Koenraad M Van Doorslaer
- Assistant Professor, Virology
- Assistant Professor, BIO5 Institute
- Assistant Professor, Cancer Biology - GIDP
- Assistant Professor, Genetics - GIDP
- Assistant Professor, Immunobiology
Dr. Van Doorslaer received his BS and MS from the University of Leuven, Belgium. He left the land of beer and chocolate to pursue a PhD in the lab of Dr. Robert Burk, at the Albert Einstein college of Medicine in New York. For his post-doctoral work, he joined the lab of Dr. McBride located within the National Institute of Allergy and Infectious Disease at the NIH.
Dr. Van Doorslaer recently joined the faculty of the School of Animal and Comparative Biomedical Sciences at the University of Arizona. His current research combines thorough evolutionary analysis with state-of-the-art molecular techniques to understand why certain DNA viruses are oncogenic. His lab is particularly interested in a subset of human oncogenic papillomaviruses.
- Ph.D. Biomedical Sciences
- Albert Einstein College of Medicine, Bronx, New York, United States
- The evolution of papillomavirus carcinogenicity”
- M.S. Biomedical Sciences
- University of Leuven, Leuven, Belgium
- The evolution of papillomavirus carcinogenicity
- B.S. Biomedical Sciences
- University of Leuven, Leuven, Belgium
- University of Arizona, Tucson, Arizona (2016 - Ongoing)
- University of Arizona, Tucson, Arizona (2016 - Ongoing)
- National Institutes of Health (2016)
- National Institutes of Health (2011 - 2016)
- Albert Einstein College of Medicine (2007 - 2011)
- University of Leuven (2004 - 2005)
- Graham Brown Cancer center, University of Louisville (2004 - 2005)
- Award for best oral presentation at 2017 DNA tumor virus meeting
- International papillomavirus Society, Summer 2017
Virology, evolution of host-pathogen interactions
Papillomaviruses (PVs) are a diverse family of dsDNA viruses infecting most, if not all, amniotes. Papillomaviruses infect cutaneous or mucosal epithelia. While most infections are self-limiting, persistent infection with specific human papillomaviruses has been shown to be the causative agent for cervical cancer. All established oncogenic HPV types belong to a single viral genus (the Alphapapillomaviridae). Of note, phylogenetically, these oncogenic HPV types cluster into a so-called high-risk (HR) clade, indicating an evolutionary relationship between these viruses. Importantly, not all HPV types within this HR clade are associated with cancer.I am intrigued by the observation that only a limited subset of human papillomaviruses is oncogenic. Throughout my studies I have used a combination of biochemical assays and computational analyses to understand why evolutionarily related viruses differ in their ability to cause cancer in humans.It is improbable that the ability to cause cancer provides papillomaviruses with an evolutionary advantage. It is likely that many of the viral functions linked to oncogenesis were evolutionarily beneficial as papillomavirus adapted to novel environmental niches on the host (e.g. external genitalia vs. cervix). Papillomaviruses have evolved to usurp the cellular machinery to complete their life-cycle. The papillomaviral lifecycle perturbs the normal differentiation cycle of the infected cell, forcing cells to divide far beyond their normal lifespan. It is feasible that the continued insult provided by replicating viruses eventually results in malignant transformation of the infected cell. However, while persistent infection is key to viral oncogenesis, many long-term persisting viruses do not cause cancer. By carefully interrogating the differences between these viruses, I believe it will be possible to elucidate which viral phenotypes are associated with oncogenic progression. The pathways targeted by these viruses may represent powerful targets for therapeutic intervention.
Directed ResearchACBS 492 (Spring 2021)
Medical+Molecular VirolIMB 533 (Spring 2021)
Medical+Molecular VirolMCB 433 (Spring 2021)
Medical+Molecular VirolMIC 433 (Spring 2021)
Prins+Molec MechanismsIMB 565 (Spring 2021)
ResearchCBIO 900 (Spring 2021)
Research ConferenceCBIO 695A (Spring 2021)
ThesisMCB 910 (Spring 2021)
ResearchCBIO 900 (Fall 2020)
Research ConferenceCBIO 695A (Fall 2020)
ThesisMCB 910 (Fall 2020)
Honors ThesisMCB 498H (Spring 2020)
Medical+Molecular VirolIMB 533 (Spring 2020)
Medical+Molecular VirolMCB 433 (Spring 2020)
Medical+Molecular VirolMIC 433 (Spring 2020)
Medical+Molecular VirolMIC 533 (Spring 2020)
Prins+Molec MechanismsIMB 565 (Spring 2020)
ResearchCBIO 900 (Spring 2020)
Research ConferenceCBIO 695A (Spring 2020)
Honors ThesisMCB 498H (Fall 2019)
Introduction to ResearchMCB 795A (Fall 2019)
Lab Research RotationGENE 795A (Fall 2019)
ResearchCBIO 900 (Fall 2019)
Research ConferenceCBIO 695A (Fall 2019)
Directed RsrchMCB 392 (Spring 2019)
Honors Independent StudyMCB 499H (Spring 2019)
Independent StudyMIC 499 (Spring 2019)
Master's ReportABS 909 (Spring 2019)
Medical+Molecular VirolIMB 533 (Spring 2019)
Medical+Molecular VirolMCB 433 (Spring 2019)
Medical+Molecular VirolMIC 433 (Spring 2019)
Medical+Molecular VirolMIC 533 (Spring 2019)
Prins+Molec MechanismsIMB 565 (Spring 2019)
ResearchCBIO 900 (Spring 2019)
Research ConferenceCBIO 695A (Spring 2019)
Honors Independent StudyMCB 499H (Fall 2018)
Internship in Applied BiosciABS 593A (Summer I 2018)
Directed ResearchBME 492 (Spring 2018)
Honors Independent StudyMCB 399H (Spring 2018)
Independent StudyACBS 599 (Spring 2018)
Prins+Molec MechanismsIMB 565 (Spring 2018)
Directed ResearchBME 492 (Fall 2017)
Independent StudyMIC 399 (Fall 2017)
Independent StudyMIC 499 (Fall 2017)
Independent StudyMIC 499 (Spring 2017)
Medical+Molecular VirolIMB 533 (Spring 2017)
Medical+Molecular VirolMCB 433 (Spring 2017)
