Papillomaviruses infect and replicate in keratinocytes, but viral proteins are initially expressed at low levels and there is no effective and quantitative method to determine the efficiency of infection on a cell-to-cell basis. Here we describe human papillomavirus (HPV) genomes that express marker proteins (antibiotic resistance genes and Green Fluorescent Protein), and can be used to elucidate early stages in HPV infection of primary keratinocytes. To generate these recombinant genomes, the late region of the oncogenic HPV18 genome was replaced by CpG free marker genes. Insertion of these exogenous genes did not affect early replication, and had only minimal effects on early viral transcription. When introduced into primary keratinocytes, the recombinant marker genomes gave rise to drug-resistant keratinocyte colonies and cell lines, which maintained the extrachromosomal recombinant genome long-term. Furthermore, the HPV18 "marker" genomes could be packaged into viral particles (quasivirions) and used to infect primary human keratinocytes in culture. This resulted in the outgrowth of drug-resistant keratinocyte colonies containing replicating HPV18 genomes. In summary, we describe HPV18 marker genomes that can be used to quantitatively investigate many aspects of the viral life cycle.

It is becoming clear that, in addition to gene gain, the loss of genes may be an important evolutionary mechanism for many organisms. However, gene loss is often associated with an increased mutation rate, thus quickly erasing evidence from the genome. The analysis of evolutionarily related sequences can provide empirical evidence for gene loss events. This paper analyzes the sequences of over 300 genetically distinct papillomaviruses and provides evidence for a role of gene loss during the evolution of certain papillomavirus genomes. Phylogenetic analysis suggests that the viral E6 gene was lost at least twice. Despite belonging to distant papillomaviral genera, these viruses lacking a canonical E6 protein may potentially encode a highly hydrophobic protein from an overlapping open reading frame, which we designate E10. Evolutionary pressure working on this alternative frame, may explain why, despite having lost the E6 open reading frame between 20 and 60 million years ago, evidence of an E6-like protein is conserved.

Oncogenic human papillomaviruses (HPVs) replicate in differentiating epithelium, causing 5% of cancers worldwide. Like most other DNA viruses, HPV infection initiates after trafficking viral genome (vDNA) to host cell nuclei. Cells possess innate surveillance pathways to detect microbial components or physiological stresses often associated with microbial infections. One of these pathways, cGAS/STING, induces IRF3-dependent antiviral interferon (IFN) responses upon detection of cytosolic DNA. Virion-associated vDNA can activate cGAS/STING during initial viral entry and uncoating/trafficking, and thus cGAS/STING is an obstacle to many DNA viruses. HPV has a unique vesicular trafficking pathway compared to many other DNA viruses. As the capsid uncoats within acidic endosomal compartments, minor capsid protein L2 protrudes across vesicular membranes to facilitate transport of vDNA to the Golgi. L2/vDNA resides within the Golgi lumen until G2/M, whereupon vesicular L2/vDNA traffics along spindle microtubules, tethering to chromosomes to access daughter cell nuclei. L2/vDNA-containing vesicles likely remain intact until G1, following nuclear envelope reformation. We hypothesize that this unique vesicular trafficking protects HPV from cGAS/STING surveillance. Here, we investigate cGAS/STING responses to HPV infection. DNA transfection resulted in acute cGAS/STING activation and downstream IFN responses. In contrast, HPV infection elicited minimal cGAS/STING and IFN responses. To determine the role of vesicular trafficking in cGAS/STING evasion, we forced premature viral penetration of vesicular membranes with membrane-perturbing cationic lipids. Such treatment renders a non-infectious trafficking-defective mutant HPV infectious, yet susceptible to cGAS/STING detection. Overall, HPV evades cGAS/STING by its unique subcellular trafficking, a property that may contribute to establishment of infection.

