Human malignancies associated with persistent HPV infection.

Human papillomavirus (HPV)-associated malignancies account for ~5% of human cancers worldwide. Thirteen, or more, HPV types are oncogenic, but infection with these viruses is common and usually cleared within 2 years. Only infections that become persistent are associated with the development of cancer, often occurring several decades later. These cancers mostly arise in 6 different anatomical regions: 5 are anogenital (anus, cervix, penis, vagina, and vulva) and the sixth is the oropharynx. Oncogenic HPVs promote cellular proliferation and genomic instability, but the anatomical niche of the target tissue also plays an important role in the development of cancer. Cells that reside in transitional regions between different types of epithelia, such as in the anus, cervix, and oropharynx, are particularly vulnerable to oncogenesis.

Regulation of Human Papillomavirus 18 Genome Replication, Establishment, and Persistence by Sequences in the Viral Upstream Regulatory Region.

During persistent human papillomavirus infection, the viral genome replicates as an extrachromosomal plasmid that is efficiently partitioned to daughter cells during cell division. We have previously shown that an element which overlaps the human papillomavirus 18 (HPV18) transcriptional enhancer promotes stable DNA replication of replicons containing the viral replication origin. Here, we perform comprehensive analyses to elucidate the function of this maintenance element. We conclude that no unique element or binding site in this region is absolutely required for persistent replication and partitioning and instead propose that the overall chromatin architecture of this region is important to promote efficient use of the replication origin. These results have important implications for the genome partitioning mechanism of papillomaviruses. IMPORTANCE Persistent infection with oncogenic human papillomaviruses (HPVs) is responsible for ∼5% of human cancers. The viral DNA replicates as an extrachromosomal plasmid and is partitioned to daughter cells in dividing keratinocytes. Using a complementation assay that allows us to separate viral transcription and replication, we provide insight into viral sequences that are required for long-term replication and persistence in keratinocytes. Understanding how viral genomes replicate persistently for such long periods of time will guide the development of antiviral therapies.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression.

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression.

The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA.

The papillomavirus E2 protein executes numerous essential functions related to viral transcription, replication of viral DNA, and viral genome maintenance. Because E2 lacks enzymatic activity, many of these functions are mediated by interactions with host cellular proteins. Unbiased proteomics approaches have successfully identified a number of E2-host protein interactions. We have extended such studies and have identified and validated the cellular proteins structural maintenance of chromosome 5 (SMC5) and SMC6 as interactors of the viral E2 protein. These two proteins make up the core components of the SMC5/6 complex. The SMC5/6 complex is a member of the conserved structural maintenance of chromosomes (SMC) family of proteins, which are essential for genome maintenance. We have examined the role of SMC5/6 in various E2 functions. Our data suggest that SMC6 is not required for E2-mediated transcriptional activation, E1/E2-mediated transient replication, or differentiation-dependent amplification of viral DNA. Our data, however, suggest a role for SMC5/6 in viral genome maintenance.IMPORTANCE The high-risk human papillomaviruses (HPVs) are the etiological cause of cervical cancer and the most common sexually transmitted infection. While the majority of infections may be asymptomatic or cause only benign lesions, persistent infection with the oncogenic high-risk HPV types may lead to serious diseases, such as cervical cancer, anogenital carcinoma, or head and neck oropharyngeal squamous cell carcinoma. The identification of virus-host protein interactions provides insights into the mechanisms of viral DNA persistence, viral genome replication, and cellular transformation. Elucidating the mechanism of early events in the virus replication cycle as well as of integration of viral DNA into host chromatin may present novel antiviral strategies and targets for counteracting persistent infection. The E2 protein is an important viral regulatory protein whose functions are mediated through interactions with host cell proteins. Here we explore the interaction of E2 with SMC5/6 and the functional consequences.

The Epstein-Barr Virus Episome Maneuvers between Nuclear Chromatin Compartments during Reactivation.

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle.