Medical+Molecular VirolMIC 433 (Spring 2017)
Medical+Molecular VirolMIC 533 (Spring 2017)
Prins+Molec MechanismsIMB 565 (Spring 2017)
- Van Doorslaer, K. M., & Sparza, M. (2019). Evolution of DNA viruses. Encyclopedia of Virology
- Fontenele, R. S., Salywon, A. M., Majure, L. C., Cobb, I. N., Bhaskara, A., Avalos-Calleros, J. A., Argüello-Astorga, G. R., Schmidlin, K., Khalifeh, A., Smith, K., Schreck, J., Lund, M. C., Köhler, M., Wojciechowski, M. F., Hodgson, W. C., Puente-Martinez, R., Van Doorslaer, K., Kumari, S., Vernière, C., , Filloux, D., et al. (2020). A Novel Divergent Geminivirus Identified in Asymptomatic New World Cactaceae Plants. Viruses, 12(4).More infoCactaceae comprise a diverse and iconic group of flowering plants which are almost exclusively indigenous to the New World. The wide variety of growth forms found amongst the cacti have led to the trafficking of many species throughout the world as ornamentals. Despite the evolution and physiological properties of these plants having been extensively studied, little research has focused on cactus-associated viral communities. While only single-stranded RNA viruses had ever been reported in cacti, here we report the discovery of cactus-infecting single-stranded DNA viruses. These viruses all apparently belong to a single divergent species of the family and have been tentatively named Opuntia virus 1 (OpV1). A total of 79 apparently complete OpV1 genomes were recovered from 31 different cactus plants (belonging to 20 different cactus species from both the Cactoideae and Opuntioideae clades) and from nine cactus-feeding cochineal insects ( sp.) sampled in the USA and Mexico. These 79 OpV1 genomes all share > 78.4% nucleotide identity with one another and < 64.9% identity with previously characterized geminiviruses. Collectively, the OpV1 genomes display evidence of frequent recombination, with some genomes displaying up to five recombinant regions. In one case, recombinant regions span ~40% of the genome. We demonstrate that an infectious clone of an OpV1 genome can replicate in and In addition to expanding the inventory of viruses that are known to infect cacti, the OpV1 group is so distantly related to other known geminiviruses that it likely represents a new geminivirus genus. It remains to be determined whether, like its cactus hosts, its geographical distribution spans the globe.
- Jackson, R., Maarsingh, J. D., Herbst-Kralovetz, M. M., & Van Doorslaer, K. (2020). 3D Oral and Cervical Tissue Models for Studying Papillomavirus Host-Pathogen Interactions. Current protocols in microbiology, 59(1), e129.More infoHuman 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.
- King, K., & Van Doorslaer, K. M. (2018). Building (Viral) phylogenetic trees using a maximum likelihood approach. Current Protocols in Microbiology.
- O'Brien, H., Alvin, J. W., Menghani, S. V., Sanchez-Rosario, Y., Van Doorslaer, K., & Johnson, M. D. (2020). Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors. mSphere, 5(3).More infoCopper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems ( operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In (the pneumococcus), CopY is the copper repressor for the operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the box. Using this motif, we sought to determine whether genes outside the operon are also regulated by the CopY repressor, which was previously shown in We found that CopY did not bind to operators upstream of these candidate genes During this process, we found that the box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant ( ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs. Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.
- Pace, J., Youens-Clark, K., Freeman, C., Hurwitz, B., & Van Doorslaer, K. (2020). PuMA: A papillomavirus genome annotation tool. Virus evolution, 6(2), veaa068.More infoHigh-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).
- Payne, N., Kraberger, S., Fontenele, R. S., Schmidlin, K., Bergeman, M. H., Cassaigne, I., Culver, M., Varsani, A., & Van Doorslaer, K. (2020). Novel Circoviruses Detected in Feces of Sonoran Felids. Viruses, 12(9).More infoSonoran felids are threatened by drought and habitat fragmentation. Vector range expansion and anthropogenic factors such as habitat encroachment and climate change are altering viral evolutionary dynamics and exposure. However, little is known about the diversity of viruses present in these populations. Small felid populations with lower genetic diversity are likely to be most threatened with extinction by emerging diseases, as with other selective pressures, due to having less adaptive potential. We used a metagenomic approach to identify novel circoviruses, which may have a negative impact on the population viability, from confirmed bobcat () and puma () scats collected in Sonora, Mexico. Given some circoviruses are known to cause disease in their hosts, such as porcine and avian circoviruses, we took a non-invasive approach using scat to identify circoviruses in free-roaming bobcats and puma. Three circovirus genomes were determined, and, based on the current species demarcation, they represent two novel species. Phylogenetic analyses reveal that one circovirus species is more closely related to rodent associated circoviruses and the other to bat associated circoviruses, sharing highest genome-wide pairwise identity of approximately 70% and 63%, respectively. At this time, it is unknown whether these scat-derived circoviruses infect felids, their prey, or another organism that might have had contact with the scat in the environment. Further studies should be conducted to elucidate the host of these viruses and assess health impacts in felids.
