Ezechukwu, Nichelle Cox, Hyun Chung, Jenna Cormier, Meidrah Tyler, Victoria Vidal, Billy Ballard, Roland Pattillo, Jay Vadgama. Upregulation of epigenetic changes acquired by HPV16 E6/E7 oncoproteins in mouse keratinocytes: Targeting ATM/PI3K [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3326.

Stochastic Modeling of the Co-Regulation between Early and E8 Promoters in Human Papillomavirus.

High risk HPV can induce cervical and oropharyngeal cancerous Iesions. The initial phase of the infection is characterized by a fine regulation of the viral DNA replication, in order to maintain 10-100 DNA copies per cell. Such regulation is primarily controlled by El and E2 proteins produced by the early promoter. The recently discovered E8 promoter is capable to co-regulate the early one in order to maintain a low and constant viral DNA copy number.The aim of this study is to develop a novel stochastic mathematical model of the co-regulation between the E8 and the early promoter, with the main purpose to rigorously show the E8 promoter capability to finely regulate the HPV transcripts which control the DNA replication in the first stages of the infection.The model, condensing the biological knowledge present in literature, describes the interaction between the two promoters and shows how the E8 co-regulation is capable to reject the stochastic noise of E2 gene expression to a higher extent than the early promoter negative auto-feedback. This proves the capability of the E8 promoter to finely control the HPV genomes copy number.

Susceptibility of epithelial cells cultured from different regions of human cervix to HPV16-induced immortalization.

Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for cervical cancer. Greater than 90% of these cancers originate in the cervical transformation zone (TZ), a narrow region of metaplastic squamous epithelium that develops at the squamocolumnar junction between the ectocervix and endocervix. It is unclear why the TZ has high susceptibility to malignant transformation and few studies have specifically examined cells from this region. We hypothesized that cells cultured from TZ are more susceptible to cellular immortalization, an alteration that contributes to malignant development. We cultured primary epithelial cells from each region of human cervix (ectocervix, endocervix and TZ) and measured susceptibility to immortalization after transfection with the complete HPV-16 genome or infection of HPV16 E6/E7 retroviruses. Cells cultured from each cervical region expressed keratin markers (keratin 14 and 18) that confirmed their region of origin. In contrast to our prediction, cells from TZ were equally susceptible to immortalization as cells from ectocervix or endocervix. Thus, increased susceptibility of the TZ to cervical carcinogenesis is not due to increased frequency of immortalization by HPV-16. We developed a series of HPV16-immortalized cell lines from ectocervix, endocervix and TZ that will enable comparisons of how these cells respond to factors that promote cervical carcinogenesis.

Adenosine causes read-through into the late region of the HPV16 genome in a guanosine-dependent manner

Adenosine plays an important role in cell death and differentiation as well as in tumorigenesis and the intra- and extra-cellular levels range from nanomolar to millimolar levels under various physiological or pathophysiological conditions. Here we report that adenosine can activate HPV16 late gene expression in a dose- and time-dependent manner, but only in the presence of guanosine. This activation occurred within hours after addition of the nucleosides and was primarily dependent on the ENT1 nucleoside transporter protein. Induction of HPV16 late gene expression was mainly the result of increased read-through at the early HPV16 polyadenylation signal into the late region of the HPV16 genome, thereby producing HPV16 late L2 mRNAs. The effect of guanosine and adenosine on HPV16 late gene expression was mediated by the increased binding to HPV16 mRNAs and nuclear export of the cellular HuR protein. Our results demonstrate that nucleosides can affect HPV16 gene expression.

Presence of Human Papillomavirus (HPV) Apolipoprotein B Messenger RNA Editing, Catalytic Polypeptide-Like 3 (APOBEC)-Related Minority Variants in HPV-16 Genomes From Anal and Cervical Samples but Not in HPV-52 and HPV-58.