We have shown previously that Sp100 (a component of the ND10 nuclear body) represses transcription, replication and establishment of incoming human papillomavirus (HPV) DNA in the early stages of infection. In this follow up study, we show that Sp100 does not substantially regulate viral infection in the maintenance phase, however at late stages of infection Sp100 interacts with amplifying viral genomes to repress viral processes. We find that Sp100 localizes to HPV16 replication foci generated in primary keratinocytes, to HPV31 replication foci that form in differentiated cells, and to HPV16 replication foci in CIN 1 cervical biopsies. To analyze this further, Sp100 was down regulated by siRNA treatment of differentiating HPV31 containing cells and levels of viral transcription and replication were assessed. This revealed that Sp100 represses viral transcription and replication in differentiated cells. Analysis of Sp100 binding to viral chromatin showed that Sp100 bound across the viral genome, and that binding increased at late stages of infection. Therefore, Sp100 represses the HPV life cycle at both early and late stages of infection.

The Promise of Proteomics in the Study of Oncogenic Viruses.

Oncogenic viruses are responsible for about 15% human cancers. This article explores the promise and challenges of viral proteomics in the study of the oncogenic human DNA viruses, HPV, McPyV, EBV and KSHV. These viruses have coevolved with their hosts and cause persistent infections. Each virus encodes oncoproteins that manipulate key cellular pathways to promote viral replication and evade the host immune response. Viral proteomics can identify cellular pathways perturbed by viral infection, identify cellular proteins that are crucial for viral persistence and oncogenesis, and identify important diagnostic and therapeutic targets.

The Papillomavirus Episteme: a major update to the papillomavirus sequence database.

The Papillomavirus Episteme (PaVE) is a database of curated papillomavirus genomic sequences, accompanied by web-based sequence analysis tools. This update describes the addition of major new features. The papillomavirus genomes within PaVE have been further annotated, and now includes the major spliced mRNA transcripts. Viral genes and transcripts can be visualized on both linear and circular genome browsers. Evolutionary relationships among PaVE reference protein sequences can be analysed using multiple sequence alignments and phylogenetic trees. To assist in viral discovery, PaVE offers a typing tool; a simplified algorithm to determine whether a newly sequenced virus is novel. PaVE also now contains an image library containing gross clinical and histopathological images of papillomavirus infected lesions. Database URL: https://pave.niaid.nih.gov/.

ICTV Virus Taxonomy Profile: Papillomaviridae.

The Papillomaviridae is a family of small, non-enveloped viruses with double-stranded DNA genomes of 5 748 to 8 607 bp. Their classification is based on pairwise nucleotide sequence identity across the L1 open reading frame. Members of the Papillomaviridae primarily infect mucosal and keratinised epithelia, and have been isolated from fish, reptiles, birds and mammals. Despite a long co-evolutionary history with their hosts, some papillomaviruses are pathogens of their natural host species. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Papillomaviridae, which is available at http://www.ictv.global/report/papillomaviridae.

The Ancient Evolutionary History of Polyomaviruses.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Mechanisms and strategies of papillomavirus replication.

Animal and human papillomaviruses (HPVs) replicate persistently in specific types of stratified epithelia of their host. After the initial infection, the viral genome replicates at low levels in the dividing cells of the epithelium, and these cells form a reservoir of infection that can last for decades. When the infected cells differentiate, viral genomes replicate to high levels to form progeny virus that is released from the surface of the epithelium. This complex life cycle requires several different modes of viral DNA replication, but papillomaviruses are masters at hijacking key cellular processes to facilitate their own reproduction.

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.

A proteomic approach to discover and compare interacting partners of papillomavirus E2 proteins from diverse phylogenetic groups.

Papillomaviruses are a very successful group of viruses that replicate persistently in localized regions of the stratified epithelium of their specific host. Infection results in pathologies ranging from asymptomatic infection, benign warts, to malignant carcinomas. Despite this diversity, papillomavirus genomes are small (7-8 kbp) and contain at most eight genes. To sustain the complex papillomaviral life cycle, each viral protein has multiple functions and interacts with and manipulates a plethora of cellular proteins. In this study, we use tandem affinity purification and MS to identify host factors that interact with 11 different papillomavirus E2 proteins from diverse phylogenetic groups. The E2 proteins function in viral transcription and replication and correspondingly interact with host proteins involved in transcription, chromatin remodeling and modification, replication, and RNA processing.

