Human Papillomaviruses - Knowns, Mysteries, and Unchartered Territories

Abstract

There has been an explosion in the number of papillomaviruses that have been identified and fully sequenced. Yet only a minute fraction of these has been studied in any detail. Most of our molecular research efforts have focused on the E6 and E7 proteins of “high-risk”, cancer-associated human papillomaviruses (HPVs). Interactions of the high-risk HPV E6 and E7 proteins with their respective cellular targets, the p53 and the retinoblastoma tumor suppressors, have been investigated in minute detail. Some have thus questioned if research on papillomaviruses remains an exciting and worthwhile area of investigation. However, fundamentally new insights on the biological activities and cellular targets of the high-risk HPV E6 and E7 proteins have been discovered and previously unstudied HPVs have been newly associated with human diseases. HPV infections continue to be an important cause of human morbidity and mortality and since there are no antivirals to combat HPV infections, research on HPVs should remain attractive to new investigators and biomedical funding agencies, alike.

Introduction

In addition to the capsid proteins, all papillomaviruses express the E1 and E2 proteins that are essential for viral replication, viral genome partitioning during mitosis of the host cell, and transcriptional regulation of viral gene expression. In addition, most papillomaviruses also express a series of accessory proteins that function to enable the viral life cycle by rewiring important signaling circuits of the infected host cell. Amongst these, the E6 and E7 proteins of the “high-risk”, cancer-associated human papillomaviruses (HPVs) have been studied in detail. Despite what some say that we have arrived at a definitive mechanistic understanding of how these proteins and by extension the E6 and E7 proteins of other papillomaviruses function and that there is not much new to learn, many mysteries remain. Moreover, some papillomaviruses encode accessory proteins that remain essentially unstudied, and thus represent “unchartered territories”. The following is a biased and by no means complete selection of some of the mysteries and unchartered territories of papillomaviral accessory proteins that are ready for mechanistic investigation. For comprehensive discussions on the cellular targets of the E6 and E7 proteins, the role of HPVs in epithelial differentiation, and the link between papillomaviruses and epithelial stem cell dynamics, readers are referred to the following reviews^1–5.

Knowns

Many of the early studies in the papillomavirus field focused on animal PVs. The bovine papillomavirus type 1 (BPV1) served as the go-to model for investigations on the mechanisms of viral replication, the regulation of viral gene transcription, and cellular transformation. The cotton tail rabbit papillomavirus (CRPV, now referred to as SfPV1) causes cutaneous papillomas, which can progress to frank carcinomas⁶ and thus provided the first animal model to study PV pathogenesis and carcinogenesis^7,8.

PVs are small viruses with double-stranded, circular DNA genomes of less than 10,000 base pairs. Only one of the two strands is known to be transcribed. The genomic organization of these viruses is quite simple, and PV genomes consist of two regions that encode proteins and a non-coding regulatory region that contains various binding sites for viral and cellular transcription factors as well as the viral origin of replication. The early coding region contains two to nine open reading frames (ORFs) that encode nonstructural proteins whereas the late region encodes the major and minor capsid proteins, L1 and L2, respectively. All known PVs encode the core viral transcription and replication factors, E1 and E2. Most PVs also encode additional accessory proteins, including E4, E5, E6, E7, E9, and/or E10⁹, which a colleague has referred to as “evolutionary embellishments”¹⁰.

Pioneering studies by Stefania Jablonska and Gerard Orth linked HPV infections to cutaneous squamous cell carcinomas (cSCCs) that emerge in individuals afflicted by a rare hereditary skin condition, epidermodysplasia verruciformis (EV)¹¹. After the isolation of HPV16 sequences from a human cervical carcinoma by Harald zur Hausen’s group¹², several other HPVs were isolated from cervical cancers^13,14. It is now clear that infections with a small group of “high-risk” HPVs contribute to almost 5% of all human cancers worldwide. In addition to cervical carcinomas, high-risk HPV infections cause other anogenital tract cancers as well as a growing number of head and neck cancers, particularly oropharyngeal carcinomas^15,16. Remarkably, HPV-associated cancers minimally express only two small viral proteins, E6 and E7, and the cancers remain “addicted” to expressing these two proteins¹⁷. Consequently, the biological activities of the high-risk HPV E6 and E7 proteins have been investigated most thoroughly.

An efficacious prophylactic vaccine became available within less than two decades after these HPVs were linked to cervical cancers^18,19. Even a single administration shows great promise in preventing new infections by the specific HPV types that are targeted by the vaccine²⁰. After the commercial launch of the HPV vaccine, a common sentiment (even by fellow virologists) was that mechanistic studies with HPVs were no longer needed because the cancer-causing HPVs were about to be eradicated. Sadly, this sentiment persists and, in the US, research grant applications to the National Institutes of Health (NIH) that focus on delineating oncogenic activities of HPVs are no longer routinely reviewed by virology-based review groups but are punted to general cancer panels where they often fail to generate enthusiasm. Consequently, there is a dearth of young investigators who dare to venture into studying HPV biology. This is even though the vaccine has not yet reached the medically underserved populations where the incidence of HPV-associated cancers is highest²¹ and vaccination rates remain low in many countries²². Moreover, many regions of the world currently experience an unabated, steep increase in HPV-associated oropharyngeal tumors^23,24. Despite these sobering circumstances, there are no antivirals to combat HPV infections. Hence, it should be obvious that research on HPVs, even on the well-studied E6 and E7 proteins of the “high-risk” HPVs, remains necessary from a biomedical perspective. Moreover, mechanistic studies on HPV pathogenesis will continue to provide new and unexpected insights into the fascinating biology of these viruses and how they interact with and subvert their hosts.

How known are the “knowns” of the papillomavirus E6 and E7 proteins?

The sequences of >400 HPVs are available through the PAVE database^25,26 and only a few mucosal “high-risk” alpha HPVs, mostly HPV16 and HPV18, have been studied in any detail. Mechanistic studies have mostly focused on their E6 and E7 proteins because they are known cancer drivers^27,28. High-risk HPVs also encode E5 proteins, multi-pass transmembrane proteins that have been implicated in multiple biological processes, most frequently as activators of receptor tyrosine kinase signaling²⁹. Many of these studies were likely motivated by results obtained with the BPV1 E5 protein. BPV1 E5 encodes a single-pass disulfide-linked dimeric transmembrane protein³⁰, which forces the dimerization of receptor tyrosine kinases thereby activating them in the absence of ligand binding and is largely unrelated to high-risk HPV E5 proteins³¹. Unlike E6 and E7, expression of the high-risk HPV E5 proteins is not necessary for the maintenance of the transformed phenotype, but high-risk HPV E5 proteins may play important roles in blunting host cellular innate and adaptive immune responses^32,33. Cutaneous beta and gamma HPVs do not encode E5 proteins, whereas the low-risk alpha HPVs as well as some animal PVs, can encode multiple E5 proteins that share only limited sequence similarities to the high-risk HPV E5 proteins (Figure 1; Table 1). Hence, much remains to be learned about the biological activities of the various PV E5 proteins³⁴.