- Uhlorn, B. L., Jackson, R., Li, S., Bratton, S. M., Van Doorslaer, K., & Campos, S. K. (2020). Vesicular trafficking permits evasion of cGAS/STING surveillance during initial human papillomavirus infection. PLoS pathogens, 16(11), e1009028.More infoOncogenic 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.
- Kim, W. J., Mai, A., Weyand, N. J., Rendón, M. A., Van Doorslaer, K., & So, M. (2019). Neisseria gonorrhoeae evades autophagic killing by downregulating CD46-cyt1 and remodeling lysosomes. PLoS pathogens, 15(2), e1007495.More infoThe Gram-negative human pathogen N. gonorrhoeae (Ngo) quickly attaches to epithelial cells, and large numbers of the bacteria remain on the cell surface for prolonged periods. Ngo invades cells but few viable intracellular bacteria are recovered until later stages of infection, leading to the assumption that Ngo is a weak invader. On the cell surface, Ngo quickly recruits CD46-cyt1 to the epithelial cell cortex directly beneath the bacteria and causes its cleavage by metalloproteinases and Presenilin/γSecretease; how these interactions affect the Ngo lifecycle is unknown. Here, we show Ngo induces an autophagic response in the epithelial cell through CD46-cyt1/GOPC, and this response kills early invaders. Throughout infection, the pathogen slowly downregulates CD46-cyt1 and remodeling of lysosomes, another key autophagy component, and these activities ultimately promote intracellular survival. We present a model on the dynamics of Ngo infection and describe how this dual interference with the autophagic pathway allows late invaders to survive within the cell.
- O\textquoterightBrien, H., Alvin, J. W., Menghani, S. V., Van, D. K., & Johnson, M. (2019). Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY. bioRxiv.
- Pace, J., Youens-Clark, K. .., Freeman, C., Hurwitz, B., & Van Doorslaer, K. (2019). PuMA: a papillomavirus genome annotation tool. bioRxiv.
- Van Arsdale, A., Patterson, N. E., Maggi, E. C., Agoni, L., Van Doorslaer, K., Harmon, B., Nevadunsky, N., Kuo, D. Y., Einstein, M. H., Lenz, J., & Montagna, C. (2020). Insertional oncogenesis by HPV70 revealed by multiple genomic analyses in a clinically HPV-negative cervical cancer. Genes, chromosomes & cancer, 59(2), 84-95.More infoCervical 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.
- Van Doorslaer, K., & Dillner, J. (2019). The Launch of an International Animal Papillomavirus Reference Center. Viruses, 11(1).More infoThe is a family of DNA viruses [...].
- King, K. M., & Van Doorslaer, K. (2018). Building (Viral) Phylogenetic Trees Using a Maximum Likelihood Approach. Current protocols in microbiology, 51(1), e63.More infoPhylogenetic analyses allow for inferring a hypothesis about the evolutionary history of a set of homologous molecular sequences. This hypothesis can be used as the basis for further molecular and computational studies. In this unit, we offer one specific method to construct a Maximum Likelihood phylogenetic tree. We outline how to identify homologous sequences and construct a multiple sequence alignment. Following alignment, sequences are screened for potentially confounding factors such as recombination and genetic saturation. Finally, a Maximum Likelihood phylogenetic tree can be constructed implementing a rigorously tested model of evolution. The workflow outlined in this unit provides sufficient background for inferring a robust phylogenetic tree starting from a particular gene of interest. © 2018 by John Wiley & Sons, Inc.
- Pastrana, D. V., Peretti, A., Welch, N. L., Borgogna, C., Olivero, C., Badolato, R., Notarangelo, L. D., Gariglio, M., FitzGerald, P. C., McIntosh, C. E., Reeves, J., Starrett, G. J., Bliskovsky, V., Velez, D., Brownell, I., Yarchoan, R., Wyvill, K. M., Uldrick, T. S., Maldarelli, F., , Lisco, A., et al. (2018). Metagenomic Discovery of 83 New Human Papillomavirus Types in Patients with Immunodeficiency. mSphere, 3(6).More infoSeveral 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 were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus 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. Although some members of the viral family cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed () 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 HPV types and suggest that WHIM syndrome patients are uniquely susceptible to HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual HPV skin warts observed in WHIM patients.
- Smeele, Z. E., Burns, J. M., Van Doorslaer, K. M., Fontenele, R. S., Waits, K., Stainton, D., Shero, M. R., Beltran, R. S., Kirkham, A. L., Berngartt, R., Kraberger, S., & Varsani, A. (2018). Diverse papillomaviruses identified in Weddell seals. Journal of General Virology.
- Van Doorslaer, K., Chen, Z., Bernard, H. U., Chan, P. K., DeSalle, R., Dillner, J., Forslund, O., Haga, T., McBride, A. A., Villa, L. L., Burk, R. D., & Ictv Report Consortium, . (2018). ICTV Virus Taxonomy Profile: Papillomaviridae. The Journal of general virology, 99(8), 989-990.More infoThe 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.
- Van Doorslaer, K., Kraberger, S., Austin, C., Farkas, K., Bergeman, M., Paunil, E., Davison, W., & Varsani, A. (2018). Fish polyomaviruses belong to two distinct evolutionary lineages. The Journal of general virology, 99(4), 567-573.More infoThe 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.