PSORS2 is due to mutations in CARD14.

Psoriasis is a common, immune-mediated genetic disorder of the skin and is associated with arthritis in approximately 30% of cases. Previously, we localized PSORS2 (psoriasis susceptibility locus 2) to chromosomal region 17q25.3-qter after a genome-wide linkage scan in a family of European ancestry with multiple cases of psoriasis and psoriatic arthritis. Linkage to PSORS2 was also observed in a Taiwanese family with multiple psoriasis-affected members. In caspase recruitment domain family, member 14 (CARD14), we identified unique gain-of-function mutations that segregated with psoriasis by using genomic capture and DNA sequencing. The mutations c.349G>A (p.Gly117Ser) (in the family of European descent) and c.349+5G>A (in the Taiwanese family) altered splicing between CARD14 exons 3 and 4. A de novo CARD14 mutation, c.413A>C (p.Glu138Ala), was detected in a child with sporadic, early-onset, generalized pustular psoriasis. CARD14 activates nuclear factor kappa B (NF-kB), and compared with wild-type CARD14, the p.Gly117Ser and p.Glu138Ala substitutions were shown to lead to enhanced NF-kB activation and upregulation of a subset of psoriasis-associated genes in keratinocytes. These genes included chemokine (C-C motif) ligand 20 (CCL20) and interleukin 8 (IL8). CARD14 is localized mainly in the basal and suprabasal layers of healthy skin epidermis, whereas in lesional psoriatic skin, it is reduced in the basal layer and more diffusely upregulated in the suprabasal layers of the epidermis. We propose that, after a triggering event that can include epidermal injury, rare gain-of-function mutations in CARD14 initiate a process that includes inflammatory cell recruitment by keratinocytes. This perpetuates a vicious cycle of epidermal inflammation and regeneration, a cycle which is the hallmark of psoriasis.

Brd4 is displaced from HPV replication factories as they expand and amplify viral DNA.

Replication foci are generated by many viruses to concentrate and localize viral DNA synthesis to specific regions of the cell. Expression of the HPV16 E1 and E2 replication proteins in keratinocytes results in nuclear foci that recruit proteins associated with the host DNA damage response. We show that the Brd4 protein localizes to these foci and is essential for their formation. However, when E1 and E2 begin amplifying viral DNA, Brd4 is displaced from the foci and cellular factors associated with DNA synthesis and homologous recombination are recruited. Differentiated HPV-infected keratinocytes form similar nuclear foci that contain amplifying viral DNA. We compare the different foci and show that, while they have many characteristics in common, there is a switch between early Brd4-dependent foci and mature Brd4-independent replication foci. However, HPV genomes encoding mutated E2 proteins that are unable to bind Brd4 can replicate and amplify the viral genome. We propose that, while E1, E2 and Brd4 might bind host chromatin at early stages of infection, there is a temporal and functional switch at later stages and increased E1 and E2 levels promote viral DNA amplification, displacement of Brd4 and growth of a replication factory. The concomitant DNA damage response recruits proteins required for DNA synthesis and repair, which could then be utilized for viral DNA replication. Hence, while Brd4 can enhance replication by concentrating viral processes in specific regions of the host nucleus, this interaction is not absolutely essential for HPV replication.

The Papillomavirus Episteme: a central resource for papillomavirus sequence data and analysis.

The goal of the Papillomavirus Episteme (PaVE) is to provide an integrated resource for the analysis of papillomavirus (PV) genome sequences and related information. The PaVE is a freely accessible, web-based tool (http://pave.niaid.nih.gov) created around a relational database, which enables storage, analysis and exchange of sequence information. From a design perspective, the PaVE adopts an Open Source software approach and stresses the integration and reuse of existing tools. Reference PV genome sequences have been extracted from publicly available databases and reannotated using a custom-created tool. To date, the PaVE contains 241 annotated PV genomes, 2245 genes and regions, 2004 protein sequences and 47 protein structures, which users can explore, analyze or download. The PaVE provides scientists with the data and tools needed to accelerate scientific progress for the study and treatment of diseases caused by PVs.