Figure 1: Examples of Papillomavirus E5 proteins.

The bovine papillomavirus 1 (BPV1) E5 protein has been studied in the greatest detail and encodes a single-pass transmembrane protein. The beta, delta, zeta, and some epsilon E5s are also predicted to encode single-pass membrane proteins but there is limited sequence similarity between the E5 proteins encoded by the different groups. Some epsilon E5 proteins do not have recognizable transmembrane domains. In contrast, the alpha and gamma E5s encode multi-pass transmembrane proteins. Potential transmembrane sequences as predicted by the CCTOP server (https://cctop.ttk.hu/) are highlighted in crimson. See Table 1 for a full listing of papillomaviruses that encode E5 proteins.

Table 1:

HPVs and animal PVs that encode E5 proteins.

E5 Protein Family	Virus	Species	Host Common Name	GenBank ID	Notes
E5_Alpha	HPV16	Alphapapillomavirus 9	Human	K02718
	HPV18	Alphapapillomavirus 7	Human	X05015
	HPV31	Alphapapillomavirus 9	Human	J04353
	HPV33	Alphapapillomavirus 9	Human	M12732
	HPV34	Alphapapillomavirus 11	Human	X74476
	HPV35	Alphapapillomavirus 9	Human	X74477
	HPV39	Alphapapillomavirus 7	Human	M62849
	HPV45	Alphapapillomavirus 7	Human	X74479
	HPV52	Alphapapillomavirus 9	Human	X74481
	HPV58	Alphapapillomavirus 9	Human	D90400
	HPV59	Alphapapillomavirus 7	Human	X77858
	HPV67	Alphapapillomavirus 9	Human	D21208
	HPV68	Alphapapillomavirus 7	Human	DQ080079
	HPV70	Alphapapillomavirus 7	Human	U21941
	HPV73	Alphapapillomavirus 11	Human	X94165
	HPV85	Alphapapillomavirus 7	Human	AF131950
	HPV97	Alphapapillomavirus 7	Human	DQ080080
	HPV177	Alphapapillomavirus 11	Human	KR816168
E5_Beta	HPV2	Alphapapillomavirus 4	Human	X55964
	HPV3	Alphapapillomavirus 2	Human	X74462
	HPV10	Alphapapillomavirus 2	Human	X74465
	HPV27	Alphapapillomavirus 4	Human	X74473
	HPV28	Alphapapillomavirus 2	Human	U31783
	HPV29	Alphapapillomavirus 2	Human	U31784
	HPV57	Alphapapillomavirus 4	Human	X55965
	HPV61	Alphapapillomavirus 3	Human	U31793
	HPV62	Alphapapillomavirus 3	Human	AY395706
	HPV71	Alphapapillomavirus 14	Human	AB040456
	HPV72	Alphapapillomavirus 3	Human	X94164
	HPV77	Alphapapillomavirus 2	Human	Y15175
	HPV78	Alphapapillomavirus 2	Human	KC138720
	HPV81	Alphapapillomavirus 3	Human	AJ620209
	HPV83	Alphapapillomavirus 3	Human	AF151983
	HPV84	Alphapapillomavirus 3	Human	AF293960
	HPV86	Alphapapillomavirus 3	Human	AF349909
	HPV87	Alphapapillomavirus 3	Human	AJ400628
	HPV89	Alphapapillomavirus 3	Human	AF436128
	HPV90	Alphapapillomavirus 14	Human	AY057438
	HPV94	Alphapapillomavirus 2	Human	AJ620211
	HPV102	Alphapapillomavirus 3	Human	DQ080083
	HPV106	Alphapapillomavirus 14	Human	DQ080082
	HPV114	Alphapapillomavirus 3	Human	GQ244463
	HPV117	Alphapapillomavirus 2	Human	GQ246950
	HPV125	Alphapapillomavirus 2	Human	FN547152
	HPV160	Alphapapillomavirus 2	Human	AB745694
	CgPV1	Alphapapillomavirus 14	Colobus monkey	GU014532
	NasiPV1	Unclassified	Narrow-ridged finless porpoise	OQ274126	Encodes E5_Beta and E5_Unclassified
E5_Delta	HPV6	Alphapapillomavirus 10	Human	X00203	Encodes E5_Delta and E5_Gamma
	HPV7	Alphapapillomavirus 8	Human	X74463
	HPV11	Alphapapillomavirus 10	Human	M14119	Encodes E5_Delta and E5_Gamma
	HPV13	Alphapapillomavirus 10	Human	X62843	Encodes E5_Delta and E5_Gamma
	HPV32	Alphapapillomavirus 1	Human	X74475
	HPV40	Alphapapillomavirus 8	Human	X74478
	HPV43	Alphapapillomavirus 8	Human	AJ620205
	HPV44	Alphapapillomavirus 10	Human	U31788	Encodes E5_Delta and E5_Gamma
	HPV74	Alphapapillomavirus 10	Human	AF436130	Encodes E5_Delta and E5_Gamma
	HPV91	Alphapapillomavirus 8	Human	AF419318
	PpPV1	Alphapapillomavirus 10	Pygmy chimpanzee	X62844	Encodes E5_Delta and E5_Gamma
E5_Epsilon	BPV32	Unclassified	Domestic cow	MW401529
	BPV40	Unclassified	Domestic cow	MW428425
	MfPV3	Alphapapillomavirus 12	Cynomolgus macaque	EF558839	Encodes E5_Epsilon and E5_Zeta
	MfPV4	Alphapapillomavirus 12	Cynomolgus macaque	EF558841	Encodes E5_Epsilon and E5_Zeta
	MfPV5	Alphapapillomavirus 12	Cynomolgus macaque	EF558843	Encodes E5_Epsilon and E5_Zeta
	MfPV6	Alphapapillomavirus 12	Cynomolgus macaque	EF558840	Encodes E5_Epsilon and E5_Zeta
	MfPV7	Alphapapillomavirus 12	Cynomolgus macaque	EF558838	Encodes E5_Epsilon and E5_Zeta
	MfPV8	Alphapapillomavirus 12	Cynomolgus macaque	EF558842	Encodes E5_Epsilon and E5_Zeta
	MfPV9	Alphapapillomavirus 12	Cynomolgus macaque	EU490516	Encodes E5_Epsilon and E5_Zeta
	MfPV10	Alphapapillomavirus 12	Cynomolgus macaque	EU490515	Encodes E5_Epsilon and E5_Zeta
	MfPV11	Alphapapillomavirus 12	Cynomolgus macaque	GQ227670	Encodes E5_Epsilon and E5_Zeta
	MfuPV1	Unclassified Alphapapillomavirus	Japanese macaque	KT944080	Encodes E5_Epsilon and E5_Zeta
	MmPV1	Alphapapillomavirus 12	Rhesus macaque	M60184	Encodes E5_Epsilon and E5_Zeta
	MmPV6	Unclassified Alphapapillomavirus	Rhesus macaque	MH745748
	NasiPV3	Unclassified	Narrow-ridged finless porpoise	OQ274128
	PhPV1	Alphapapillomavirus 12	Hamadryas baboon	JF304764	Encodes E5_Epsilon and E5_Zeta
	PtroPV1	Unclassified	Chimpanzee	OP934207
E5_Gamma	HPV6	Alphapapillomavirus 10	Human	X00203	Encodes E5_Delta and E5_Gamma
	HPV11	Alphapapillomavirus 10	Human	M14119	Encodes E5_Delta and E5_Gamma
	HPV13	Alphapapillomavirus 10	Human	X62843	Encodes E5_Delta and E5_Gamma
	HPV44	Alphapapillomavirus 10	Human	U31788	Encodes E5_Delta and E5_Gamma
	HPV74	Alphapapillomavirus 10	Human	AF436130	Encodes E5_Delta and E5_Gamma
	PpPV1	Alphapapillomavirus 10	Pygmy chimpanzee	X62844	Encodes E5_Delta and E5_Gamma
E5_Zeta	MfPV3	Alphapapillomavirus 12	Cynomolgus macaque	EF558839	Encodes E5_Epsilon and E5_Zeta
	MfPV4	Alphapapillomavirus 12	Cynomolgus macaque	EF558841	Encodes E5_Epsilon and E5_Zeta
	MfPV5	Alphapapillomavirus 12	Cynomolgus macaque	EF558843	Encodes E5_Epsilon and E5_Zeta
	MfPV6	Alphapapillomavirus 12	Cynomolgus macaque	EF558840	Encodes E5_Epsilon and E5_Zeta
	MfPV7	Alphapapillomavirus 12	Cynomolgus macaque	EF558838	Encodes E5_Epsilon and E5_Zeta
	MfPV8	Alphapapillomavirus 12	Cynomolgus macaque	EF558842	Encodes E5_Epsilon and E5_Zeta
	MfPV9	Alphapapillomavirus 12	Cynomolgus macaque	EU490516	Encodes E5_Epsilon and E5_Zeta
	MfPV10	Alphapapillomavirus 12	Cynomolgus macaque	EU490515	Encodes E5_Epsilon and E5_Zeta
	MfPV11	Alphapapillomavirus 12	Cynomolgus macaque	GQ227670	Encodes E5_Epsilon and E5_Zeta
	MfuPV1	Unclassified Alphapapillomavirus	Japanese macaque	KT944080	Encodes E5_Epsilon and E5_Zeta
	MmPV1	Alphapapillomavirus 12	Rhesus macaque	M60184	Encodes E5_Epsilon and E5_Zeta
	PhPV1	Alphapapillomavirus 12	Hamadryas baboon	JF304764	Encodes E5_Epsilon and E5_Zeta
E5_Unclassified	HPV54	Alphapapillomavirus 13	Human	U37488
	AaPV1	Deltapapillomavirus 1	European elk	M15953
	BPV1	Deltapapillomavirus 4	Domestic cow	X02346
	BPV2	Deltapapillomavirus 4	Domestic cow	M20219
	BPV13	Deltapapillomavirus 4	Domestic cow	JQ798171
	BPV14	Deltapapillomavirus 4	Domestic cow	KP276343
	BgPV1	Deltapapillomavirus 4	Yak	JX174437
	CcaPV1	Deltapapillomavirus 5	Western roe deer	EF680235
	EbonPV2	Unclassified	Dwarf bonneted bat	OL704824
	GcPV1	Unclassified Deltapapillomavirus	Giraffe	KX954132
	MmPV2	Unclassified Alphapapillomavirus	Rhesus macaque	MG837557
	NasiPV1	Unclassified	Narrow-ridged finless porpoise	OQ274126	Encodes E5_Beta and E5_Unclassified
	OaPV1	Deltapapillomavirus 3	Domestic sheep	U83594
	OaPV2	Deltapapillomavirus 3	Domestic sheep	U83595
	OaPV4	Unclassified Deltapapillomavirus	Domestic sheep	KX954121
	OcPV1	Kappapapillomavirus 1	New Zealand white rabbit	AF227240
	OjohPV1	Unclassified Deltapapillomavirus	Okapi	MH049343
	OvPV1	Deltapapillomavirus 2	American white-tailed deer	M11910
	RalPV1	Unclassified Deltapapillomavirus	Visayan spotted deer	KT626573
	RtPV1	Deltapapillomavirus 1	Reindeer	AF443292
	SfPV1	Kappapapillomavirus 2	Cottontail rabbit	K02708