The aim of this study was to investigate the presence of minority variants (MVs) in high-risk human papillomavirus (HPV) types (HPV-16, -52, and -58) from cervical and anal smears. Whole HPV genome ultra-deep sequencing (UDS) was performed on cervical and anal smears collected during patient follow-up. Bioinformatics analyses were performed using Bowtie2 (Geneious). We assessed 55 HPV-16-positive, 20 HPV-52-positive, and 17 HPV-58-positive samples, with significant differences in patient characteristics for the 2 anatomic sites. HPV-16 MVs were detected in 20 samples (36%), with no difference between cervical and anal samples. We did not find an association between the presence of MVs and cytovirological parameters. Seven HPV-16 genomes (13%) were apolipoprotein B messenger RNA editing, catalytic polypeptide-like 3 (APOBEC) edited. Among the cervical HPV-16-positive samples, most MVs (55%) resulted from APOBEC-related mutations. MVs were detected in 10 HPV-52-positive (50%) and 12 HPV-58-positive (71%) samples, with no difference between cervical and anal samples. No APOBEC-related mutations were found on HPV-58 or HPV-52 genomes. Overall, high-risk HPV MVs were found in about half of all cases in both anal and cervical samples. Interestingly, we reported for the first time a differential impact of APOBEC3 mutagenic activity depending on high-risk HPV type.

HPV6 and HPV11 Genome Methylation in Condyloma AccuminatumMeasured by Bisulfite Sequencing

To the Editor: Condyloma acuminatum (CA, or genital warts) is a highly contagious and common sexually transmitted disease and caused by the infection of some types of human papillomavirus (HPV), especially types 6 and 11.1 Methylation is the most popular modification in human genome and has been observed to be correlated with human diseases.2 Moreover, it was known that methylation of partial region in multiple HPV genomes, including 16, 18, 30, and 45, was correlated with cervical cancer,3,4 another disease in women caused by HPV infection. All these facts made us hypothesize that methylation of HPV genome might also be correlated with CA. However, the HPV methylation status in CA is unknown. In this pilot study, we investigated the HPV genome methylation status in patients with CA by bisulfite sequencing. To this end, multiple genital skin tissues from typical male patients with CA with giant genital warts were collected from surgery and genotyped for HPV infection by Polymerase Chain Reaction (PCR) and xMAP (Luminex, Austin, TX; primer and probe set available on request). All patients were diagnosed based on typical clinical presentations5 and further confirmed by biopsy findings. From these samples, 4 patients with HPV6 infection and 3 with HPV11 were chosen for this study. DNA was isolated by standard phenol–chloroform method and treated with freshly prepared bisulfite using the EZ DNA methylation kit (Zymo Research, Orange, CA) according to the manufacturer's protocol. In bisulfite treatment, unmethylated C was converted into U, which was then converted to T in following PCR, whereas methylated C remained unmodified. Because HPV genome methylation in cervical tissue showed a gene-specific manner,3,4 which meant that the methylation in one segment could represent the situation in whole gene, we designed one PCR assay (see Table S1, Supplemental Digital Content 1, https://links.lww.com/AJDP/A65) for each protein-coding gene and upstream regulatory region by Methprimer6 (http://www.urogene.org/methprimer/index1.html). All segments were amplified by Taq DNA polymerase (Takara, Dalian, China) and directly sequenced in AB 3730 sequencer (Thermo Fisher, Waltham, MA) after purification, which was an accurate approach, and had been widely used in previous studies.7 This study was approved by the Institutional Ethics Committee, and informed consent was obtained from all individuals. In this study, 76 and 54 CpG sites were surveyed for HPV6 and 11 genomes, respectively, which account for ∼35–48% of total CpG sites, and the chromatograms for each CpG sites were inspected visually. None of the CpG sites displayed a clear C peak, which indicated that no methylation was observed in HPV6 and 11 genomes from CA tissues, and was consistent with recent report in HPV11 and 6 from respiratory8 and laryngeal9 papillomas tissues, respectively. Although the normal skin tissue was not available for control (persons with HPV infection but without CA symptom) in this study, we still could deduce that the methylation in normal genital tissue was also absent because of the hypomethylation of HPV genome in cervix without neoplasia3,4 and the lack of genes coding for DNA methyltransferase in HPV genome. Another concern was the relatively small sample size and sample bias (only male patients included) in this study. Because most patients were prone to receive pharmacotherapy instead of surgery, it was difficult to obtain more tissues. Further investigation with larger sample size and female patients might generalize our conclusion. In another side, because DNA methylation level was relatively similar among the different patients with same disease, it could be speculated that other patients with CA might also present hypomethylation in HPV genome. All these facts indicated that the methylation of HPV genome was not changed during the development of CA, which might indicate that the methyltransferase system still worked properly in CA tissues.