Polyomaviruses are non-enveloped viruses with circular double-stranded DNA genomes (~4-7 kb). Initially identified in mammals, polyomaviruses have now been identified in birds and a few fish species. Although fragmentary polyomavirus-like sequences have been detected as apparent 'hitchhikers' in shotgun genomics datasets of various arthropods, the possible diversity of these viruses in invertebrates remains unclear. Scorpions are predatory arachnids that are among the oldest terrestrial animals. Using high-throughput sequencing and traditional molecular techniques we determine the genome sequences of eight novel polyomaviruses in scorpions (Centruroides sculpturatus) from the greater Phoenix area, Arizona, USA. Analysis of Centruroides transcriptomic datasets elucidated the splicing of the viral late gene array, which is more complex than that of vertebrate polyomaviruses. Phylogenetic analysis provides further evidence of co-divergence of polyomaviruses with their hosts, suggesting that at least one ancestral species of polyomaviruses was circulating amongst the primitive common ancestors of arthropods and chordates.

Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e., cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data (https://viz.datascience.arizona.edu/3DEpiEx/).

Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts.IMPORTANCE Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients.

The complete genome of a novel papillomavirus was isolated from a cutaneous papillomatous lesion of a North American porcupine (Erethizon dorsatum) using multiply primed rolling-circle amplification (RCA). The nucleotide sequence, genome organization, and phylogenetic position of the Erethizon dorsatum papillomavirus type 1 (EdPV-1) were determined. EdPV-1 is only distantly related to other benign cutaneous papillomavirus sequences and is the first member of the novel Sigma papillomavirus genus.

High-throughput sequencing technologies provide unprecedented power to identify novel viruses from a wide variety of (environmental) samples. The field of 'viral metagenomics' has dramatically expanded our understanding of viral diversity. Viral metagenomic approaches imply that many novel viruses will not be described by researchers who are experts on (the genomic organization of) that virus family. We have developed the papillomavirus annotation tool (PuMA) to provide researchers with a convenient and reproducible method to annotate and report novel papillomaviruses. PuMA currently correctly annotates 99% of the papillomavirus genes when benchmarked against the 655 reference genomes in the papillomavirus episteme. Compared to another viral annotation pipeline, PuMA annotates more viral features while being more accurate. To demonstrate its general applicability, we also developed a preliminary version of PuMA that can annotate polyomaviruses. PuMA is available on GitHub (https://github.com/KVD-lab/puma) and through the iMicrobe online environment (https://www.imicrobe.us/#/apps/puma).

Fish papillomaviruses form a newly discovered group broadly recognized as the Secondpapillomavirinae subfamily. This study expands the documented genomes of the fish papillomaviruses from six to 16, including one from the Antarctic emerald notothen, seven from commercial market fishes, one from data mining of sea bream sequence data, and one from a western gull cloacal swab that is likely diet derived. The genomes of secondpapillomaviruses are ∼6 kilobasepairs (kb), which is substantially smaller than the ∼8 kb of terrestrial vertebrate papillomaviruses. Each genome encodes a clear homolog of the four canonical papillomavirus genes, E1, E2, L1, and L2. In addition, we identified open reading frames (ORFs) with short linear peptide motifs reminiscent of E6/E7 oncoproteins. Fish papillomaviruses are extremely diverse and phylogenetically distant from other papillomaviruses suggesting a model in which terrestrial vertebrate-infecting papillomaviruses arose after an evolutionary bottleneck event, possibly during the water-to-land transition.

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2's chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.

Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e. , cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data ( https://viz.datascience.arizona.edu/3DEpiEx/ ).

In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

In this paper we describe the characterization of the genomes of two sea turtle papillomaviruses, Chelonia mydas PV (CmPV-1) and Caretta caretta PV (CcPV-1). The isolation and sequencing of the first non-avian reptilian PVs extend the evolutionary history of PVs to include all amniotes. PVs have now been described in mammals, birds and non-avian reptiles. The chelonian PVs form a distinct clade most closely related to the avian PVs. Unlike the avian PVs, both chelonian PVs have canonical E6 and E7 ORFs, indicating that these genes were present in the common ancestor to mammalian and non-mammalian amniote PVs. Rates of evolution among the non-mammalian PVs were generally slower than those estimated for mammalian PVs, perhaps due to lower metabolic rates among the ectothermic reptiles.