Much progress has been made in delineating the biological activities of the high-risk HPV E6 and E7 proteins. The high-risk HPV E7 proteins bind and destabilize the retinoblastoma tumor suppressor protein, pRB^35,36, thus “liberating” the E2F transcriptional activator and inducing aberrant proliferation. The mechanism of pRB destabilization remains unknown for most high-risk HPV E7 proteins. E7 expression causes activation of the p53 tumor suppressor. This cellular defense response is short-circuited by the E6 protein and the associated E6-AP (UBE3A) ubiquitin ligase which target p53 for degradation through the proteasome³⁷. Combined inhibition of the pRB and p53 tumor suppressors opens the gates for unchecked aberrant cellular proliferation¹⁷. E6 also activates telomerase and in combination with E7-mediated pRB inactivation causes an extension of cellular lifespan, thereby facilitating cellular immortalization^38,39. Since E6 and E7 expression interfere with genomic stability, high-risk HPV E6/E7-expressing cells are prone to acquiring additional chromosomal aberrations that facilitate malignant progression⁴⁰. Variations of this enticing and seductive model of HPV carcinogenesis are widely published in review articles and textbooks even though it is incomplete, at best⁴¹. In addition to regulating E2F transcription factor activity, pRB also has additional, less extensively studied “non-canonical” biological activities that are relevant to its tumor suppressor function⁴² and these will also be inhibited as a result of destabilization by the high-risk HPV E7 proteins.

Similarly, the assumption that there is “one best replication strategy” for HPVs and that the E6 and E7 proteins by different HPV genera share similar biological activities and target the same cellular proteins has not been proven correct^43–45. The E7 proteins of the low-risk mucosal alpha HPVs and the cutaneous beta HPVs that were originally linked to cSCCs in EV patients bind pRB with lower efficiency than high-risk HPVs and they do not destabilize pRB^46,47. The E6 proteins of these viruses do not target p53 for degradation⁴⁸ and cannot efficiently stimulate telomerase activity to enable cellular immortalization^27,49. Some cutaneous gamma HPVs encode E7 proteins that bind pRB through different sequences which does not result in efficient activation of E2F target genes^50,51. The cellular targets of the E6 proteins of beta HPVs associated with cSCCs in EV patients are distinct from those of the high-risk alpha HPV E6 proteins and impact pathways that regulate keratinocyte differentiation and genomic stability^43–45. Thus, there is still much that remains to be learned regarding the “known” biological activities of the E5, E6, and E7 proteins and how they enable the viral life cycle.