The high-risk HPV infection and urinary system tumor

AbstractHPV is classified into high-risk and low-risk types depending on its probability of leading to tumorigenesis. Many studies have shown that HPV infection, especially the infection caused by the high-risk type, is always related to prostate cancer, bladder cancer, penile cancer, testicular cancer, and other urinary system tumors. However, previous studies differed in sexual openness and racial genetic susceptibility of the study object, sample size, and experimental methods. Hence, the correlation between high-risk HPV infection and urinary system tumors remains controversial. The early open reading frame of the HPV genome is composed of E1–E7, among which E6 and E7 are the key transfer proteins. The combination of these proteins with oncogene and anti-oncogene may be one of the mechanisms leading to tumorigenesis.

STAT3 activation by E6 is essential for the differentiation-dependent HPV18 life cycle.

Human papillomaviruses (HPV) activate a number of host factors to control their differentiation-dependent life cycles. The transcription factor signal transducer and activator of transcription (STAT)-3 is important for cell cycle progression and cell survival in response to cytokines and growth factors. STAT3 requires phosphorylation on Ser727, in addition to phosphorylation on Tyr705 to be transcriptionally active. In this study, we show that STAT3 is essential for the HPV life cycle in undifferentiated and differentiated keratinocytes. Primary human keratinocytes containing high-risk HPV18 genomes display enhanced STAT3 phosphorylation compared to normal keratinocytes. Expression of the E6 oncoprotein is sufficient to induce the dual phosphorylation of STAT3 at Ser727 and Tyr705 by a mechanism requiring Janus kinases and members of the MAPK family. E6-mediated activation of STAT3 induces the transcription of STAT3 responsive genes including cyclin D1 and Bcl-xL. Silencing of STAT3 protein expression by siRNA or inhibition of STAT3 activation by small molecule inhibitors, or by expression of dominant negative STAT3 phosphorylation site mutants, results in blockade of cell cycle progression. Loss of active STAT3 impairs HPV gene expression and prevents episome maintenance in undifferentiated keratinocytes and upon differentiation, lack of active STAT3 abolishes virus genome amplification and late gene expression. Organotypic raft cultures of HPV18 containing keratinocytes expressing a phosphorylation site STAT3 mutant display a profound reduction in suprabasal hyperplasia, which correlates with a loss of cyclin B1 expression and increased differentiation. Finally, increased STAT3 expression and phosphorylation is observed in HPV positive cervical disease biopsies compared to control samples, highlighting a role for STAT3 activation in cervical carcinogenesis. In summary, our data provides evidence of a critical role for STAT3 in the HPV18 life cycle.