- Van Doorslaer, K., Chen, D., Chapman, S., Khan, J., & McBride, A. A. (2017). Persistence of an Oncogenic Papillomavirus Genome Requires cis Elements from the Viral Transcriptional Enhancer. mBio, 8(6).More infoHuman papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication.IMPORTANCE Human papillomaviruses infect cutaneous and mucosal epithelial cells of the host, and this results in very-long-lived, persistent infection. The viral genomes are small, circular, double-stranded DNA molecules that replicate extrachromosomally in concert with cellular DNA. This replication strategy requires that the virus has a robust mechanism to partition and retain the viral genomes in dividing cells. This has been difficult to study, because viral transcription, replication, and partitioning are regulated by the same viral proteins and involve overlapping elements in the viral genome. We developed a complementation assay that allows us to separate these functions and define the elements required for long-term replication and stable maintenance replication of the HPV genome. This has important implications, as disruption of viral maintenance replication can eliminate viral genomes from infected cells, thus curing persistent HPV infection.
- Van Doorslaer, K., Li, Z., Xirasagar, S., Maes, P., Kaminsky, D., Liou, D., Sun, Q., Kaur, R., Huyen, Y., & McBride, A. A. (2017). The Papillomavirus Episteme: a major update to the papillomavirus sequence database. Nucleic acids research, 45(D1), D499-D506.More infoThe 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/.
- Van Doorslaer, K., Ruoppolo, V., Schmidt, A., Lescroël, A., Jongsomjit, D., Elrod, M., Kraberger, S., Stainton, D., Dugger, K. M., Ballard, G., Ainley, D. G., & Varsani, A. (2017). Unique genome organization of non-mammalian papillomaviruses provides insights into the evolution of viral early proteins. Virus evolution, 3(2), vex027.More infoThe family Papillomaviridae contains more than 320 papillomavirus types, with most having been identified as infecting skin and mucosal epithelium in mammalian hosts. To date, only nine non-mammalian papillomaviruses have been described from birds (n = 5), a fish (n = 1), a snake (n = 1), and turtles (n = 2). The identification of papillomaviruses in sauropsids and a sparid fish suggests that early ancestors of papillomaviruses were already infecting the earliest Euteleostomi. The Euteleostomi clade includes more than 90 per cent of the living vertebrate species, and progeny virus could have been passed on to all members of this clade, inhabiting virtually every habitat on the planet. As part of this study, we isolated a novel papillomavirus from a 16-year-old female Adélie penguin (Pygoscelis adeliae) from Cape Crozier, Ross Island (Antarctica). The new papillomavirus shares ∼64 per cent genome-wide identity to a previously described Adélie penguin papillomavirus. Phylogenetic analyses show that the non-mammalian viruses (expect the python, Morelia spilota, associated papillomavirus) cluster near the base of the papillomavirus evolutionary tree. A papillomavirus isolated from an avian host (Northern fulmar; Fulmarus glacialis), like the two turtle papillomaviruses, lacks a putative E9 protein that is found in all other avian papillomaviruses. Furthermore, the Northern fulmar papillomavirus has an E7 more similar to the mammalian viruses than the other avian papillomaviruses. Typical E6 proteins of mammalian papillomaviruses have two Zinc finger motifs, whereas the sauropsid papillomaviruses only have one such motif. Furthermore, this motif is absent in the fish papillomavirus. Thus, it is highly likely that the most recent common ancestor of the mammalian and sauropsid papillomaviruses had a single motif E6. It appears that a motif duplication resulted in mammalian papillomaviruses having a double Zinc finger motif in E6. We estimated the divergence time between Northern fulmar-associated papillomavirus and the other Sauropsid papillomaviruses be to around 250 million years ago, during the Paleozoic-Mesozoic transition and our analysis dates the root of the papillomavirus tree between 400 and 600 million years ago. Our analysis shows evidence for niche adaptation and that these non-mammalian viruses have highly divergent E6 and E7 proteins, providing insights into the evolution of the early viral (onco-)proteins.
- Warren, C. J., Westrich, J. A., Doorslaer, K. V., & Pyeon, D. (2017). Roles of APOBEC3A and APOBEC3B in Human Papillomavirus Infection and Disease Progression. Viruses, 9(8).More infoThe apolipoprotein B messenger RNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) family of cytidine deaminases plays an important role in the innate immune response to viral infections by editing viral genomes. However, the cytidine deaminase activity of APOBEC3 enzymes also induces somatic mutations in host genomes, which may drive cancer progression. Recent studies of human papillomavirus (HPV) infection and disease outcome highlight this duality. HPV infection is potently inhibited by one family member, APOBEC3A. Expression of APOBEC3A and APOBEC3B is highly elevated by the HPV oncoproteins E6 and E7 during persistent virus infection and disease progression. Furthermore, there is a high prevalence of APOBEC3A and APOBEC3B mutation signatures in HPV-associated cancers. These findings suggest that induction of an APOBEC3-mediated antiviral response during HPV infection may inadvertently contribute to cancer mutagenesis and virus evolution. Here, we discuss current understanding of APOBEC3A and APOBEC3B biology in HPV restriction, evolution, and associated cancer mutagenesis.
- Buck, C. B., Van Doorslaer, K., Peretti, A., Geoghegan, E. M., Tisza, M. J., An, P., Katz, J. P., Pipas, J. M., McBride, A. A., Camus, A. C., McDermott, A. J., Dill, J. A., Delwart, E., Ng, T. F., Farkas, K., Austin, C., Kraberger, S., Davison, W., Pastrana, D. V., & Varsani, A. (2016). The Ancient Evolutionary History of Polyomaviruses. PLoS pathogens, 12(4), e1005574.More infoPolyomaviruses 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.