Embryonic mesoderm and endoderm induction requires the actions of non-embryonic Nodal-related ligands and Mxtx2.

Vertebrate mesoderm and endoderm formation requires signaling by Nodal-related ligands from the TGFß superfamily. The factors that initiate Nodal-related gene transcription are unknown in most species and the relative contributions of Nodal-related ligands from embryonic, extraembryonic and maternal sources remain uncertain. In zebrafish, signals from the yolk syncytial layer (YSL), an extraembryonic domain, are required for mesoderm and endoderm induction, and YSL expression of nodal-related 1 (ndr1) and ndr2 accounts for a portion of this activity. A variable requirement of maternally derived Ndr1 for dorsal and anterior axis formation has also been documented. Here we show that Mxtx2 directly activates expression of ndr2 via binding to its first intron and is required for ndr2 expression in the YSL. Mxtx2 is also required for the Nodal signaling-independent expression component of the no tail a (ntla) gene, which is required for posterior (tail) mesoderm formation. Therefore, Mxtx2 defines a new pathway upstream of Nodal signaling and posterior mesoderm formation. We further show that the co-disruption of extraembryonic Ndr2, extraembryonic Ndr1 and maternal Ndr1 eliminates endoderm and anterior (head and trunk) mesoderm, recapitulating the loss of Nodal signaling phenotype. Therefore, non-embryonic sources of Nodal-related ligands account for the complete spectrum of early Nodal signaling requirements. In summary, the induction of mesoderm and endoderm depends upon the combined actions of Mxtx2 and Nodal-related ligands from non-embryonic sources.

Hitchhiking on host chromatin: how papillomaviruses persist.

Persistent viruses need mechanisms to protect their genomes from cellular defenses and to ensure that they are efficiently propagated to daughter host cells. One mechanism by which papillomaviruses achieve this is through the association of viral genomes with host chromatin, mediated by the viral E2 tethering protein. Association of viral DNA with regions of active host chromatin ensures that the virus remains transcriptionally active and is not relegated to repressed heterochromatin. In addition, viral genomes are tethered to specific regions of host mitotic chromosomes to efficiently partition their DNA to daughter cells. Vegetative viral DNA replication also initiates at specific regions of host chromatin, where the viral E1 and E2 proteins initiate a DNA damage response that recruits cellular DNA damage and repair proteins to viral replication foci for efficient viral DNA synthesis. Thus, these small viruses have capitalized on interactions with chromatin to efficiently target their genomes to beneficial regions of the host nucleus. This article is part of a Special Issue entitled: Chromatin in time and space.

Phosphorylation regulates binding of the human papillomavirus type 8 E2 protein to host chromosomes.

The papillomavirus E2 proteins are indispensable for the viral life cycle, and their functions are subject to tight regulation. The E2 proteins undergo posttranslational modifications that regulate their properties and roles in viral transcription, replication, and genome maintenance. During persistent infection, the E2 proteins from many papillomaviruses act as molecular bridges that tether the viral genomes to host chromosomes to retain them within the host nucleus and to partition them to daughter cells. The betapapillomavirus E2 proteins bind to pericentromeric regions of host mitotic chromosomes, including the ribosomal DNA loci. We recently reported that two residues (arginine 250 and serine 253) within the chromosome binding region of the human papillomavirus type 8 (HPV8) E2 protein are required for this binding. In this study, we show that serine 253 is phosphorylated, most likely by protein kinase A, and this modulates the interaction of the E2 protein with cellular chromatin. Furthermore, we show that this phosphorylation occurs in S phase, increases the half-life of the E2 protein, and promotes chromatin binding from S phase through mitosis.