A44 Complete HPV genomes from cervical samples using next-generation sequencing in Luxembourg

A44 Complete HPV genomes from cervical samples using next-generation sequencing in Luxembourg

Patients with integrated HPV16 in head and neck cancer show poor survival

ObjectivesWe previously reported identifying three categories of HPV16-positive head and neck tumors based on The Cancer Genome Atlas (TCGA) RNA and DNA sequence data. Category 1 had truly integrated HPV16 genomes, category 2 had simple episomal genomes, and category 3 had novel episomes that were a hybrid between viral and human DNA. Using our categorization, we investigated in this study survival of patients with integrated HPV16 tumors versus patients with episomal HPV16 tumors. Materials and methodsThe TCGA RNA-Seq sequence reads were used to quantify HPV E2 and E7 gene expression, which was used as a marker for HPV integration. ResultsThe results demonstrate that integration is associated with poor survival; those patients with integrated HPV tumors fared no better than non-HPV tumors in their five-year survival. Integrated HPV in tumors was found strikingly to be prevalent in patients born earlier while episomal HPV was prevalent in patients born later. We also observed a fairly constant incidence of all HPV forms among head and neck cancer patients over the last eight years of this study (2006–2013). ConclusionWe propose our characterization of HPV integrated and episomal state is more accurate than previous studies that may have mischaracterized the hybrid HPV-human DNA episomes as integrated. The state of integrated HPV is associated with a poor clinical outcome. Results suggest that the incidence of integrated HPV among all HPV forms peaked and is decreasing. We discuss the importance of our findings for the management of HPV positive head and neck cancer.

Human Papillomavirus Type 16 Integration Analysis by Real-time PCR Assay in Associated Cancers

Human papillomavirus (HPV) is a common viral infection worldwide associated with a variety of cancers. The integration of the HPV genome in these patients causes chromosomal instability and triggers carcinogenesis. The aim of this study was to investigate the HPV-16 genome physical status in four major cancers related to HPV infection. Formalin-fixed paraffin-embedded blocks from our previous projects on head and neck, colorectal, penile, and cervical cancers were collected, and HPV-16–positive specimens were used for further analysis. The DNA extraction copy number of E2 and E7 genes was calculated by qualitative real-time PCR method. Serially diluted standards that were cloned in PUC57 plasmid were used. Standard curve and melting curve analysis was used for quantification. Of the 672 specimens studied, 76 (11.3%) were HPV-16 positive. We found that 35.6% (16/45) were integrated. Statistical analysis showed that there were significant correlations between integration of HPV-16 and cervical cancer end-stage carcinogenesis (P < .0001), episomal form, and ASCUS lesions (P = .045). Significant correlation in penile cancer patients was seen between the episomal form and high-grade cancer stage (P = .037). Integration is a major factor in the carcinogenesis mechanism of HPV and has different prevalence in various cancers with a higher rate in progression except in penile cancer.

The Intersection of HPV Epidemiology, Genomics and Mechanistic Studies of HPV-Mediated Carcinogenesis.

Of the ~60 human papillomavirus (HPV) genotypes that infect the cervicovaginal epithelium, only 12–13 “high-risk” types are well-established as causing cervical cancer, with HPV16 accounting for over half of all cases worldwide. While HPV16 is the most important carcinogenic type, variants of HPV16 can differ in their carcinogenicity by 10-fold or more in epidemiologic studies. Strong genotype-phenotype associations embedded in the small 8-kb HPV16 genome motivate molecular studies to understand the underlying molecular mechanisms. Understanding the mechanisms of HPV genomic findings is complicated by the linkage of HPV genome variants. A panel of experts in various disciplines gathered on 21 November 2016 to discuss the interdisciplinary science of HPV oncogenesis. Here, we summarize the discussion of the complexity of the viral–host interaction and highlight important next steps for selected applied basic laboratory studies guided by epidemiological genomic findings.

High Whole-Genome Sequence Diversity of Human Papillomavirus Type 18 Isolates.