The complete genomic DNA of a novel papillomavirus (PV) was isolated from a basosquamous carcinoma on the wing of an Egyptian fruit bat (Rousettus aegyptiacus). Initial short sequences of the E1 and L1 genes of this virus were retrieved by PCR with degenerate papillomavirus-specific primers, and the entire R. aegyptiacus papillomavirus type 1 (RaPV-1) DNA was then amplified by long template PCR, cloned and sequenced with a transposon insertion method. The RaPV-1 genome counts 7970 basepairs and contains the typical papillomavirus open reading frames (ORF) (E1, E2, E4, E6, E7, L1 and L2). Based on a concatenated alignment of the E1, E2, L1 and L2 open reading frames of RaPV-1 and 46 other human and animal papillomavirus type species, a neighbor-joining phylogenetic tree was constructed. This phylogenetic analysis shows that RaPV-1 has a close-to-root position in the papillomavirus evolutionary tree. Since RaPV-1 is only distantly related to other papillomaviruses (with maximally 50% nucleotide sequence identity across the L1 open reading frame), it cannot be assigned to one of the existing papillomavirus genera and therefore represents the first member of a novel, as yet unnamed, close-to-root papillomavirus genus. This is the first time a papillomavirus has been isolated and characterized from a member of the Chiroptera order.

Anellovirus infections are highly prevalent in mammals, however, prior to this study only a handful of anellovirus genomes had been identified in members of the Felidae family. Here we characterise anelloviruses in pumas (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis), caracals (Caracal caracal) and domestic cats (Felis catus). The complete anellovirus genomes (n = 220) recovered from 149 individuals were diverse. ORF1 protein sequence similarity network analysis coupled with phylogenetic analysis, revealed two distinct clusters that are populated by felid-derived anellovirus sequences, a pattern mirroring that observed for the porcine anelloviruses. Of the two-felid dominant anellovirus groups, one includes sequences from bobcats, pumas, domestic cats and an ocelot, and the other includes sequences from caracals, Canada lynx, domestic cats and pumas. Coinfections of diverse anelloviruses appear to be common among the felids. Evidence of recombination, both within and between felid-specific anellovirus groups, supports a long coevolution history between host and virus.

The family Cactaceae comprises a diverse group of typically succulent plants that are native to the American continent but have been introduced to nearly all other continents, predominantly for ornamental purposes. Despite their economic, cultural, and ecological importance, very little research has been conducted on the viral community that infects them. We previously identified a highly divergent geminivirus that is the first known to infect cacti. Recent research efforts in non-cultivated and asymptomatic plants have shown that the diversity of this viral family has been under-sampled. As a consequence, little is known about the effects and interactions of geminiviruses in many plants, such as cacti. With the objective to expand knowledge on the diversity of geminiviruses infecting cacti, we used previously acquired high-throughput sequencing results to search for viral sequences using BLASTx against a viral RefSeq protein database. We identified two additional sequences with similarity to geminiviruses, for which we designed abutting primers and recovered full-length genomes. From 42 cacti and five scale insects, we derived 42 complete genome sequences of a novel geminivirus species that we have tentatively named Opuntia virus 2 (OpV2) and 32 genomes of an Opuntia-infecting becurtovirus (which is a new strain of the spinach curly top Arizona virus species). Interspecies recombination analysis of the OpV2 group revealed several recombinant regions, in some cases spanning half of the genome. Phylogenetic analysis demonstrated that OpV2 is a novel geminivirus more closely related to viruses of the genus Curtovirus, which was further supported by the detection of three recombination events between curtoviruses and OpV2. Both OpV2 and Opuntia becurtoviruses were identified in mixed infections, which also included the previously characterized Opuntia virus 1. Viral quantification of the co-infected cactus plants compared with single infections did not show any clear trend in viral dynamics that might be associated with the mixed infections. Using experimental Rhizobium-mediated inoculations, we found that the initial accumulation of OpV2 is facilitated by co-infection with OpV1. This study shows that the diversity of geminiviruses that infect cacti is under-sampled and that cacti harbor diverse geminiviruses. The detection of the Opuntia becurtoviruses suggests spill-over events between viruses of cultivated species and native vegetation. The threat this poses to cacti needs to be further investigated.

Human papillomavirus (HPV) infection occurs in differentiating epithelial tissues. Cancers caused by high-risk types (e.g., HPV16 and HPV18) typically occur at oropharyngeal and anogenital anatomical sites. The HPV life cycle is differentiation-dependent, requiring tissue culture methodology that is able to recapitulate the three-dimensional (3D) stratified epithelium. Here we report two distinct and complementary methods for growing differentiating epithelial tissues that mimic many critical morphological and biochemical aspects of in vivo tissue. The first approach involves growing primary human epithelial cells on top of a dermal equivalent consisting of collagen fibers and living fibroblast cells. When these cells are grown at the liquid-air interface, differentiation occurs and allows for epithelial stratification. The second approach uses a rotating wall vessel bioreactor. The low-fluid-shear microgravity environment inside the bioreactor allows the cells to use collagen-coated microbeads as a growth scaffold and self-assemble into 3D cellular aggregates. These approaches are applied to epithelial cells derived from HPV-positive and HPV-negative oral and cervical tissues. The second part of the article introduces potential downstream applications for these 3D tissue models. We describe methods that will allow readers to start successfully culturing 3D tissues from oral and cervical cells. These tissues have been used for microscopic visualization, scanning electron microscopy, and large omics-based studies to gain insights into epithelial biology, the HPV life cycle, and host-pathogen interactions. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Establishing human primary cell-derived 3D organotypic raft cultures Support Protocol 1: Isolation of epithelial cells from patient-derived tissues Support Protocol 2: Growth and maintenance of primary human epithelial cells in monolayer culture Support Protocol 3: PCR-based HPV screening of primary cell cultures Basic Protocol 2: Establishing human 3D cervical tissues using the rotating wall vessel bioreactor Support Protocol 4: Growth and maintenance of human A2EN cells in monolayer culture Support Protocol 5: Preparation of the slow-turning lateral vessel bioreactor Support Protocol 6: Preparation of Cytodex-3 microcarrier beads Basic Protocol 3: Histological assessment of 3D organotypic raft tissues Basic Protocol 4: Spatial analysis of protein expression in 3D organotypic raft cultures Basic Protocol 5: Immunofluorescence imaging of RWV-derived 3D tissues Basic Protocol 6: Ultrastructural visualization and imaging of RWV-derived 3D tissues Basic Protocol 7: Characterization of gene expression by RT-qPCR.

The Polyomaviridae is a diverse family of circular double-stranded DNA viruses. Polyomaviruses have been isolated from a wide array of animal hosts. An understanding of the evolutionary and ecological dynamics of these viruses is essential to understanding the pathogenicity of polyomaviruses. Using a high throughput sequencing approach, we identified a novel polyomavirus in an emerald notothen (Trematomus bernacchii) sampled in the Ross sea (Antarctica), expanding the known number of fish-associated polyomaviruses. Our analysis suggests that polyomaviruses belong to three main evolutionary clades; the first clade is made up of all recognized terrestrial polyomaviruses. The fish-associated polyomaviruses are not monophyletic, and belong to two divergent evolutionary lineages. The fish viruses provide evidence that the evolution of the key viral large T protein involves gain and loss of distinct domains.

Cervical carcinogenesis, the second leading cause of cancer death in women worldwide, is caused by multiple types of human papillomaviruses (HPVs). To investigate a possible role for HPV in a cervical carcinoma that was HPV-negative by PCR testing, we performed HPV DNA hybridization capture plus massively parallel sequencing. This detected a subgenomic, URR-E6-E7-E1 segment of HPV70 DNA, a type not generally associated with cervical cancer, inserted in an intron of the B-cell lymphoma/leukemia 11B (BCL11B) gene in the human genome. Long range DNA sequencing confirmed the virus and flanking BCL11B DNA structures including both insertion junctions. Global transcriptomic analysis detected multiple, alternatively spliced, HPV70-BCL11B, fusion transcripts with fused open reading frames. The insertion and fusion transcripts were present in an intraepithelial precursor phase of tumorigenesis. These results suggest oncogenicity of HPV70, identify novel BCL11B variants with potential oncogenic implications, and underscore the advantages of thorough genomic analyses to elucidate insights into HPV-associated tumorigenesis.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.