- Harrison, J. S., Jacobs, T. M., Houlihan, K., Van Doorslaer, K., & Kuhlman, B. (2016). UbSRD: The Ubiquitin Structural Relational Database. Journal of molecular biology, 428(4), 679-687.More infoThe structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein-protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein-protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd.
- Van Doorslaer, K. M., Porter, S., McKinney, C., Stepp, W. H., & McBride, A. A. (2016). Novel recombinant papillomavirus genomes expressing selectable genes.. Scientific Reports, 6.More infoPapillomaviruses 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.
- Van Doorslaer, K., & McBride, A. A. (2016). Molecular archeological evidence in support of the repeated loss of a papillomavirus gene. Scientific Reports, 6.More infoIt 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.
- Van Doorslaer, K., & McBride, A. A. (2016). Molecular archeological evidence in support of the repeated loss of a papillomavirus gene. Scientific reports, 6, 33028.More infoIt 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.
- Van Doorslaer, K., Chen, Z., & McBride, A. A. (2016). Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples. Current Protocols in Microbiology, 14B--9.More infoPathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting viru
- Van Doorslaer, K., Chen, Z., & McBride, A. A. (2016). Detection and Genotyping of Human Papillomaviruses from Archival Formalin-Fixed Tissue Samples. Current protocols in microbiology, 43, 14B.9.1-14B.9.20.More infoPathology departments routinely process and store formalin-fixed, paraffin-embedded (FFPE) tissue samples for clinical diagnosis. These collections often contain decades' worth of samples and represent a treasure trove of specimens that can be analyzed for retrospective epidemiological studies, diagnostics, and pathogen discovery. Accurate amplification and sequencing of DNA from these samples is critical for the usability of these FFPE samples. Here we present a collection of protocols that describe extraction of DNA from FFPE tissues, PCR amplification of human papillomavirus DNA, and subsequent genotyping of the infecting virus. © 2016 by John Wiley & Sons, Inc.
- Van Doorslaer, K., Li, Z., Xirasagar, S., Maes, P., Kaminsky, D., Liou, D., Sun, Q., Kaur, R., Huyen, Y., & McBride, A. A. (2016). The Papillomavirus Episteme: a major update to the papillomavirus sequence database. Nucleic Acids Research, gkw879.More infoThe 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/.
- Van Doorslaer, K., Porter, S., McKinney, C., Stepp, W. H., & McBride, A. A. (2016). Novel recombinant papillomavirus genomes expressing selectable genes. Scientific reports, 6, 37782.More infoPapillomaviruses 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.
- Harrison, J. S., Jacobs, T. M., Houlihan, K., Van Doorslaer, K., & Kuhlman, B. (2015). Data in support of UbSRD: The Ubiquitin Structural Relational Database. Data in brief, 5, 605-15.More infoThis article provides information to support the database article titled "UbSRD: The Ubiquitin Structural Relational Database" (Harrison et al., 2015)  . The ubiquitin-like homology fold (UBL) represents a large family that encompasses both post-translational modifications, like ubiquitin (UBQ) and SUMO, and functional domains on many biologically important proteins like Parkin, UHRF1 (ubiquitin-like with PDB and RING finger domains-1), and Usp7 (ubiquitin-specific protease-7) (Zhang et al., 2015; Rothbart et al., 2013; Burroughs et al., 2012; Wauer et al., 2015) , , , . The UBL domain can participate in several unique protein-protein interactions (PPI) since protein adducts can be attached to and removed from amino groups of lysine side chains and the N-terminus of proteins. Given the biological significance of UBL domains, many have been characterized with high-resolution techniques, and for UBQ and SUMO, many protein complexes have been characterized. We identified all the UBL domains in the PDB and created a relational database called UbSRD (Ubiquitin Structural Relational Database) by using structural analysis tools in the Rosetta (Leaver et al., 2013; O'Meara et al., 2015; Leaver-fay et al., 2011) , , , . Querying UbSRD permitted us to report many quantitative properties of UBQ and SUMO recognition at different types interfaces (noncovalent: NC, conjugated: CJ, and deubiquitanse: DB). In this data article, we report the average number of non-UBL neighbors, secondary structure of interacting motifs, and the type of inter-molecular hydrogen bonds for each residue of UBQ and SUMO. Additionally, we used PROMALS3D to generate a multiple sequence alignment used to construct a phylogram for the entire set of UBLs (Pei and Grishin, 2014) . The data described here will be generally useful to scientists studying the molecular basis for recognition of UBQ or SUMO.
- Jang, M. K., Anderson, D. E., van Doorslaer, K., & McBride, A. A. (2015). A proteomic approach to discover and compare interacting partners of papillomavirus E2 proteins from diverse phylogenetic groups. Proteomics, 15(12), 2038-50.More infoPapillomaviruses 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.
- Van Doorslaer, K., DeSalle, R., Einstein, M. H., & Burk, R. D. (2015). Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche. PLoS pathogens, 11(6), e1004980.More infoIn 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.