Persistent infections - a question of target cells, cell competition, and basal cell identity?

HPV infections of the anogenital tract are very frequent. Most spontaneously resolve within a few months^52,53 but some result in long-term persistent infections. Persistent cervical infections with high-risk HPVs constitute the major risk factor for the development of high-grade premalignant lesions and cancers^15,54. Studies aimed at identifying the factors that contribute to the establishment of long-term, persistent HPV infections have only recently been initiated^54–57. The immune status of the host plays an important role, as evidenced by the increased incidence of high-risk HPV-associated cervical and anal cancers in HIV-positive individuals⁵⁸ and the emergence of beta HPV-associated cSCCs in immunosuppressed organ transplant patients⁵⁹. The E2, E5, E6, and E7 proteins have each been implicated in blinding the host’s innate and/or adaptive immune system^33,60–62. Whether or not persistently infected tissues constantly shed infectious HPVs or whether virus production is only activated occasionally and if so, what the triggers are has not been elucidated. Additionally, whether there are differences between persistently infected males and females is also unknown.

Stratified epithelia are dynamic, and most keratinocytes commit to differentiation and are replaced every few weeks, with only a few long-lived cells retained in the basal layer of stratified epithelia for the long term⁶³. Even so, a key hallmark of many HPVs is their ability to establish persistent, long-term infections in the proliferative basal layer⁵. How papillomaviruses promote or retain basal cell identity, which is necessary for the retention of infected cells in the basal layer, has not yet been fully elucidated.

One theory has been that HPVs specifically infect a stem cell population in basal epithelia and a specific cell population within the squamocolumnar transformation zone of the cervix that may be at a higher risk for the development of persistent high-risk HPV infections⁶⁴. The HPV16 E6 and E7 proteins may cause aberrant stem cell mobilization^4,65 and similar findings have been reported for the E6 and E7 proteins of some cutaneous HPVs⁶⁶. It will be challenging to experimentally determine whether the cell initially infected by HPV is a stem cell, and it is hard to imagine that a virus that could only persistently infect a minuscule subset of basal cells would be as highly prevalent as HPV.

An alternative and more likely model is that instead of targeting stem cells for initial infection, HPV-encoded proteins could reprogram infected keratinocytes to adopt a basal epithelial cell identity. Much like HPV proteins activating cell growth pathways, it is plausible that HPVs have evolved to act on pathways that control epithelial homeostasis to drive cells into a more stem-like state. Two such pathways that are central regulators of epithelial stemness are the Notch and Hippo/YAP signaling pathways.

Notch signaling is a well-known driver of keratinocyte differentiation⁶⁷. There is already strong evidence that cutaneous HPV E6 proteins inactivate Notch. Several groups determined that cutaneous HPV E6 proteins inhibit differentiation by binding to the transcriptional co-regulator mastermind like 1 (MAML1), thereby repressing Notch signaling^68–70. Like cutaneous HPV E6, the Mus musculus papillomavirus 1 (MmuPV1) E6, inhibits Notch signaling by binding MAML1⁷¹, and it promotes basal identity in mouse lesions⁷². HPV16 E6 may also affect Notch signaling indirectly as a consequence of p53 inactivation. NOTCH1 is a transcriptional target of p53, and p53 inactivation downregulates NOTCH1 expression and downstream Notch signaling^73,74. The role of HPV16 E6 in downregulating NOTCH1 mRNA levels is dependent on its ability to degrade p53⁷⁵, but how well these effects on Notch are conserved in other mucosal HPV E6 proteins remains to be determined. Except for the MmuPV1 E6 experiments⁷⁵, the studies that have tested the effects of E6 on Notch have mostly been performed in two-dimensional cell culture systems. It will be important to determine whether the Notch-suppressive effects of cutaneous HPV E6 proteins are associated with an ability to drive basal identity in lesions or three-dimensional tissue models.

The YAP1 oncoprotein is a potent driver of epithelial cell stemness and self-renewal^76–81. YAP1 activation is sufficient to rapidly drive non-viral squamous epithelial cancers at mucosal sites in several mouse models^82–85. YAP1 activity is suppressed by an active Hippo kinase cascade. Remarkably, the three mitogenic pathways that are most differentially altered with high mutation rates in HPV-negative HNSCC and low mutation rates in HPV-positive HNSCC are p53 (targeted by E6), cell cycle/RB1 (targeted by E7), and Hippo/YAP^86,87. Recent mechanistic insight into how HPV oncoproteins activate YAP1 comes from research on the host cell targets of HPV E7 proteins. Highly conserved sequences in the C-terminus of HPV E7 enable E7 to bind directly and with nanomolar affinity to the tumor suppressor PTPN14⁸⁸. PTPN14 is a suppressor of YAP1^89–92, and high-risk HPV E7 proteins activate YAP1 dependent on their ability to target PTPN14 for degradation⁸⁶. Consistent with the findings that YAP1 drives an epithelial stemness gene expression program, high-risk HPV E7-expressing keratinocytes retain their basal cell identity as a consequence of PTPN14 degradation and YAP activation in engineered three-dimensional stratified epithelial tissues⁸⁶. Independent confirmation of the concept that PTPN14 inactivation drives basal epithelial identity comes from an analysis that used large, genome-wide biobank sequencing datasets for unbiased identification of drivers of basal cell carcinoma. Remarkably, these analyses revealed that individuals with germline mutations in PTPN14 are highly predisposed to develop basal cell carcinoma, and likely, cervical cancer⁹³. There are several reports that HPV E6 proteins can also activate YAP1 or promote its nuclear localization^94–97 and it will be important to determine whether or how this contributes to the maintenance of basal cell identity.

Other recent experiments have used co-culture experiments to measure cell competition in two-dimensional cultures. In such experiments, high-risk HPV E6- but not E7-expressing basal keratinocytes have been shown to “outcompete” uninfected basal keratinocytes^68,72,97. However, it is unclear whether the cell competition assays measure the same properties of keratinocytes that enable cells to be maintained in the basal layer. Induction of cell competition may allow for the lateral expansion of a pool of infected basal cells that are spawned from the initially infected cell. For the high-risk HPV16 E6 protein, induction of cell competition has been linked to p53 binding⁶⁸ and E6AP activity⁹⁷. These findings are consistent with reports that p53 promotes cell death in dense epithelial cultures^98,99. In experiments with the MmuPV1 E6 proteins, an intact binding site for the transcriptional co-activator of Notch signaling, MAML1, was required for cell competition⁷².

It will be interesting to determine how intertwined these two activities of E6 and E7 are, which activities of E6 and/or E7 direct basal identity and cell competition, whether inhibiting these activities will block HPV persistence, and whether specific subsets of HPVs, such as high-risk HPVs, have a unique ability to promote basal identity and cell competition.

Why are HPV16 and other high-risk HPVs such successful, highly prevalent viruses?

Even though HPV16 has been more extensively studied than any other HPV, we know embarrassingly little about what makes it such a successful and thus frequently detected virus. HPV16 is the most prevalent HPV in cervical carcinoma and is even more predominant in other anogenital tract carcinomas and head and neck cancers. Because cancer formation often represents a dead end for the viral life cycle, it is not obvious why despite the frequent cancer association, HPV16 is such a “successful” and prevalent virus. The argument that cancer association should negatively affect evolutionary fitness can also be made for other high-risk HPVs.

Perhaps the high-risk HPVs and HPV16 in particular, are evolutionarily, nimbler, and thus they can more rapidly adapt to individual hosts. When Mirabello and colleagues sequenced HPV16 genomes from HPV16-infected individuals, they detected an astonishing number of sequence variants¹⁰⁰. A subsequent large-scale sequencing study of HPV31-infected individuals yielded similar results¹⁰¹. HPV31 is a high-risk HPV¹⁰² and like HPV16 is a member of the alpha 9 species. In general, the detected variants represented a majority of the HPV species that were detected in a particular individual. Hence, they did not represent some minor, biologically irrelevant species, derived from a rare, abortive “accident” during genome replication^100,101. The mechanisms by which these variants arise, however, have not been fully explored. Like other intracellular pathogens, some HPVs potently trigger the expression of members of the apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) family of cytidine deaminases¹⁰³. These are important components of the host cell’s arsenal of defense mechanisms and function as potent mutagens and garble the infecting pathogen’s genetic information¹⁰⁴. Unlike many other pathogens, HPVs do not appear to mute this cellular defense response¹⁰⁵, and many of the HPV16 variants detected in this study are consistent with APOBEC3 activity^100,101. Those mutations that do not follow the APOBEC mutational signature may have arisen due to replication errors. HPV genomes are synthesized at sites of double-strand DNA breaks and may involve a more error-prone recombination-based replication mechanism, which could represent another important source for the generation of these variants^106–108.

Some of the nucleotide changes present in these variants result in alterations of the coding sequence of the viral ORFs, including E6 and, remarkably to a far lesser extent, E7^100,101. It will be interesting to determine whether some of these variants may be more or less carcinogenic¹⁰⁹ and whether the presence of a specific variant may predict an increased or decreased risk for malignant progression. Moreover, it will be fascinating to investigate whether some of these HPV16 variants may have emerged and/or accumulated to be optimally compatible with the specific genetic makeup of an individual host. Consistent with this idea, it is well known that there are HPV16 variants that track with specific ethnic groups^110,111.

Are there additional “high-risk” HPVs?

There are currently 12 mucosal alpha HPVs (types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59) that have been designated “carcinogenic to humans” by the World Health Organization/International Agency for Research on Cancer (WHO/IARC) as of 2012 with several other HPVs listed as “probably” or “possibly” carcinogenic¹¹². Seven of the 12 carcinogenic HPVs (types 16, 18, 31, 33, 45, 52, and 58) are included in the current 9-valent prophylactic HPV vaccine¹¹³. As the transmission of HPV vaccine types is decimated in vaccinated populations, it will be important to determine whether other HPVs will fill the void, thus becoming more prevalent over time and potentially needing to be included in future vaccine preparations. This will necessitate the use of less biased survey mechanisms, such as next-generation sequencing (NGS), that can reliably detect any HPV type in a given specimen.

The existence of additional high-risk HPVs was demonstrated by the recent identification of HPV42 as the oncogenic driver of digital papillary adenocarcinoma. This was achieved by analyzing non-human reads from NGS data of human skin cancers¹¹⁴. HPV42 was isolated 30 years earlier from a vulvar papilloma and even though the authors noted that “Although associated with benign genital lesions, it shows characteristics so far found in HPVs associated with either invasive carcinomas or nongenital HPVs only”¹¹⁵ it was not further studied at that time.

There are likely other HPVs that deserve to be evaluated for their carcinogenic potential. These may include the gamma 6 HPVs, HPV101¹¹⁶, HPV103¹¹⁶, and HPV108¹¹⁷, and based on their genomic organization also HPV214¹¹⁸, HPV226¹¹⁹, and HPV-MW02C24ANR¹²⁰. Most gamma HPVs infect cutaneous epithelia and have received almost no experimental attention because they are widely regarded as biomedically irrelevant normal skin commensals. The gamma 6 HPVs as well as HPV214 and 226, however, have all been isolated from the anogenital tract mucosa, with HPV101 and 108 having been isolated from high- and low-grade premalignant cervical lesions, respectively. Unlike all the other known >430 fully sequenced HPVs currently listed in the PAVE database, the gamma 6 HPVs do not contain E6 ORFs, but they encode in its place E10, a unique, 37 amino acid, hydrophobic, putatively helical transmembrane protein¹²¹ (Figure 2A).

Figure 2: E10 proteins encoded by HPVs and animal PVs.

HPV101, HPV103 HPV108 are classified as gamma 6 PVs, whereas HPV214, HPV226, and HPV-mw02c24anr are currently unclassified gamma HPVs.

Identical residues are highlighted by black boxes and chemically similar residues by gray boxes. Potential transmembrane sequences as predicted by the CCTOP server (https://cctop.ttk.hu/) are indicated by crimson bars. (A). Many bovine papillomaviruses (BPVs) as well as a PV isolated from a domestic goat (Capra hircus Papillomavirus 2; ChPV2), raindeer (Rangifer tarandus Papillomavirus 2; RtPV2), cottontail rabbit (Sylvilagus floridanus Papillomavirus 1; SfPV1 aka CRPV), and New Zealand white rabbit (Oryctolagus cuniculus Papillomavirus 1; OcPV1) also encode E10 proteins. The E10s encoded by BPV20, BPV41, ChPV2, RtPV2, SfPV1 and OcPV1 are overprinted on the E6 ORF. Amino acid residues conserved amongst all the animal PV encoded E10s are highlighted by pale green boxes, overprinted E10 residues conserved amongst BPV20, BPV40, ChPV2, and RtPV2 are highlighted by olive-colored boxes, and residues conserved between SfPV1 and OcPV1 E10s by moss green boxes. Chemically similar amino acid residues are highlighted by gray boxes. Potential transmembrane sequences as predicted by the CCTOP server (https://cctop.ttk.hu/) are indicated by crimson bars. (B). A papillomavirus isolated from the Javan rusa (Rusa timorensis papillomavirus 2; RtiPV2) also encodes an E10 protein but it shares no extensive sequence similarity to other E10s and is not predicted to contain a transmembrane region (C).

Even though HPV5 and HPV8 were the first HPVs found to be associated with human cancers, the potential association of cutaneous beta and, potentially, some gamma HPVs with cSCCs in immune-competent, and not genetically predisposed patients remains tenuous. One provocative study has suggested the interesting possibility that infection with some cutaneous HPVs may in fact protect from human skin carcinogenesis¹²². It is currently unknown whether the cutaneous HPVs may also be classified as low- or high-risk similar to the mucosal HPVs. While some of the E6 and E7 proteins of the cutaneous beta HPVs have transforming activities in various cell- and animal-based model systems, the molecular mechanisms by which these viruses may contribute to cSCC development have not been fully delineated^123,124. This matter is complicated by the fact that cutaneous HPVs are detected in bona fide precancerous lesions but not in most cancer cells, and hence they may only contribute to cancer initiation, but not to cancer maintenance. Such a “hit-and-run” carcinogenesis model is hard to prove, but a fascinating animal experiment has provided some experimental support for this concept. Transgenic mice where HPV38 E6 and E7 expression was targeted to the basal epithelial cells were much more highly susceptible to UV carcinogenesis than control mice, and the mutations in the lesions were similar to those seen in human cSCCs. Most importantly, the tumors persisted when the HPV 38 E6/E7 expression cassette was deleted using the cre-lox recombination system. This result is consistent with the model that at least some cutaneous HPVs can sensitize cells to UV-induced genomic instability and contribute to skin carcinogenesis through a “hit-and-run” mechanism¹²⁵. Induction of genome instability is a well-recognized “enabling characteristic” that can facilitate the acquisition of cancer hallmarks,¹²⁶ but is only one of the potential “hit-and-run” mechanisms by which viruses can contribute to cancer development¹²⁷. Hence, it would not be surprising if there are additional HPVs that contribute to cancer initiation, but unlike the high-risk alpha HPVs, may not be necessary for tumor maintenance.

Are proteins the only important cellular targets and effectors of papillomaviruses?

Most of the research on papillomavirus-host interactions has focused on associated cellular proteins and modulation of protein-coding RNAs. However, most of the human transcriptome is noncoding¹²⁸. RNA sequencing analyses have documented that HPVs can alter the expression of host cell non-coding RNAs. One class of non-coding RNA that has been investigated in some detail in the context of HPV infections are microRNAs (miRNAs). Whether some HPVs express miRNAs has been debated^129–131. Modulation of cellular miRNA expression in HPV-expressing cells, however, has been extensively documented, but the effect of dysregulated miRNAs on viral and host cellular gene expression is not yet fully understood^132,133. Moreover, miRNAs are also secreted in extracellular vesicles (EVs) and the miRNA content of EVs is altered in HPV oncogene-expressing cells¹³⁴. Whether and how this may affect gene expression in neighboring, uninfected cells and/or tumor stromal cells will be an interesting area of future research.

In addition to miRNA, HPVs also affect the expression of other non-coding RNAs, including circular (circ)RNAs and long noncoding (lnc)RNAs. HPV16 produces a circRNA, which unlike most cellular circRNAs has coding capacity and can encode the E7 protein¹³⁵. The level of expression and thus the biological relevance of this circRNA has been debated^136,137, but the dysregulation of cellular circRNAs in human carcinogenesis has been well-documented¹³⁸. LncRNAs are biochemically versatile molecules that can interact with other RNAs, as well as DNA and proteins, and have been implicated in many biological processes¹³⁹. For example, the HPV16 E7 protein forms a complex with the lncRNA HOTAIR, which recruits the PRC2 chromatin remodeling complex to silence gene expression¹⁴⁰. This suggests that HPV proteins may not only alter the expression of but also directly affect the biological activities of cellular lncRNAs. Studies on the functional interplay of HPV proteins with the various classes of non-coding RNAs will continue to yield fascinating insights into the strategies by which these viruses functionally reprogram their host cells.

Is there only a single papillomavirus that infects standard laboratory mice?

The species specificity has long been a major barrier to HPV pathogenesis research. The “classical” CRPV and BPV1 models have provided interesting insights, but rabbits and particularly cattle are not handled in typical academic animal facilities. Experiments with non-human primates are prohibitively expensive and difficult to justify ethically. The dog model provided key insights during the preclinical development of the prophylactic vaccine, but it has seen only limited use in molecular pathogenesis studies⁷. Although the multimammate mouse (Mastomys coucha) is not a traditional organism for animal work, it allows for experimental infections with wild-type and mutant PV genomes and serves as an excellent model of papillomavirus-induced skin carcinogenesis¹⁴¹.

The identification and isolation of MmuPV1 from cutaneous papillomas in a colony of nude mice provided a more convenient model in a genetically tractable, standard animal system^142,143. It enables viral pathogenesis experiments not only by experimental infections with mutant MmuPV1 genomes but also by testing the effect of viral infection in genetically manipulated mice¹⁴⁴. The MmuPV1 E6 and E7 proteins share some biological activities with certain cutaneous beta and gamma HPVs^145,146. However, MmuPV1 does not only cause cutaneous lesions and tumors as it can also infect mucosal epithelia and cause anogenital tract lesions like the well-studied mucosal high-risk alpha HPVs^147,148. Sexual transmission of MmuPV1 has been observed in laboratory settings¹⁴⁹, opening the exciting possibility of investigating means to interfere with the sexual transmission of PVs.

The major advantage of mouse models, the ability to perform experiments in inbred mouse strains with well-defined genetic backgrounds, is also a major shortcoming in that such mice do not reflect the genetic diversity of human populations. This limits efforts to discover genetic modifiers that affect disease susceptibility. In recent years, populations of outbred mice have been generated by interbreeding of multiple well-defined, inbred founder strains¹⁵⁰. MmuPV1 pathogenesis studies with such outbred mice promise to provide fascinating insights into host factors that determine disease outcomes.

Multiple papillomavirus types have been detected in most animal species. A total of 44 bovine PVs are listed in PAVE and they show a remarkable genetic diversity^25,26. It is thus hard to believe that there is only a single PV that infects Mus musculus, the common house mouse. It will be interesting to determine whether non-laboratory-confined Mus musculus harbor other PVs and if they are associated with other disease manifestations.

Unchartered territory – is it worth studying some of the “strange” animal papillomaviruses?

The list of animal PVs in the PAVE database currently stands at >280 entries^25,26. With a few exceptions that are mentioned in earlier sections, their life cycles and potential disease associations are mostly unknown. Yet the genomic organization of some of the animal PVs is fascinating. The currently unclassified PVs isolated from various fish species as well as from a toad, Rhinella marina (RmarPV1) do not encode E6 and E7 proteins, whereas the omega PVs (occurring in bears and seals) as well as the PVs isolated from cetaceans encode E6, but not E7 proteins. Others such as the unclassified PVs from geckos encode an E7, but not an E6 protein (Table 2). Furthermore, some of the animal PVs encode interesting variations of the E6 and E7 proteins. Avian PVs, for example, encode much larger E7 proteins and no or truncated E6 proteins¹⁵¹. The avian E7 proteins all contain the canonical LXCXE (L, leucine; C, cysteine; E, glutamic acid; X, any amino acid)-based binding motif for the retinoblastoma protein family members in their amino-terminal sequences and (CXXC)₂ metal binding motifs in their carboxy termini. However, the spacing between the two CXXC motifs is smaller than in E7 proteins encoded by other PVs and there is overall poor amino acid sequence conservation between the different avian PV E7 proteins. The truncated versions of E6 that are encoded by some avian PVs consist of a single (CXXC)₂-based metal binding site (Figure 3), and some contain an additional unique ORF, termed E9, that is overprinted on E1 (Figure 4; Table 3). Other animal PVs, including many bovine papillomaviruses (BPVs), contain E10 but no E6 ORFs. These E10s encode 42 amino acid hydrophobic polypeptides that share no significant amino acid similarity to the gamma 6 HPVs encoded E10s. Other animal PVs also contain E10 ORFs, but they are overprinted on E6 and can encode 42 to 50 amino acid hydrophobic polypeptides that are related to the non-overprinted BPV E10s but are distinct from the gamma 6 HPV E10s (Figure 2B; Table 4). SfPV1 and OcPV1, which contain E6-overprinted E10s also encode hydrophobic E5 ORFs (Figure 2B; Table 4). The E10 protein encoded by the Javan rusa PV2 (RtiPV2) is unique, lacks a predicted transmembrane domain, and shares no sequence similarity to the other E10s (Figure 2C; Table 4).

Table 2:

Animal PVs that do not encode E6 and/or E7 proteins.

Virus	Species	Host Common Name	GenBank ID	Notes
AmPV2	Omegapapillomavirus	Giant panda	MF327535	No E7
CstrPV1	Unclassified	Sea bass	MZ570863	No E6 or E7
CstrPV2	Unclassified	Sea bass	MZ570864	No E6 or E7
CstrPV3	Unclassified	Sea bass	MZ570865	No E6 or E7
DdPV1	Upsilonpapillomavirus 1	Common short-beaked dolphin	GU117620	No E7
DleuPV1	Unclassified	Beluga whale	OP856597	No E7
EaPV1	Dyochipapillomavirus 1	Donkey	KF741371	No E6; E7 does not contain LXCXE motif
EaPV3	Unclassified	African wild ass	OP699054	No E6; E7 does not contain LXCXE motif
HfrePV1	Unclassified	Common house gecko	MK207055	No E6
HfrePV2	Unclassified	Common house gecko	MN194600	No E6
LcamPV1	Unclassified	Red snapper	MH617579	No E6 or E7
LwPV6	Omegapapillomavirus	Weddell seal	MG571091	No E7
LwPV7	Omegapapillomavirus	Weddell seal	MG571092	No E7
MaegPV1	Unclassified	Haddock	MH616908	No E6 or E7
MaegPV2	Unclassified	Haddock	MH617143	No E6 or E7
MaegPV3	Unclassified	Haddock	MZ570859	No E6 or E7
MaegPV4	Unclassified	Haddock	MZ570860	No E6 or E7
MaegPV5	Unclassified	Haddock	MZ570861	No E6 or E7
MrPV1	Unclassified	Ricketts big-footed bat	JQ814847	No E7
NasiPV1	Unclassified	Narrow-ridged finless porpoise	OQ274126	No E7
NasiPV2	Unclassified	Narrow-ridged finless porpoise	OQ274127	No E7
NasiPV3	Unclassified	Narrow-ridged finless porpoise	OQ274128	No E7
NasiPV4	Unclassified	Narrow-ridged finless porpoise	OQ274129	No E7
NasiPV5	Unclassified	Narrow-ridged finless porpoise	OQ274130	No E7
NasiPV6	Unclassified	Narrow-ridged finless porpoise	OQ274131	No E7
OmykPV1	Unclassified	Rainbow trout	MH510267	No E6 or E7
PphPV1	Omikronpapillomavirus 1	Harbor porpoise	GU117621	No E7
PphPV2	Upsilonpapillomavirus 3	Harbor porpoise	GU117622	No E7
PphPV4	Dyopipapillomavirus 1	Harbor porpoise	GU117623	No E7
PsPV1	Omikronpapillomavirus 1	Burmeisters porpoise	AJ238373	No E7
PV-fi100-fish	Unclassified	Fish	OP933686	No E6 or E7
PV-wesgulfec-fish	Unclassified	Unidentified	MZ602149	No E6 or E7
RmarPV1	Unclassified	Cane toad	MW582900	No E6 or E7
SaPV1	Unclassified	Gilt-head bream	KX643372	No E6 or E7
SglaPV1	Unclassified	Wels catfish	MN515404	No E6 or E7
SsPV1	Dyodeltapapillomavirus 1	Domestic pig	EF395818	No E7
TberPV1	Unclassified	Emerald rockcod	MZ447865	No E6
TtPV1	Upsilonpapillomavirus 1	Bottlenosed dolphin	EU240894	No E7
TtPV2	Upsilonpapillomavirus 2	Bottlenosed dolphin	AY956402	No E7
TtPV3	Upsilonpapillomavirus 1	Bottlenosed dolphin	EU240895	No E7
TtPV4	Upsilonpapillomavirus 1	Bottlenosed dolphin	JN709469	No E7
TtPV5	Omikronpapillomavirus 1	Bottlenosed dolphin	JN709470	No E7
TtPV6	Omikronpapillomavirus 1	Bottlenosed dolphin	JN709471	No E7
TtPV7	Upsilonpapillomavirus 1	Bottlenosed dolphin	JN709472	No E7
TtPV8	Dyopipapillomavirus	Bottlenosed dolphin	MG744508	No E7
TtPV9	Omikronpapillomavirus	Bottlenosed dolphin	MG905161	No E7
UmPV1	Omegapapillomavirus 1	Polar bear	EF536349	No E7

Figure 3: Small E6 proteins encoded by avian papillomaviruses.

The amino acid sequences of E6 proteins encoded by papillomaviruses isolated from the Atlantic canary (Serinus canaria papillomavirus 1; ScPV1), mallard (Anas platyrhynchos Papillomavirus 1 and 2; AplaPV1 and AplaPV2), yellow-necked francolin (Francolinus leucoscepus Papillomavirus 1; FlPV1), Atlantic puffin (Fratercula arctica Papillomavirus 1; FarcPV1), American herring gull (Larus smithsonianus Papillomavirus 1; LsmiPV1), black-legged kittiwake (Rissa tridactyla Papillomavirus 1, RtriPV1), and Adelie penguin (Pygoscelis adeliae papillomavirus 1 and 2; PaPV1 and PaPV2) are shown and compared to the amino (E6N)- and carboxyl-terminal (E6C) regions of HPV16 E6. Identical residues are highlighted by black boxes and chemically similar residues by gray boxes. The CXXC (C=cysteine, X=any amino acid) metal binding motifs are highlighted in red.

Figure 4. Unique E9 proteins encoded by avian papillomaviruses.

The amino acid sequences of E9 proteins encoded by papillomavirus isolated from the gray parrot (Psittacus erithacus Papillomavirus 1; PePV1), yellow-necked francolin (Francolinus leucoscepus Papillomavirus 1; FlPV1), common chaffinch (Fringilla coelebs Papillomavirus 1; FcPV1), mallard (Anas platyrhynchos Papillomavirus 1; AplaPV1), black-legged kittiwake (Rissa tridactyla Papillomavirus 1, RtriPV1), Atlantic puffin (Fratercula arctica Papillomavirus 1; FarcPV1), American herring gull (Larus smithsonianus Papillomavirus 1; LsmiPV1), Atlantic canary (Serinus canaria papillomavirus 1; ScPV1), and Adelie penguin (Pygoscelis adeliae papillomavirus 1 and 2; PaPV1 and PaPV1), are shown. Identical residues are highlighted by black boxes and chemically similar residues by gray boxes. The E9 ORFs are overprinted on the E1 ORF. See Table 3 for a full list of papillomaviruses with E9 ORFs.

Table 3:

Animal PVs that encode E9 proteins.

Virus	Species	Host Common Name	GenBank ID	Notes
AplaPV1	Unclassified	Mallard	MK620303	Overprinted on E1
FarcPV1	Unclassified	Atlantic puffin	MK620302	Overprinted on E1
FcPV1	Etapapillomavirus 1	Common chaffinch	AY057109	Overprinted on E1
FlPV1	Dyoepsilonpapillomavirus 1	Yellow-necked francolin	EU188799	Overprinted on E1
LsmiPV1	Unclassified	American herring gull	MK620304	Overprinted on E1
PaPV1	Treisepsilonpapillomavirus 1	Adelie penguin	KJ173785	Overprinted on E1
PaPV2	Unclassified Treisepsilonpapillomavirus	Adelie penguin	MF168943	Overprinted on E1
PePV1	Thetapapillomavirus 1	Gray parrot	AF502599	Overprinted on E1
RtriPV1	Unclassified	Black-legged kittiwake	MK620305	Overprinted on E1
ScPV1	Unclassified Etapapillomavirus	Atlantic canary	MF564196	Overprinted on E1

Table 4:

HPVs and animal PVs that encode E10 proteins.

Virus	Species	Host Common Name	GenBank ID	Notes
HPV101	Gammapapillomavirus 6	Human	DQ080081	Does not encode E6
HPV103	Gammapapillomavirus 6	Human	DQ080078	Does not encode E6
HPV108	Gammapapillomavirus 6	Human	FM212639	Does not encode E6
HPV214	Unclassified Gammapapillomavirus	Human	MF509819	Does not encode E6
HPV226	Unclassified Gammapapillomavirus	Human	MG813996	Does not encode E6
HPV-mw02c24a	Unclassified Gammapapillomavirus	Human	MF588696	Does not encode E6
BPV3	Xipapillomavirus 1	Domestic cow	AF486184	Does not encode E6
BPV4	Xipapillomavirus 1	Domestic cow	X05817	Does not encode E6
BPV6	Xipapillomavirus 1	Domestic cow	AJ620208	Does not encode E6
BPV9	Xipapillomavirus 1	Domestic cow	AB331650	Does not encode E6
BPV10	Xipapillomavirus 1	Domestic cow	AB331651	Does not encode E6
BPV11	Xipapillomavirus 1	Domestic cow	AB543507	Does not encode E6
BPV12	Xipapillomavirus 2	Domestic cow	JF834523	Does not encode E6
BPV15	Xipapillomavirus 1	Domestic cow	KM983393	Does not encode E6
BPV20	Unclassified Xipapillomavirus	Domestic cow	KU519395	Overprinted on E6
BPV23	Unclassified Xipapillomavirus	Domestic cow	KX098515	Does not encode E6
BPV24	Unclassified Xipapillomavirus	Domestic cow	MG602223	Does not encode E6
BPV26	Unclassified Xipapillomavirus	Domestic cow	MG281846	Does not encode E6
BPV28	Unclassified Xipapillomavirus	Domestic cow	LC500686	Does not encode E6
BPV29	Unclassified Xipapillomavirus	Domestic cow	LC514113	Does not encode E6
BPV30	Unclassified	Domestic cow	OL672227	Does not encode E6
BPV31	Unclassified	Domestic cow	MW390885	Does not encode E6
BPV35	Unclassified	Domestic cow	MW404256	Does not encode E6
BPV36	Unclassified	Domestic cow	MW404257	Does not encode E6
BPV37	Unclassified	Domestic cow	MW404258	Does not encode E6
BPV38	Unclassified	Domestic cow	MW404259	Does not encode E6
BPV39	Unclassified	Domestic cow	MW404260	Does not encode E6
BPV40	Unclassified	Domestic cow	MW428425	Encodes E5_Epsilon
BPV41	Unclassified	Domestic cow	MW428427	Overprinted on E6
BPV42	Unclassified	Domestic cow	MW428428	Does not encode E6
BPV43	Unclassified	Domestic cow	MW428429	Does not encode E6
ChPV2	Unclassified	Domestic goat	MN148899	Overprinted on E6
OcPV1	Kappapapillomavirus 1	New Zealand white rabbit	AF227240	Overprinted on E6; Encodes E5_Unclassified
RtPV2	Xipapillomavirus 3	Reindeer	KC810012	Overprinted on E6
RtiPV2	Unclassified	Javan rusa	KT852571
SfPV1	Kappapapillomavirus 2	Cottontail rabbit	K02708	Overprinted on E6; Encodes E5_Unclassified

One might argue that studies on these accessory proteins encoded by animal PVs are difficult to justify since there are no obvious biomedical applications. If the past has taught us something, however, then studying obscure phenomena in non-human model organisms can lead to surprising and transformative discoveries such as RNA interference and CRISPR-based gene editing.

Concluding Remarks

It appears obvious that the gloomy predictions of the upcoming demise of papillomavirus research are greatly exaggerated. The field will continue to flourish as long as investigators continue to upend some of the “knowns”, start addressing some of the “mysteries”, and also dare to boldly explore the “unchartered territories”. In the current era of low funding rates and an apparent general lack of interest in cultivating viral oncology research with funding agencies, this may be a tall order. Funders may realize that the availability of a prophylactic vaccine, however efficacious it may be, has not and will not put an immediate end to the human suffering caused by HPV infections. As a community, we can start this process by embracing new, “disruptive” ideas and approaches and support and inspire young investigators to enter and established researchers to remain in this field. Continued research into the “curious” life cycle of various papillomaviruses will provide us with important insights into epithelial cell biology and point to signaling circuits and mechanisms that are generally relevant to our mechanistic understanding of diseases and cancers even those that are not caused by papillomaviruses.

Data Availability Statement

The sequence data that support the findings of this study are available in PAVE at https://pave.niaid.nih.gov/.