p63 Is Necessary for the Activation of Human Papillomavirus Late Viral Functions upon Epithelial Differentiation

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k High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population.H uman papillomaviruses (HPVs) are double-stranded DNA viruses comprised of more than 100 subtypes, some of which, designated high risk, cause cervical and approximately one quarter of head and neck squamous cell carcinomas (HNSCCs) (1,20,21). HPV is associated primarily with HNSCCs that occur in the oropharynx, which includes the back of the tongue, walls of the throat, the soft palate, and the tonsils. Approximately 60% of oropharyngeal SCCs are caused by one of the high-risk types of HPV, and the vast majority of those are positive for HPV type 16 (HPV16) (10). Importantly, these HNSCCs are a distinct type distinguishable from HPV-negative tumors which have alcohol and tobacco use as the predominant risk factors (1).HPV is the causative agent of nearly all cervical cancers and is best studied in that disease. Although HPV infections are common, with up to 90% of women estimated to be infected in their lifetime, most infections are cleared by the immune system and do not progress to cancer. A small fraction of HPV infections, however, lead to low-grade and high-grade squamous intraepithelial lesions and cervical cancer. Persistent infection is the most significant risk factor for developing cervical cancer. While the exact progression of events is still not entirely understood, replication, persistence, integration of the viral genome, and increased expression of the viral E6 and E7 oncogenes are important steps in malignant pro...

Section: Discussionmentioning

confidence: 99%

The Fanconi Anemia Pathway Limits Human Papillomavirus Replication

Hoskins

Morreale

Werner

et al. 2012

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“…Lentivirus was produced as previously described (40). Plasmids carrying the short hairpin RNA (shRNA) sequences for Rad51 (TRCN0000018876 and TRCN0000018879) or BRCA1 (TRCN0000009824 and TRCN0000039833) or a scramble nontarget control shRNA cloned into the pLKO.1-puro background were obtained from the UNC Lentiviral Core Facility (Chapel Hill, NC).…”

Section: Methodsmentioning

confidence: 99%

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

Chappell

Gautam

et al. 2016

High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCEProductive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. H uman papillomaviruses (HPVs) are small double-stranded DNA viruses approximately 8 kb in size that exhibit a preferential tropism for epithelial cells. High-risk mucosal HPV subtypes are the causative agents of cervical cancer and have been increasingly associated with anogenital, oropharyngeal, and head and neck cancers (1). The life cycle of HPV is intimately linked to the differentiation of its host cell, the keratinocyte (2). After exposure through a microwound in the stratified ep...

“…Late gene expression is a second late viral event that is associated with differentiation status (4,28). Activation of the HPV-31 late promoter induces expression of transcripts encoding E1ˆE4, E1, E2, and E5 and can occur independently of amplification, which acts primarily to increase total transcript levels (28).…”

mentioning

confidence: 99%

Human Papillomaviruses Modulate MicroRNA 145 Expression To Directly Control Genome Amplification

Gunasekharan

Laimins

2013

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H igh-risk human papillomaviruses (HPVs) are the causative agents of cervical and most other anogenital malignancies. Infection by HPVs occurs in the basal layers of stratified epithelia that become exposed due to microabrasions. Following entry, viral genomes are established as low-copy nuclear episomes at approximately 10 to 100 copies per cell (1, 2). In differentiated suprabasal layers, high levels of late gene expression are induced along with genome amplification and virion assembly (3). Two major viral promoters, designated early and late, control transcription during differentiation, but additional regulation also occurs through alternative splicing and other posttranscriptional mechanisms (1, 2, 4). One important mechanism of posttranscriptional control is mediated by small RNAs that are 22 nucleotides in size, referred to as microRNAs (miRNAs). miRNAs bind to partially complementary target sites in mRNA that are often located in the 3=UTR (untranslated regions) but can also be found in coding sequences and the 5=UTR (5). Binding by miRNAs to target sequences results in either the degradation of the target mRNAs or translational repression of the encoded protein (6). A single miRNA can repress the expression of over 100 genes, and a single gene can be repressed by multiple miRNAs. miRNAs often target multiple genes in a pathway and can act cooperatively with other miRNAs (7). Many miRNAs target developmental (8-10) and differentiation pathways (11)(12)(13)(14), as well as cell proliferation and transformation pathways (15-17). Herpesviruses and other small DNA viruses encode their own miRNAs (18); however, this is not the case for HPVs (19). Instead, HPVs modulate expression of host miRNAs to control various aspects of their viral life cycle (20, 21).Several miRNAs have been shown to be modulated by HPV proteins and to play key roles in the viral life cycle. miRNA 203 (miR-203) is a key regulator of epithelial differentiation and is downregulated in HPV-positive cells upon differentiation. This repression allows for the maintenance of high levels of p63, which is normally repressed by miR-203 in differentiating, infected suprabasal cells and is important for maintaining cells in an active state in the cell cycle (21). Additional studies demonstrated miR-218 to be downregulated by HPV E6, which correlates with increased levels of LAMB3, a translational target of this miRNA (22). A number of reports have documented changes in levels of cellular microRNAs in HPV-positive cells using microarray analyses, but only a limited number of studies has examined the significance of these changes in the viral life cycle (23-25).Since HPVs modulate the expression of cellular microRNAs to regulate aspects of their differentiation-dependent life cycle, we sought to identify cellular miRNAs that were altered by HPV proteins in a more physiological context than that reflected in monolayer cultures alone. For this analysis, we performed deep sequencing using organotypic raft cultures of a matched set of normal (viv) and H...