- Warren, C. J., Van Doorslaer, K., Pandey, A., Espinosa, J. M., & Pyeon, D. (2015). Role of the host restriction factor APOBEC3 on papillomavirus evolution. Virus evolution, 1(1).More infoMore than 270 different types of papillomaviruses have been discovered in a wide array of animal species. Despite the great diversity of papillomaviruses, little is known about the evolutionary processes that drive host tropism and the emergence of oncogenic genotypes. Although host defense mechanisms have evolved to interfere with various aspects of a virus life cycle, viruses have also coevolved copious strategies to avoid host antiviral restriction. Our and other studies have shown that the cytidine deaminase APOBEC3 family members edit HPV genomes and restrict virus infectivity. Thus, we hypothesized that host restriction by APOBEC3 served as selective pressure during papillomavirus evolution. To test this hypothesis, we analyzed the relative abundance of all dinucleotide sequences in full-length genomes of 274 papillomavirus types documented in the Papillomavirus Episteme database (PaVE). Here, we report that TC dinucleotides, the preferred target sequence of several human APOBEC3 proteins (hA3A, hA3B, hA3F, and hA3H), are highly depleted in papillomavirus genomes. Given that HPV infection is highly tissue-specific, the expression levels of APOBEC3 family members were analyzed. The basal expression levels of all APOBEC3 isoforms, excluding hA3B, are significantly higher in mucosal skin compared with cutaneous skin. Interestingly, we reveal that Alphapapillomaviruses (alpha-PVs), a majority of which infects anogenital mucosa, display the most dramatic reduction in TC dinucleotide content. Computer modeling and reconstruction of ancestral alpha-PV genomes suggest that TC depletion occurred after the alpha-PVs diverged from their most recent common ancestor. In addition, we found that TC depletion in alpha-PVs is greatly affected by protein coding potential. Taken together, our results suggest that PVs replicating in tissues with high APOBEC3 levels may have evolved to evade restriction by selecting for variants that contain reduced APOBEC3 target sites in their genomes.
- Harari, A., Wood, C. E., Van Doorslaer, K., Chen, Z., Domaingue, M. C., Elmore, D., Koenig, P., Wagner, J. D., Jennings, R. N., & Burk, R. D. (2013). Condylomatous genital lesions in cynomolgus macaques from Mauritius. Toxicologic pathology, 41(6), 893-901.More infoGenital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction-based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.
- Van Doorslaer, K. (2013). Evolution of the papillomaviridae. Virology, 445(1-2), 11-20.More infoViruses belonging to the Papillomaviridae family have been isolated from a variety of mammals, birds and non-avian reptiles. It is likely that most, if not all, amniotes carry a broad array of viral types. To date, the complete genomic sequence of more than 240 distinct viral types has been characterized at the nucleotide level. The analysis of this sequence information has begun to shed light on the evolutionary history of this important virus family. The available data suggests that many different evolutionary mechanisms have influenced the papillomavirus phylogenetic tree. Increasing evidence supports that the ancestral papillomavirus initially specialized to infect different ecological niches on the host. This episode of niche sorting was followed by extensive episodes of co-speciation with the host. This review attempts to summarize our current understanding of the papillomavirus evolution.
- Van Doorslaer, K., Tan, Q., Xirasagar, S., Bandaru, S., Gopalan, V., Mohamoud, Y., Huyen, Y., & McBride, A. A. (2013). The Papillomavirus Episteme: a central resource for papillomavirus sequence data and analysis. Nucleic acids research, 41(Database issue), D571-8.More infoThe 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.
- Machado, F. S., Rodriguez, N. E., Adesse, D., Garzoni, L. R., Esper, L., Lisanti, M. P., Burk, R. D., Albanese, C., Van Doorslaer, K., Weiss, L. M., Nagajyothi, F., Nosanchuk, J. D., Wilson, M. E., & Tanowitz, H. B. (2012). Recent developments in the interactions between caveolin and pathogens. Advances in experimental medicine and biology, 729, 65-82.More infoThe role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.
- Van Doorslaer, K., & Burk, R. D. (2012). Association between hTERT activation by HPV E6 proteins and oncogenic risk. Virology, 433(1), 216-9.More infoExpression of activated telomerase and subversion of the p16/pRb pathway is sufficient and essential for the in vitro immortalization of primary keratinocytes. Most cancers-including cervical carcinoma-over-express hTERT, the catalytic domain of the telomerase complex. Only a limited set of viruses within the Alphapapillomavirus genus are oncogenic. The viral functions responsible for this distinction are not well understood. The human papillomavirus type 16 E6 protein activates the hTERT promoter. We used a luciferase-based assay to test the ability of 29 viral types, representing all current species within the Alphapapillomavirus genus, to activate the hTERT promoter. We show that oncogenic types specifically activate the hTERT promoter, while non-oncogenic types do not. Statistical analysis supports the notion that activation of the hTERT promoter is uniquely associated with oncogenic types, independent of evolutionary relationships. This finding begins to shed light on the viral phenotypes correlated with oncogenic potential.
- Smith, B., Chen, Z., Reimers, L., van Doorslaer, K., Schiffman, M., Desalle, R., Herrero, R., Yu, K., Wacholder, S., Wang, T., & Burk, R. D. (2011). Sequence imputation of HPV16 genomes for genetic association studies. PloS one, 6(6), e21375.More infoHuman Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity.
- Van Doorslaer, K., Bernard, H. U., Chen, Z., de Villiers, E. M., zur Hausen, H., & Burk, R. D. (2011). Papillomaviruses: evolution, Linnaean taxonomy and current nomenclature. Trends in microbiology, 19(2), 49-50; author reply 50-1.
- Wood, C. E., Tannehill-Gregg, S. H., Chen, Z., Doorslaer, K. v., Nelson, D. R., Cline, J. M., & Burk, R. D. (2011). Novel betapapillomavirus associated with hand and foot papillomas in a cynomolgus macaque. Veterinary pathology, 48(3), 731-6.More infoBetapapillomavirus is a genus of papillomaviruses (PVs) commonly found in human skin and associated with both benign and malignant skin lesions. Only 2 previous beta-PVs have been fully characterized in nonhuman species. This report describes a novel beta-PV, named Macaca fascicularis PV type 2 (MfPV2), isolated from exophytic skin papillomas on the hands and feet of a 2-year-old male cynomolgus monkey (M. fascicularis). On histology the papillomas were composed of diffusely thickened epidermis with superficial foci of cytomegaly, cytoplasmic pallor, marginalized chromatin, and rare eosinophilic intranuclear inclusion bodies. Positive immunostaining for p16 and the proliferation marker Ki67 was present multifocally within affected epidermis, most prominently within basal-type cells. Complete sequence identity (100%) was noted between PV genomes fully sequenced from hand and foot lesions. The MfPV2 genome was 7632 base pairs in length and included putative open reading frames (ORFs) for E1, E2, E4, E6, E7, L1, and L2 genes, similar to other PVs. The closest relatives to MfPV2 based on the L1 ORF sequence were all beta-PVs. These included human PV (HPV) 9, HPV115, HPV76, HPV75, and MfPV1 (60-70% pairwise identity for all), the latter of which was also isolated from hand and foot papillomas in a cynomolgus macaque. Phylogenetic analysis placed MfPV2 in a new species group (beta-6), distinct from HPVs (beta-1 to beta-5) and MfPV1 (beta-1). These findings characterize a new nonhuman beta-PV and provide additional support for the idea that tissue tropism among ancestral primate PVs developed prior to divergence of certain Old World primate lineages.
- Bernard, H. U., Burk, R. D., Chen, Z., van Doorslaer, K., zur Hausen, H., & de Villiers, E. M. (2010). Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology, 401(1), 70-9.More infoWe present an expansion of the classification of the family Papillomaviridae, which now contains 29 genera formed by 189 papillomavirus (PV) types isolated from humans (120 types), non-human mammals, birds and reptiles (64, 3 and 2 types, respectively). To accommodate the number of PV genera exceeding the Greek alphabet, the prefix "dyo" is used, continuing after the Omega-PVs with Dyodelta-PVs. The current set of human PVs is contained within five genera, whereas mammalian, avian and reptile PVs are contained within 20, 3 and 1 genera, respectively. We propose standardizations to the names of a number of animal PVs. As prerequisite for a coherent nomenclature of animal PVs, we propose founding a reference center for animal PVs. We discuss that based on emerging species concepts derived from genome sequences, PV types could be promoted to the taxonomic level of species, but we do not recommend implementing this change at the current time.
- Connolly, E. C., Van Doorslaer, K., Rogler, L. E., & Rogler, C. E. (2010). Overexpression of miR-21 promotes an in vitro metastatic phenotype by targeting the tumor suppressor RHOB. Molecular cancer research : MCR, 8(5), 691-700.More infoMetastasis is a multistep process that involves the deregulation of oncogenes and tumor suppressors beyond changes required for primary tumor formation. RHOB is known to have tumor suppressor activity, and its knockdown is associated with more aggressive tumors as well as changes in cell shape, migration, and adhesion. This study shows that oncogenic microRNA, miR-21, represses RHOB expression by directly targeting the 3' untranslated region. Loss of miR-21 is associated with an elevation of RHOB in hepatocellular carcinoma cell lines Huh-7 and HepG2 and in the metastatic breast cancer cell line MDA-MB-231. Using in vitro models of distinct stages of metastasis, we showed that loss of miR-21 also causes a reduction in migration, invasion, and cell elongation. The reduction in migration and cell elongation can be mimicked by overexpression of RHOB. Furthermore, changes in miR-21 expression lead to alterations in matrix metalloproteinase-9 activity. Therefore, we conclude that miR-21 promotes multiple components of the metastatic phenotype in vitro by regulating several important tumor suppressors, including RHOB.
- Van Doorslaer, K., & Burk, R. D. (2010). Evolution of human papillomavirus carcinogenicity. Advances in virus research, 77, 41-62.More infoMembers of the Alphapapillomavirus genus are the causative agent for virtually all cases of cervical cancer. However, strains (commonly referred to as types) within this genus span the entire range of pathogenicity from highly carcinogenic (e.g., HPV16, odds ratio = 281.9, responsible for 50% of all cervical cancers), moderately carcinogenic (e.g., HPV31) to not carcinogenic (e.g., HPV71). The persistent expression of the viral oncoproteins (E6 and E7) from HPV16 has been shown to be necessary and sufficient to transform primary human keratinocytes in vitro. A plethora of functions have been described for both oncoproteins, and through functional comparisons between HPV16 and HPV6, a subset of these functions have been suggested to be oncogenic. However, extrapolating functional differences from these comparisons is unlikely to tease apart the fine details. In this review, we argue that a thorough understanding of the molecular mechanisms differentiating oncogenic from nononcogenic types should be obtained by performing functional assays in an evolutionary and epidemiological framework. We continue by interpreting some recent results using this paradigm and end by suggesting directions for future inquiries.
- Van Doorslaer, K., Reimers, L. L., Studentsov, Y. Y., Einstein, M. H., & Burk, R. D. (2010). Serological response to an HPV16 E7 based therapeutic vaccine in women with high-grade cervical dysplasia. Gynecologic oncology, 116(2), 208-12.More infoInfection with oncogenic human papillomaviruses has been linked to the development of cervical neoplasia and cancer. The exclusive expression of E7, a viral oncogene, in infected cells makes this protein an ideal target for immunotherapy. We recently reported on the results of a trial in women with cervical carcinoma-in-situ using HspE7, a protein vaccine consisting of full length HPV16 E7 linked to a heat shock protein from M. bovis. The stimulating effects of HspE7 on specific cytotoxic T lymphocytes have been demonstrated in vitro and in (pre-)clinical trials. The induction of a B-cell response by HspE7 and its association with clinical outcome is unknown, and is the purpose of this study.
- Van Doorslaer, K. M. (2019, September). Papillomavirus evolution and immunity. LSU invited seminar. LSU: LSU.
- Van Doorslaer, K. M. (2019, july). Papillomaviruses in cancer. DNA tumor virus meeting.
- Van Doorslaer, K. M. (2018, August). Fish Associated Viruses Provide Insights into the Evolution of Viral Oncogenes. DNA Tumor Virus Meeting. Madison, WI.
- Van Doorslaer, K. M. (2018, August). Viral transcriptional enhancer cis elements are necessary for persistent HPV18 replication. DNA Tumor Virus Meeting. Madison, WI.
- Van Doorslaer, K. M. (2018, July). Fish Associated Viruses Provide Insights into the Evolution of Viral Oncogenes. American Society for Virology Annual Meeting.
- Van Doorslaer, K. M. (2018, July). The Papillomavirus Episteme. American Society for Virology Annual Meeting. College Park, MD.
- Van Doorslaer, K. M. (2018, July). Viral transcriptional enhancer cis elements are necessary for persistent HPV18 replication. American Society for Virology Annual Meeting. College Park, MD.
- Van Doorslaer, K. M. (2018, June). Fish associated viruses provide insights into the evolution of viral oncogenes. Virus Genomics and evolution. Cambridge, UK.
- Van Doorslaer, K. M. (2018, October). Non-mammalian viruses provide insights into the evolution of viral oncogenes. 32nd International Papillomavirus Conference. Sydney, Aus.
- Van Doorslaer, K. M. (2018, October). Proposing a new scheme for papillomavirus classification. 32nd International Papillomavirus Conference. Sydney Australia.
- Van Doorslaer, K. M. (2018, October). PuMA: Towards an automated annotation tool. 32nd International Papillomavirus Conference. Sydney, Australia.
- Van Doorslaer, K. M. (2017, April). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. 56th Annual Meeting of the Arizona/Southern Nevada ASM Branch. Tucson, AZ: ASM local Branch.
- Van Doorslaer, K. M. (2017, April). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. 56th Annual meeting of the Arizona/Southern Nevada ASM Branch. Tucson, AZ, USA: Arizona/Southern Nevada ASM Branch.
- Van Doorslaer, K. M. (2017, April). Papillomaviruses as tools to study evolution. MicroLunch, Cross Campus Microbiology Focus Group, University of Arizona. Tucson, AZ, USA: MicroLunch, Cross Campus Microbiology Focus Group, University of Arizona.
- Van Doorslaer, K. M. (2017, Feb). Papillomaviruses; replication and cancer. MARC Biomedical Research Colloquium. Tucson, AZ, USA: MARC Biomedical Research program, University of Arizona.
- Van Doorslaer, K. M. (2017, Feb). Papillomaviruses; replication and cancer. UA Microbiolgy Club meeting, University of Arizona. Tucson, AZ, USA: UA Microbiolgy Club, University of Arizona.
- Van Doorslaer, K. M. (2017, July). Genomic plasticity near the root of the papillomavirus evolutionary tree. International DNA tumor virus meeting. Birmingham, UK: International DNA tumor virus meeting.
- Van Doorslaer, K. M. (2017, March). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. CIDD seminar (Penn State University). Hershey, PA, USA: Penn State University.
- Van Doorslaer, K. M. (2017, March). Use of comparative virology to understand papillomavirus oncogenicity. Frontiers in Immunobiology & Immunopathogenesis Symposium 2017. Tucson, AZ, USA: Dept. of Immunobiology, University of Arizona.
- Van Doorslaer, K. M. (2017, November). Evolutionary clues to papillomavirus oncogenicity. UA-ASU Virology Symposium. Phoenix, AZ, USA: University of Arizona and Arizona State University.
- Van Doorslaer, K. M. (2017, November). Papillomaviruses; challenges for cancer control. Arizona Wellbeing Commons - Cancer Prevention, Detection, Management and Treatment Division Meeting. Phoenix, AZ, USA: Arizona Wellbeing Commons.
- Van Doorslaer, K. M. (2016, Nov). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. Cancer Biology Program Seminar, University of Arizona Cancer Center. Tucson, AZ, USA: Arizona Cancer Center.
- Van Doorslaer, K. M. (2016, Nov). In vitro tools to study the papillomavirus lifecycle. Head and Neck Cancer collaborative seminar, University of Arizona Cancer Center. Tucson, AZ, USA: Arizona Cancer Center.
- Van Doorslaer, K. M. (2016, Oct). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. Inaugural CME collaborative seminar Arizona State University. Phoenix, AZ, USA: Arizona State University.
- Van Doorslaer, K. M. (2016, Oct). Comparative Genomics as a Tool to Understand Papillomavirus Evolution and Disease. MicroLunch, Cross Campus Microbiology Focus Group, University of Arizona. Tucson, AZ, USA: Arizona Cancer Center.
- Jackson, J., Jandova, J., King, K., & Van Doorslaer, K. M. (2017, Nov). Detecting papillomavirus recombination in vitro. ABRCMS. Phoenix, AZ: ASM.