Towards a Multi-Level and a Multi-Disciplinary Approach to DNA Oncovirus Virulence Samuel Alizon, Ignacio Bravo, Paul Farrell, Sally Roberts

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Towards a multi-level and a multi-disciplinary approach to DNA oncovirus virulence Samuel Alizon, Ignacio Bravo, Paul Farrell, Sally Roberts

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Samuel Alizon, Ignacio Bravo, Paul Farrell, Sally Roberts. Towards a multi-level and a multi- disciplinary approach to DNA oncovirus virulence. Philosophical Transactions of the Royal Society B: Biological Sciences, Royal Society, The, 2019, Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses, 374 (1773), pp.20190041. 10.1098/rstb.2019.0041. hal-02114622

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Samuel Alizon1,∗, Ignacio G. Bravo1,∗, Paul J. Farrell2, Sally Roberts3

1 Laboratoire MIVEGEC (UMR CNRS 5290, IRD 224, UM), Montpellier, France 2 Section of Virology, Imperial College, London, UK 3 Institute of Cancer and Genomic Sciences, University of Birmingham, UK ∗ Corresponding authors: [email protected] and [email protected]

Abstract One out of 10 cancers is estimated to arise from infections by a handful of oncogenic viruses. These infectious cancers constitute an opportunity for primary prevention through immunization against the viral infection, for early screening through molecular detection of the infectious agent, and potentially for speciﬁc treatments, by targeting the virus as a marker of cancer cells. Accomplishing these objectives will require a detailed understanding of the natural history of infections, the mechanisms by which the viruses contribute to disease, the mutual adaptation of viruses and hosts, and the possible viral evolution in the absence and in the presence of the public health interventions conceived to target them. This issue showcases the current developments in experimental tissue-like and animal systems, mathematical models and evolutionary approaches to understand DNA oncoviruses. Our global aim is to provide proximate explanations to the present-day interface and interactions between virus and host, as well as ultimate explanations about the adaptive value of these interactions and about the evolutionary pathways that have led to the current malignant phenotype of oncoviral infections. Cite as Alizon S, Bravo IG, Farrell PJ, Roberts S (2019) Towards a multi-level and a multi-disciplinary approach to DNA oncovirus virulence. Phil Trans R Soc Lond B. 374(1773):20190041, DOI: 10.1098/rstb.2019.0041

1 DNA oncoviruses: low infec- public health concern. This medical importance tion virulence and high disease has resulted in a substantial body of fundamental research leading to the discovery of the different burden viruses, to a detailed description of the virocellular interactions and to their identification as oncogenic Certain infections can cause cancers in humans, biological agents to humans (we will hereafter refer and indeed between 15% and 20% of all cancers to “virocell” as the metabolically active vegetative in humans have a direct infectious origin. Some stage in the viral life cycle [18]). In a few cases, of the most oncogenic biological agents to humans applied research has led to development of diagnos- are a handful of DNA viruses. In 2012, cancers tic, therapeutic and even prophylactic approaches caused by these DNA viruses represented 56% of for these viral infections. Oncogenic human papil- the 2.2 million new cases of cancers attributed to lomaviruses (HPVs) and anogenital cancers serve infectious agents. This proportion was even higher as paradigmatic example of this successful story, (> 80%) in regions of the world with low Human from the early identification of cervical cancer epi- Development Index [12, 28]. Thus, cancers caused demiology to match that of sexually transmitted by DNA oncoviruses impose a substantial disease infections, to the development of prophylactic vac- burden that becomes greater in developing coun- cines against the most oncogenic HPVs [13]. Yet, tries. The human morbidity linked to the asso- for the vast majority of oncogenic DNA viruses we ciated diseases makes DNA oncoviruses a major

1 still know relatively little about how these viruses work and refer to them as co-factors. Third, the enter, manipulate and take over the infected cell, notion of “environment” for a viral infection must how they interact with the host’s immune system be understood as a Russian-doll hierarchical inte- during the acute and the chronic phases of the in- gration across levels, from the virocell to the ecol- fection, how they maintain intra-host and popula- ogy: cell type diversity may display different per- tion diversity, and how the viral populations may missivity to the infection and different potential respond to the public health interventions imple- for malignisation (in the case of HPV, see [20]); tis- mented to tackle these infections. Ultimately, for sue diversity may differentially foster malignancy all oncogenic DNA viruses, we still ignore why [22]; organ diversity may introduce within-patient these highly prevalent infections are largely asymp- spatial structuring of the viral population [4]; indi- tomatic, yet in a small fraction of the cases they vidual behaviour may strongly impact viral circu- can progress to malignancies. lation; biological and physicochemical agents may The socioeconomic burden caused by DNA on- modify the host-pathogen interaction; and human covirus infections does not arise from a high mor- population structure will undoubtedly pattern vi- tality rate among the infected persons, but rather ral population structure. In summary, to under- from the extremely high prevalence of these infec- stand DNA oncovirus virulence, it is necessary to tions. For most DNA oncoviruses, the accumu- adopt a multi-scale approach and bridge the cellu- lated probability for a human to have ever been in- lar and the population levels. fected during lifetime approaches one, i.e. at some point in their lives virtually all humans will have 2 The challenge of defining a been infected by some oncogenic papillomavirus, polyomavirus or herpesvirus. On the other hand, common playground: from most of these infections are asymptomatic or clin- molecules to ecology, from ically irrelevant, and only a small fraction leads research protocols to clinical eventually to a cancer. Taking the best-known case of oncogenic HPVs, the prevalence of cervical infec- guidelines and public health in- tion in women below 25 years of age is 29.7%, while terventions the world average cumulative risk at 75 years old for a woman to develop cervical cancer is 1.36% The questions raised by oncogenic DNA viruses de- (data extracted from the HPV information cen- mand responses from the microscopic, molecular tre, https://www.hpvcentre.net [7]). This sharp dimension to the macroscopic, ecological dimen- contrast between high prevalence and low morbid- sion. The need to integrate so many levels for un- ity generates a situation that may appear confus- derstanding the multidimensional problem of in- ing to the general public, since at the individual fectious cancers is often hindered by the lack of a level the risk of developing cancer following an in- shared scientific culture: the burden of proof is dif- fection is low, but at the population level the cu- ferent in experimental and modelling approaches; mulative burden is high. This low virulence per in- the number of degrees of freedom, and thus the fection is a key feature of the problem. First, as for strength of any inferred association, is different in any rare event, the potential role of stochasticity is in vitro techniques and in epidemiology; a deeper high. This has been exemplified in the case of HPV understanding of the underlying evolutionary pro- infections to explain why some lesions regress nat- cesses may not necessarily bring along an imme- urally and other do not [31], or in the case of her- diate impact in cancer treatment; difficult clini- pesvirus infections to explain the role of asymmet- cal decisions must often be taken using partial in- ric segregation of viral genomes during cell division formation and resorting to phenomenon-directed [10]. Second, virulence is always the result of the knowledge, without the option to wait for a future interaction between the virus genotype, the host better understanding of the molecular basis of the genotype and their “environment”. This tripartite disease. The framework summarised by Nikolaas interplay is referred to as G*G*E interactions in Tinbergen [33] to assemble research approaches ecology and evolution, while epidemiologists often and comprehension levels is a powerful intellectual pinpoint individual edges in this interaction net- tool to conceptualise, build and share knowledge

2 across fields (Table 1). It may help us succeed in infections while others are tissue-restricted, but building a common understanding and creating a in both cases the local spatial structure strongly shared perspective for scientists with largely diver- shapes their infection fitness. Also, in most cases gent conceptions of science. DNA oncoviruses establish very long relationships From a practical side, bringing together fields with their hosts, leading to chronic infections punc- that study the same entity from different perspec- tuated by episodes of reactivation [35]. The vi- tives can directly help researchers, e.g. by using rocellular activity during the latent or the silent techniques and borrowing concepts as inspiration. phases of the chronic infection sharply differs from But the potential for this dialogue for DNA on- that in the acute phase, even for viruses with small coviruses is even bigger because we are currently genomes and limited coding potential, and the po- witnessing advances in different fields, from tissue- tential for malignancy strongly depends on the cel- like cell cultures to deep sequencing, that make lular genomic changes associated to this chronic cross-fertilization between fields extremely valu- infection. Bridging spatio-temporal scales seems able, as illustrated already by some pioneering re- necessary for at least three levels. First, an indi- search. For instance, mathematical modelling can vidual cell may acquire (epi)genotypic or pheno- be a means to infer biological quantities that are typic mutations allowing barriers and restraints to difficult to measure. This is routinely done in epi- malignancy to be overcome, but cancer is not an demiology and also for rapidly-evolving viruses, unicellular event. It is instead an organic event such as human immunodeficiency virus or hepatitis in which the cancerous lineages compete to spread C virus [9], but still rarely applied to the virocel- through population processes, but whose success lular and within-host level for oncogenic viruses is strongly dependent on the necessary coopera- by analysing viral and immunological data [19]. tion of non-cancerous cells. Second, because each Such exchange between disciplines and approaches virion can only infect a single cell, every individ- addressing either proximate or ultimate explana- ual infection is always eventually a dead end from tions is common. The challenge is to build a fer- the virus’ point of view, and this is clearer in in- tile dialogue between unveiling mechanisms and fection driven cancers, in which the transformed identify adaptations; between describing the nat- cells may not produce any viral particles. Virion ural history of the disease and understanding the production and transmission is the only key to vi- therein-intertwined evolutionary histories of hosts ral persistence, which necessarily links within-host and pathogens. Indeed, evolutionary analyses on and between-host dynamics. Finally, viral evolu- ultimate causes can shed new light on to proximate tion is perhaps the most obvious multi-scale pro- causes at the tissue level and suggest new hypothe- cess: it takes its roots in biochemical mutational ses to test, such as the study of within-cancer het- stochastic events, while its dynamics are governed erogeneity [8,1]. Conversely, a better understand- by epidemic spread first within the infected host ing of the natural history of the infections and dis- and then in the human population. eases, as well as of the virus-host interactions at DNA oncoviruses have more in common than the cell and organismic level will guide research on just causing cancers. Historically, their study has the evolution of DNA viruses and their virulence. faced a variety of obstacles, whatever the field of A promising example in this direction, is how ac- research. For biologists, the scarcity of animal counting for the well-known latency periods and and tissue-based models has limited the poten- transmission patterns shifts during varicella zoster tial for experimental studies. For epidemiologists, virus infections in the evolutionary models strongly the sharp contrast between the highly prevalent modifies our understanding of the evolution, origin asymptomatic infections and the far lower inci- and spread of the virus [36]. In summary, address- dence of virus-driven cancers has required epidemi- ing the infections and diseases caused by DNA on- ological approaches to enrol large cohorts, so that coviruses with an integrated, multilevel approach significant effects could be detected. For evolu- is needed, is timely and can represent an inspira- tionary biologists, the difficulty to estimate sub- tion for other infectious diseases. stitution rates [17] and to disentangle within-host For all oncoviruses, spatial and time scales are and between-host dynamics has left plenty of room important. Some of these viruses cause systemic for speculation and for the “conventional wisdom”

3 Table 1: The Tinbergen conceptual framework for structuring biological questions, applied to the case of oncogenic DNA viruses. We illustrate it with the example of the E6 protein from oncogenic Alpha- papillomaviruses, which interacts with and promotes degradation of the human tumour supressor p53 protein. Contemporary, syn- Historic, diachronic perspective chronic perspective Proximate explanations Mechanisms, function Natural history of the infection, ontogeny Host-parasite interactions and The connection between the viral and the their functions at the molecu- host genotypes and the clinical, pheno- lar, virocellular, organism and typic presentation of the infection, inte- population levels grating the interactions with the environment.

The E6-p53 example Mechanisms, function Natural history of the infection, ontogeny The E6 protein interacts with E6 is expressed in the early stages of the E6AP through a leucine rich infection in the parabasal and middle ep- domain and induces p53 bind- ithelial cell layer, driving cell proliferation, ing polyubiquitination and and stimulating cell cycle re-entry in the proteasomal degradation. suprabasal epithelial layers. Ultimate explanations Adaptation Evolution The problem that a structure The history of genotypic changes in the solves, and the adaptive value host and in the parasite through gen- conferred by this evolutionary erations, resulting in the current host- solution. parasite interaction phenotype.

The E6-p53 example Adaptation Evolution Degradation of p53 overcomes The gain-of-function of the E6 oncoprotein a stringent cellular checkpoint is speciﬁc to the clade of oncogenic HPVs control that blocks cell divi- and concurred with an adaptive radiation sion and limits viral replica- event triggered by the integration of a tion. The abnormally repli- proto-E5 oncogene in the viral genome. cating cell may in its turn ac- cumulate genomic defects that may eventually lead to cancer.

4 that these viruses strictly coevolve with their hu- search, the contribution by Daniel DiMaio [14] il- man host. For oncologists, it is not necessarily ap- lustrates the origins and historical twists of the parent that cancers in a single anatomical location research on HPVs and cervical cancer. This text can be different clinical entities depending on the showcases how the technical and conceptual ad- viral etiology [2]. For comparative pathologists, it vances conceived for understanding a particular may be difficult to recognise that distantly related cancer have resulted in a successful story with the viruses can cause cancers in the same anatomical identification of cytopathic changes in the infected location by analogous but not homologous mecha- cells that allow for early diagnosis, the discovery nisms [32]. Finally, as a significant side effect, the of the viral agent causing the disease, and the de- emphasis on cancer has potentially neglected many velopment of a safe vaccine that prevents infection other clinical implications of the chronic viral in- by the main oncogenic HPVs. fections, such as effects on fertility or even poten- A number of contributions in this issue ad- tial mutualistic effect [29]. These similarities call dress proximate explanations for specific questions for an effort to join forces between experts work- on virocellular mechanisms and functions: i) Evri- ing on different oncoviruses and also from experts pioti and coworkers [15] identify cellular signalling working in different fields. This is the goal of this routes converging on cyclic-AMP that seem to me- special issue, which spans from the virocellular to diate hepatitis B virus entry in human cells; ii) the the epidemiological level. need of developing novel models that bridge between the classical in vitro cell culture and the in 3 Focus of the special issue vivo animal experimentation is illustrated by the contribution from Jackson and coworkers [21], de- This issue brings together expertise and insights scribing the in vitro engineering of pseudo-organs from a variety of fields to tackle the threat posed including keratinocytes and Langerhans cells to by oncogenic DNA viruses. The individual contri- study the interaction between oncogenic HPVs butions aim at providing a timely overview of spe- and the immune system; iii) going at the in toto cific novel model developments, the ensemble ad- level, McHugh and coworkers [24] review the re- dressing several integration levels and approaches, cently available humanised mice as a tool to facil- as follows: itate the study of infections by human herpesvirus 4 (Epstein-Barr virus) and human herpesvirus 8 • Tissue models: how developments in three- (Kaposi sarcoma-associated herpesvirus); iv) at dimensional cell culture, microfluidics and the within-host level, McIlroy and coworkers [25] other experimental set-ups are improving our describe the accumulation of mutations in the ability to study the interaction between DNA genome of oncogenic polyomaviruses that are asso- oncoviruses and their host cells, and between ciated to malignant potential and that are virtually the virocells and the tissue environment. never found among circulating isolates of the same • Within-host models: how the combination of viruses; v) finally, also at the sequence level but animal models, clinical virology and mathe- focusing on changes in circulating viruses, Bridges matical modelling allows us to unveil infec- and coworkers [5] present the connection between tion dynamics polymorphisms in the Epstein-Barr virus genome and the geographical distribution of viral isolates, • Epidemiological models: how modelling and as well as their differential potential for cellular understanding human population-level pro- transformation. cesses can provide biologically relevant in- Several exciting contributions in the issue de- sights. scribe the state-of-the-art for a number of models addressing proximate explanations for the natural • Evolutionary models: how the reconstruction history of the disease. This is the case of i) the of evolutionary dynamics can provide hints manuscript by Tomassino and coworkers [34] where to guide fundamental and clinical research. the authors propose that the long-searched mech- Because historical contingency is central to un- anisms for malignisation by possibly oncogenic cu- derstand the present, also in DNA oncovirus re- taneous HPVs are analogous rather than homolo-

5 gous to those well established and present in onco- ous phenotype by oncogenic DNA virus infections: genic mucosal HPVs; ii) seroprevalence data have i) Man and coworkers [23] introduce an original been collected over decades for many DNA on- approach to enable predictions into the scientific coviruses, and often regarded as static descriptors hottest debate around vaccination against onco- of viral exposure, but two contributions in the is- genic HPV, namely the so-called type-replacement sue describe novel mathematical approaches that problem, which refers to the possibility that viral exploit within-host antibody titre evolution after lineages not targeted by the vaccines could increase cancer treatment (Piontek and coworkers [27]) and in prevalence and occupy the empty niche left by population-level analyses of antibody titre dynam- the targeted ones, provided the different HPVs are ics that reconcile DNA-based and antibody-based actually establishing competitive interactions. Fi- prevalence of oncogenic HPV infections (Brower nally, ii) Willemsen and Bravo [37] have addressed and coworkers [6]); iii) beyond the iconic exam- the reconstruction of the evolutionary history of ple of cervical cancer, Roberts and coworkers [30] the papillomaviruses, identifying the common ori- present the current knowledge on the infection of gin of the E6 and E7 powerful oncogenes and the lymphoid tissue in the oropharynx by oncogenic acquisition of their transforming activities. In- HPVs and the differential mechanisms that may triguingly, the authors show that the enhanced underlie the malignisation process in this anatom- oncogenicity of HPV16, the strongest biological ical location, a particular cancer that displays a oncogenic agent to humans, is not linked to the rapidly changing and not totally understood epi- strength of the E6 activity on p53, which is of- demiology; finally iv) Cladel and coworkers [11] ten regarded as the epitome of a viral oncoprotein summarise in their contribution the wealth of in- function. formation gained in the last years with the use of a rabbit papillomavirus animal model devel- 4 Future steps oped in their laboratory that provides intriguing results about the differences in the clinical phe- We are now witnessing the glorious era of omic notype caused by infection with viruses carrying biologic research. It had never been so easy and synonymous but largely-recoded genomes. inexpensive to generate (meta|epi)genomes, tran- The study of ultimate explanations on the func- scriptomes, proteomes and metabolomes, with im- tional adaptive value is a stimulating but delicate provements ongoing. Transforming this wealth of subject because there is always a risk to venture data into information has become rather the limit- too far away from the (limited) data. In this is- ing factor, in terms of using appropriate hardware sue, i) Murall and Alizon [26] analyse the evolu- and informatics tools, and in the lack of sound sta- tionary trade-offs associated to the viral oncopro- tistical approaches to define and test competing tein functions that on the one hand promote viral hypotheses for this large volume of data and ill- replication by stimulating cellular replication, but defined categories and redundancies. As all other that on the other hand may decrease viral fitness biologic disciplines, research on DNA oncoviruses by facilitating immune targeting or by leading to a and the diseases they cause have bloomed in the dead-end of a cancer. Further, ii) Ewald and Ewald last years, generating massive full-sequence sets for [16] elaborate on the possibility that many other large human cohorts that have refined some hy- cancers could also be of infectious origin, revisiting potheses and refuted others, identified specific sig- the adage of Francisco Duran-Reynals in early last natures of infection-driven cancers and led to dif- century, when saying that failure to demonstrate ferential treatment as a function of the viral etiol- infectious virus in a tumour does not mean that a ogy of cancer. The obvious sentences in the “future virus was not involved [3], and claiming that the directions” section for any scientific field cannot roles of the cellular stroma and the immune system but adhere to the Olympic motto of citius, altius, may prevent the identification of the viral onco- fortius: more and larger natural history studies to genic agent in the invasive, mature presentation of understand within-host ecology; larger cohorts to the cancer. understand where these viruses stand on the mu- Finally two articles of the issue address the tualist–parasite continuum; more sequence data, evolutionary, ultimate explanations of the cancer- which can be made possible with the decreased cost

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