Background: The most commonly found human papillomavirus (HPV) types in cervical cancer are HPV16 and HPV18. Genome variants of these types have been associated with differential carcinogenic potential. To date, only a handful of studies have described HPV18 whole genome sequencing results. Here we describe HPV18 variant diversity and conservation of persistent infections in a longitudinal retrospective cohort study. Methods: Cervical self-samples were obtained annually over four years and genotyped on the SPF10-DEIA-LiPA25 platform. Clearing and persistent HPV18 positive infections were selected, amplified in two overlapping fragments, and sequenced using 32 sequence primers. Results: Complete viral genomes were obtained from 25 participants with persistent and 26 participants with clearing HPV18 infections, resulting in 52 unique HPV18 genomes. Sublineage A3 was predominant in this population. The consensus viral genome was completely conserved over time in persistent infections, with one exception, where different HPV18 variants were identified in follow-up samples. Conclusions: This study identified a diverse set of HPV18 variants. In persistent infections, the consensus viral genome is conserved. The identification of only one HPV18 infection with different major variants in follow-up implies that this is a potentially rare event. This dataset adds 52 HPV18 genome variants to Genbank, more than doubling the currently available HPV18 information resource, and all but one variant are unique additions.

Human Papillomavirus E6 interaction with cellular PDZ domain proteins modulates YAP nuclear localization

HPV E6 oncoproteins associate with cellular PDZ proteins. In addition to previously identified cellular PDZ proteins, we found association of the HPV16 E6 PBM with the Dystrophin Glycoprotein Complex, LRCC1, and SLC9A3R2. HPV18 E6 had additional associations when lysates from adenomatous cell lines were used including LRPPRC, RLGAPB, EIF3A, SMC2 and 3, AMOT, AMOTL1, and ARHGEF1; some of these cellular PDZ proteins are implicated in the regulation of the YAP1 transcriptional co-activator. In keratinocytes, nuclear translocation of YAP1 was promoted by the complete HPV-16 genome, or by expression of the individual E6 or E7 oncoproteins; the activity of E6 required an intact PBM at the carboxy-terminus. This work demonstrates that E6 association with cellular PDZ proteins promotes the nuclear localization of YAP1. The ability of E6 to promote the nuclear transport of YAP1 thus identifies an E6 activity that could contribute to the transformation of cells by E6.

Classification and evolution of human papillomavirus genome variants: Alpha-5 (HPV26, 51, 69, 82), Alpha-6 (HPV30, 53, 56, 66), Alpha-11 (HPV34, 73), Alpha-13 (HPV54) and Alpha-3 (HPV61)

HPV variants from the same type can be classified into lineages and sublineages based on the complete genome differences and the phylogenetic topologies. We examined nucleotide variations of twelve HPV types within the species Alpha-5 (HPV26, 51, 69, 82), Alpha-6 (HPV30, 53, 56, 66), Alpha-11 (HPV34, 73), Alpha-13 (HPV54) and Alpha-3 (HPV61) by analyzing 1432 partial sequences and 181 complete genomes from multiple geographic populations. The inter-lineage and inter-sublineage mean differences of HPV variants ranged between 0.9–7.3% and 0.3–0.9%, respectively. The heterogeneity and phylogenies of HPV isolates indicate an independent evolutionary history for each type. The noncoding regions were the most variable regions whereas the capsid proteins were relatively conserved. Certain variant lineages and/or sublineages were geographically-associated. These data provide the basis to further classify HPV variants and should foster future studies on the evolution of HPV genomes and the associations of HPV variants with cancer risk.

Nuclear myosin 1 associates with papillomavirus E2 regulatory protein and influences viral replication

Nuclear myosin 1c (NM1) associates with RNA polymerases and is a partner in the chromatin remodeling complex B-WICH. This complex, which also contains WSTF and SNF2h proteins, is involved in transcriptional regulation. We report herein that papillomavirus protein E2 binds to NM1 and co-precipitates with the WSTF and SNF2h proteins. Our data suggest that E2 associates with the cellular B-WICH complex through binding to NM1. E2 and NM1 associate via their N-terminal domains and this interaction is ATP dependent. The cellular multifunctional protein Brd4 and beta-actin are also present in the NM1-E2 complex. NM1 downregulation by siRNA increases the replication of the BPV1 and HPV5 genomes but does not affect HPV18 genome replication. These results suggest that the B-WICH complex may play a role in the papillomavirus life cycle through NM1 and E2 protein interaction.

A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences

HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis.