A Novel In Vivo Infection Model To Study Papillomavirus-Mediated Disease of the Female Reproductive Tract.

Tractable and efficient models of papillomavirus-induced pathogenesis are limited due to the strict species-specific and tissue-specific tropism of these viruses. Here, we report a novel preclinical murine model of papillomavirus-induced cervicovaginal disease in wild-type, immunocompetent mice using the recently discovered murine papillomavirus, MmuPV1. In this model, MmuPV1 establishes persistent viral infections in the mucosal epithelia of the female reproductive tract, a necessary component needed to accurately mimic HPV-mediated neoplastic disease in humans. Persistent MmuPV1 infections were able to induce progressive neoplastic disease and carcinogenesis, either alone or in combination with previously identified cofactors of papillomavirus-induced disease. This new model will provide a much-needed platform for basic and translational studies on both papillomavirus infection and associated disease in immunocompetent mice.

HPVs are the most common sexually transmitted infection in the United States (3). There are ϳ40 mucosotropic HPV genotypes that preferentially infect the mucosal, stratified epithelia of the anogenital tract (cervix, vagina, and anus) and the oral cavity (4,5). Nearly all cervical cancers are caused by high-risk mucosotropic HPVs (e.g., HPV16, -18, and -31) (6,7). Most HPV infections are transient, but persistent infection with high-risk genotypes can lead to the progression of precancerous lesions to invasive cancer (8,9). Although HPV vaccines protect against new high-risk HPV infections, they have no effect on preexisting infections and are largely unavailable in developing nations where HPV-associated cervical cancer is often the leading cause of death by cancer in women (10,11). Consequently, there remains an urgent need to explore more effective and accessible therapeutic interventions. Meeting this need would be greatly facilitated by the availability of a preclinical model in which papillomavirus infection results in cervical cancer.
Papillomaviruses exhibit strict species-and tissue-specific tropism, characteristics that have long hindered the ability to study aspects of papillomavirus infection and disease in an efficient laboratory model. While several models have been developed in rodents and primates (12)(13)(14)(15), these models are often costly and onerous due to the scarcity of available genetically modified hosts and reagents (15,16). Recently, a murine papillomavirus (MmuPV1 or MusPV1) was isolated from skin papillomas arising in T-cell-deficient, hairless FoxN1 nu/nu (nude) mice (17). MmuPV1 presents a valuable animal virus to study papillomavirus infection and disease in laboratory mice, which are tractable, genetically manipulable, and relatively affordable (17). MmuPV1 infects both cutaneous and mucosal epithelia and subsequently promotes disease and neoplastic progression (18)(19)(20)(21)(22)(23)(24). While MmuPV1 has been largely studied thus far in the context of cutaneous disease (17,22,(24)(25)(26)(27), MmuPV1 exhibits expanded tissue tropism and causes disease in the female reproductive tracts of FoxN1 nu/nu mice (18) and carcinoma in situ in FoxN1 nu/ϩ mice (28). We recently reported that UV radiation (UVR) makes wild-type, immunocompetent mice highly susceptible to MmuPV1-induced cutaneous disease (24). Prior studies using HPV transgenic mouse models established a role for the female hormone estrogen in papillomavirus-mediated carcinogenesis in mucosal epithelia of the reproductive tract (29)(30)(31) and demonstrated that estrogen and its nuclear receptor, estrogen receptor alpha (ER␣), are necessary cofactors required for the genesis and maintenance of cervical carcinogenesis (31)(32)(33). We therefore sought to determine if MmuPV1 causes neoplastic disease in the female reproductive tract of wild-type, immunocompetent mice and whether exogenous estrogen and UVR, alone or together, contribute to neoplastic progression. While MmuPV1 has been reported not to cause cutaneous disease efficiently in mice with intact immune systems (17,24,26,27), we found that infection with MmuPV1 induced formation of low-grade precancerous lesions in the female lower reproductive tracts of wild-type immunocompetent animals after 4 months. Treatment of MmuPV1infected mice with either UVR or estrogen led to high-grade precancerous lesions, and treatment with both UVR and estrogen led to an increased burden of precancerous lesions as well as cervicovaginal cancers. Allowing MmuPV1 infection to proceed for 6 months, either alone or in combination with exogenous estrogen treatment, was sufficient to promote the development of high-grade lesions and cancers. This is the first report of in vivo cervical carcinogenesis induced by a natural papillomavirus infection in immunocompetent mice, and these results provide a novel preclinical model system for basic and translational studies on papillomavirus-induced cervicovaginal disease.

RESULTS
Development and histopathological scoring of an MmuPV1 infection model in the murine female reproductive tract. To infect the lower female murine reproductive tract with MmuPV1 (Fig. 1A), we adapted a cervicovaginal infection method originally developed by Roberts and colleagues (34). We have previously used this same method to infect mice with HPV pseudovirus (35). Female mice were first treated with medroxyprogesterone acetate (Depo-Provera) to synchronize mice in diestrus and subsequently with nonoxynol-9 (N-9), causing chemical injury to the cervicovaginal epithelium to potentiate papillomavirus entry (34). Mice were then infected in the cervicovaginal canal with 10 8 VGE (viral genome equivalents) MmuPV1. Four to six months postinfection, reproductive tracts were harvested and histopathologically evaluated.
Our well-established scoring system, described in detail by Riley et al. (31), formed the framework that guided the overall descriptions of neoplastic disease in the novel MmuPV1 infection model (Fig. 1B). We have traditionally used these histopathological scoring criteria to grade progressive neoplastic disease in estrogen-treated HPV transgenic mice (29)(30)(31)(32), in which expression of the HPV16 oncogenes E6 and/or E7 is directed to basal cells of stratified epithelia using the keratin 14 (K14) promoter (36,37). The MmuPV1 infection model involves disease caused by an active, replicating virus, thus introducing additional pathological considerations. Criteria used to score neoplastic disease include tissue organization/architecture, degree of nuclear atypia, and presence or absence of the basement membrane. During the course of neoplastic progression through successive grades of intraepithelial neoplasia, there are corresponding increases in tissue disorganization and architectural complexity, involving expansion of the basal cell layer composed of epithelial cells with higher nuclear/ cytoplasmic ratio, and increased invaginations of the basement membrane into the  underlying stroma. Squamous cell carcinomas (SCC) achieve all of these criteria and can also include invasive cancer cell clusters, some of which are associated with keratin pearls.
In scoring MmuPV1-infected reproductive tracts, the separate cervical and vaginal scores were combined into one cervicovaginal score, as most dysplastic lesions arose on a continuous epithelial surface correlating with exposure to the virus inoculum. Cytological effects of the virus are evident in the reproductive tract, which is a notable difference from histopathological features of HPV16 transgenic models that do not involve natural infection. Focal areas of MmuPV1 infection resulted in clear boundaries between epithelium with normal histopathology and regions of epithelia with histological hallmarks of virus infection (Fig. 1C, panels 1 and 3a), including karyomegaly, accumulation of amphophilic to basophilic cytoplasm, occasional perinuclear "halos" akin to koilocytes characteristic of human papillomavirus infection, and dense homogeneous chromatin similar to that seen in inclusions of other viruses (Fig. 1C, panel 2). . SCC in the infected mice predominantly demonstrated a degree of architectural complexity exceeding characteristics of a dysplastic process and that are better regarded as carcinomas with a pushing border, though occasional foci of frank invasion in the form of small invasive clusters were also identified. A spectrum of histological changes was evident in MmuPV1-infected mice, and keratin pearls were also present in cases lacking sufficient complexity to be regarded as carcinoma. There were also areas exhibiting exophytic morphology (Fig. 1D, panel 1) and intense papillation into the underlying stroma (Fig. 1D, panel 2). These additional characteristics were incorporated into our previously established scoring guide in order to analyze MmuPV1-induced disease and neoplastic progression.
MmuPV1 infects and causes neoplastic disease in the murine female reproductive tract of immunocompetent mice alone and in combination with UVB and estrogen treatment. We sought to verify if our adapted infection protocol results in infection of the cervicovaginal tract in 6-week-old immunodeficient FoxN1 nu/nu mice infected with 10 8 VGE MmuPV1 for 4 months. Consistent with previous reports (18,28), we found that MmuPV1 establishes a productive virus infection in the mucosal epithelia of the lower reproductive tract of immunodeficient mice and results in neoplastic disease (see Fig. S1 in the supplemental material). All MmuPV1-infected FoxN1 nu/nu mice (n ϭ 9) developed neoplastic disease in the cervicovaginal epithelia, and 44% (n ϭ 4/9) developed SCC. These results demonstrated that our protocol for female reproductive tract infection results in productive MmuPV1 infection, thus establishing a platform for additional studies in wild-type, immunocompetent FVB mice.
We previously reported that UV B radiation (UVB) renders wild-type immunocompetent mice highly susceptible to MmuPV1-induced cutaneous disease (24). Estrogen has also been identified as a necessary cofactor for cervical carcinogenesis in HPV16 transgenic mouse models (29,(31)(32)(33). We therefore sought to determine whether MmuPV1 causes disease in the mucosal epithelia of the female reproductive tract in wild-type, immunocompetent FVB mice, either alone or in combination with UVB and/or exogenous estrogen. Combining our optimized infection methodology with additional cofactor treatments ( Fig. 2A), we infected four different cohorts of immunocompetent female FVB mice with 10 8 VGE of MmuPV1 virus for 4 months: (i) MmuPV1 only, (ii) MmuPV1-infected mice irradiated with 1000 mJ/cm 2 UVB (MmuPV1ϩUV), (iii) MmuPV1-infected mice treated with exogenous estrogen (0.05 mg 17␤-estradiol over 60 days) (MmuPV1ϩE2), and (iv) MmuPV1-infected mice that were both UVB irradiated and treated with estrogen (MmuPV1ϩUVϩE2). Each cohort also included mockinfected controls. To evaluate reproductive tracts for persistent MmuPV1 infection, cervicovaginal lavage samples were collected from infected mice at the study endpoint and tested for the presence of MmuPV1 DNA by adapting methods described previously (21,28). In this study, a persistent MmuPV1 infection is defined as an infection that remains detectable, either by MmuPV1-specific PCR of cervicovaginal lavage samples or by directly staining positive for viral transcripts or capsid proteins in cervicovaginal tissues, several months after the mice are initially infected. Briefly, total DNA was isolated from lavage samples and amplified by PCR using primers specific to the MmuPV1 E2 gene (Fig. 2B). While we routinely observed variability in the presence of MmuPV1 DNA in mice infected with MmuPV1 only and MmuPV1ϩUV, we consistently detected viral DNA in the cervicovaginal lavage samples of mice in the MmuPV1ϩE2 and MmuPV1ϩUVϩE2 cohorts. Pretreatment with medroxyprogesterone acetate was necessary to achieve persistent infections and subsequent disease in MmuPV1ϩUVϩE2 mice (Fig. S2). These results indicate that MmuPV1 can establish persistent infections in the mucosal epithelia of wild-type, immunocompetent FVB mice.
Reproductive tracts were initially harvested 4 months postinfection, and histopathological analysis was performed to score for neoplastic disease. To ascertain whether certain anatomical sites preferentially developed MmuPV1-induced disease, separate histopathological scores were first assigned separately for the vagina and cervix (Table 1), consistent with the scoring criteria for HPV16 transgenic mice (31). We found that most lesions developed in areas that were exposed to the virus inoculum, such as cervicovaginal fornices, outer cervix, outermost regions of the cervical canal, and the vaginal canal (Fig. 2C). The endocervix and transformation zone were rarely infected and rarely developed neoplastic lesions. The overall cervicovaginal disease severity was compared between each infected cohort and its mock-infected control cohort (Table 1). Disease severity in the MmuPV1-only and MmuPV1ϩUV groups was not significantly different from mock-infected counterparts (Mock versus MmuPV1 only, P ϭ 0.219; MockϩUV versus MmuPV1ϩUV, P ϭ 0.446). However, estrogen treatment significantly increased the severity of disease in MmuPV1ϩE2 mice (MockϩE2 versus MmuPV1ϩE2, P ϭ 0.004). Likewise, overall disease severity was significantly higher in MmuPV1infected mice treated with both UVB and exogenous estrogen (MockϩUVϩE2 versus MmuPV1ϩUVϩE2, P ϭ 0.001). We observed SCC development only in MmuPV1ϩUVϩ   E2-treated mice (n ϭ 5/13 cervix, 38%; n ϭ 7/13 vagina, 54%) at the 4-month endpoint. We found C567BL/6 mice infected with MmuPV1ϩUVϩE2 to be resistant to MmuPV1induced high-grade disease in the reproductive tract, and we were unable to detect any L1-positive cells 4 months postinfection in this strain of mice (Fig. S3).
We also compared cervicovaginal disease severity (by combining cervical and vaginal scores) among MmuPV1-infected cohorts of FVB/N mice (Fig. 2D). In the MmuPV1-only cohort, 25% (n ϭ 2/8) of mice were disease free and the remaining 75% (n ϭ 6/8) developed primarily low-to moderate-grade dysplasia (grade 2). When MmuPV1-infected mice were exposed to UVB radiation (MmuPV1ϩUV), nearly 80% of mice (n ϭ 7/9) developed neoplastic disease, 22% (n ϭ 2/9) of mice developed grade 2 dysplasia, and 56% (n ϭ 5/9) developed grade 3 dysplastic lesions. The effect of UVB on disease severity was not significant compared to MmuPV1 alone (P ϭ 0.11). However, exogenous estrogen treatment significantly increased cervicovaginal disease severity compared to virus alone (MmuPV1ϩE2 versus MmuPV1 only, P ϭ 0.001). All MmuPV1ϩE2 mice (n ϭ 8) developed moderate-to high-grade precancerous lesions. Although UVB radiation alone did not significantly increase overall disease severity in MmuPV1-infected mice, it appeared to synergize with exogenous estrogen treatment to promote SCC development. Nearly 70% of MmuPV1ϩUVϩE2 mice (n ϭ 9/13) developed SCC, and the remaining mice developed moderate-to high-grade dysplasias. This increase in cervicovaginal disease severity was highly significant (P ϭ 9.53 ϫ 10 Ϫ5 ) compared to mice infected with MmuPV1 but not treated with UV and E2. The effect of UVB and estrogen appeared to be synergistic, as the severity of disease in MmuPV1ϩUVϩE2 mice was significantly higher than with either factor alone (MmuPV1ϩUVϩE2 versus MmuPV1ϩUV, P ϭ 0.002; versus MmuPV1ϩE2, P ϭ 0.011).
These results indicate that MmuPV1 can establish persistent infections in the mucosal epithelia of the female reproductive tract of wild-type, immunocompetent FVB mice that result in SCC by 4 months when infected mice are treated with UV and estrogen.
Biomarker analysis of MmuPV1-infected reproductive tract epithelia in immunocompetent mice. Focusing on the MmuPV1ϩUVϩE2 cohort, where the most severe disease developed, we analyzed markers of MmuPV1 infection. We found MmuPV1 DNA, E1^E4 transcript, and L1 capsid protein expression both in the cervicovaginal epithelia and in cancers (Fig. 3A). While markers of MmuPV1 virus infection were detected in cancers, their expression was frequently lower than in nontumor epithelia even when regions of productively infected epithelia were present within close proximity to the tumor (Fig. 3B). We have previously found that high-risk HPV induces markers of DNA synthesis (Ki67) and E2F-dependent gene expression (MCM7) in suprabasal cells throughout all layers of the stratified squamous epithelium specifically due to the ability of HPV E7 to degrade pocket proteins (38,39). We analyzed reproductive tracts where epithelia showed histological hallmarks of virus infection for Ki67 and MCM7 (Fig. 3C). To assess virus-induced changes at this interface, we further analyzed the junctions between regions with normal histopathology and those with histopathological features consistent with virus infection. Qualitatively, we saw increased numbers of Ki67-positive basal cells in infected epithelia compared to controls. However, there was a slight increase in both Ki67 and MCM7-positive cells in the parabasal and suprabasal layers. In contrast to our findings with HPV16, we did not observe Ki67-or MCM7-positive cells throughout the full thickness of the MmuPV1infected stratified epithelia under any experimental condition.
In human cervical cancers, epithelial expression of the estrogen receptor ER␣ is progressively lost during the course of HPV-induced neoplastic progression (40). To determine the status of ER␣ expression in MmuPV1-induced disease, we performed immunohistochemistry for ER␣ on MmuPV1-infected reproductive tracts (Fig. 3D). Expression of ER␣ persisted both in MmuPV1-infected epithelia and in cancer epithelial cells, consistent with estrogen-treated HPV16 transgenic mice, where ER␣ expression is retained (33).
Extended duration of infection with MmuPV1 or MmuPV1؉E2 is sufficient to drive carcinogenesis. Approximately 75% of MmuPV1ϩE2 mice developed grade 3 cervicovaginal disease at 4 months postinfection ( Fig. 2D; Table 1). We previously demonstrated that HPV16 transgenic mice develop SCC when treated with estrogen for 6 months (31). We therefore tested the hypothesis that extended treatment with estrogen increases the severity of tumorigenesis in MmuPV1-infected mice. Mice were infected with MmuPV1, followed by 6 months of exogenous estrogen treatment. Groups of mock-infected, estrogen-treated mice and mice infected with only MmuPV1 for 6 months were included as controls. Overall disease severity in MmuPV1ϩE2 (6 months) was significantly increased over mock-infected controls (P ϭ 0.01; Fig. 4A). Although overall disease severity did not significantly increase in 6-month versus 4-month estrogen-treated MmuPV1-infected mice (P ϭ 0.13; Fig. 4B), approximately 43% (n ϭ 3/7) of MmuPV1ϩE2 (6 months) developed SCC, whereas no MmuPV1ϩE2 (4 months) mice had cancer. Therefore, 6 months of exogenous estrogen treatment was sufficient to promote carcinogenesis in MmuPV1-infected mice. Surprisingly, mice infected with MmuPV1 alone for 6 months also developed high-grade disease and SCC (Fig. 4A). Allowing MmuPV1 infection to proceed for 6 months instead of 4 months significantly increased the severity of disease (P ϭ 0.05; Fig. 4B), and SCC was observed in one mouse (n ϭ 1/8, 12.5%). High-grade lesions and SCC present in MmuPV1-only and MmuPV1ϩE2 mice at 6 months postinfection were associated with productively infected epithelia (Fig. 4C). These results indicate that prolonged MmuPV1 infection can promote cervicovaginal carcinogenesis in immunocompetent mice. Furthermore, extended estrogen treatment facilitates malignant progression in MmuPV1-infected mice.

DISCUSSION
Here we report a novel infection model for papillomavirus-mediated neoplastic progression in the murine female reproductive tract with MmuPV1 (Fig. 1). We confirmed that infection of the mucosal epithelia of the female reproductive tract in immunodeficient (FoxN1 nu/nu ) mice with MmuPV1 leads to neoplastic disease and malignant progression (see Fig. S1 in the supplemental material), including progression to SCC. Importantly, we demonstrate that wild-type, immunocompetent mice on the FVB genetic background are susceptible to MmuPV1-associated neoplastic disease and carcinogenesis in the female reproductive tract (Fig. 2; Table 1). Additionally, we report that two cofactors involved in papillomavirus-mediated disease previously identified by our laboratory, UV radiation in the context of MmuPV1 infection (24) and the female hormone estrogen in the context of HPV16 transgenic mice (31)(32)(33), contribute to MmuPV1 infection and malignant progression in this cervicovaginal infection model ( Fig. 2; Table 1). Unlike cutaneous epithelia, where MmuPV1 fails to cause robust phenotypic disease in wild-type, immunocompetent mice (24), we found that female

MmuPV1-Induced Cervicovaginal Neoplasia in FVB Mice
® reproductive tract mucosal epithelia infected with the same dose of MmuPV1 can develop low to moderate levels of dysplastic disease by 4 months postinfection (Fig. 2) and high-grade lesions and SCC by 6 months postinfection (Fig. 4). Estrogen significantly increased disease severity in MmuPV1-infected mice and cooperated with UV to promote cervicovaginal carcinogenesis at 4 months postinfection. However, extended treatment with estrogen alone was sufficient to drive carcinogenesis in MmuPV1infected mice (Fig. 4). Biomarker analysis indicated that common readouts of high-risk HPV infection, such as DNA synthesis and E2F-dependent gene expression, are not similarly increased in MmuPV1-infected cervicovaginal epithelia (Fig. 3), thus identifying interesting and likely informative areas of molecular virology for future study.
Recently, Cladel et al. reported a model in which MmuPV1-infected outbred heterozygous nude mice (FoxN1 nu/ϩ ) develop carcinoma in situ in the female reproductive tract (28). Although the investigators describe FoxN1 nu/ϩ mice as immunocompetent, there is evidence that FoxN1 haploinsufficiency affects the immune system. FoxN1 gene dosage is important for postnatal thymus homeostasis, and FoxN1 nu/ϩ mice exhibit several postnatal defects such as reduced weight and thymus size, reduction of thymocyte numbers, and impaired thymic epithelial cell (TEC) differentiation and proliferation (41,42). Moreover, expression of a group of genes that regulate TEC function is sensitive to FoxN1 gene dosage (43). FoxN1 haploinsufficiency led to reduced expansion of leukemic T cells (44). Therefore, the effects of FoxN1 haploinsufficiency on thymic organogenesis and function should be critically considered given the correlation between T-cell status and MmuPV1 pathogenesis (25,26). Our study demonstrating MmuPV1-induced cervicovaginal disease in FVB mice represents the first true example of de novo papillomavirus-mediated carcinogenesis in wild-type mice that are genetically immunocompetent.
Persistent infections in HPV-infected women significantly increase the risk for highgrade disease development (45). A notable aspect of this model is that the mucosal cervicovaginal epithelium of FVB mice supported persistent MmuPV1 infection, and persistent infection correlated with the severity of subsequent disease (Fig. 2). Some mice within the MmuPV1 and MmuPV1ϩUV groups did not establish persistent infections (Fig. 2B), likely contributing to their overall disease severity being the lowest among the four treatment groups (Fig. 2D). On the other hand, all MmuPV1-infected mice treated with estrogen, either alone or in combination with UV, established persistent infections. The level of exogenous estrogen administered to mice in our study can induce persistent estrus (30), leading to persistent proliferation of epithelial cells (46), which likely provides a more permissive environment for MmuPV1 to establish its life cycle. This finding is consistent with the observation that MmuPV1 viral copy numbers are highest during estrus phase in immunodeficient mice (21). We therefore hypothesize that estrogen facilitates the establishment and/or persistence of MmuPV1 infection in the female reproductive tract.
Viral persistence was also influenced by pretreatment with medroxyprogesterone acetate and genetic background. Mice that were not pretreated with medroxyprogesterone acetate were less likely to establish persistent MmuPV1 infections and consequently developed lower-grade disease (Fig. S2). One potential explanation for this finding is that medroxyprogesterone acetate induces diestrus, thinning the stratified epithelium and increasing access to basal cells that support establishment of infections. This compound also has immunosuppressive qualities that are known to increase other microbial infections (47,48). Since the immune environment is a critical factor in MmuPV1 cutaneous pathogenesis (24)(25)(26)(27), it is possible that alteration of immune responses by medroxyprogesterone acetate facilitates MmuPV1 infection in the female reproductive tract. We also found that mice on the C57BL/6 genetic background are resistant to MmuPV1-induced cervicovaginal disease (Fig. S3). This is consistent with other reports that C57BL/6 mice are susceptible to MmuPV1-induced cutaneous disease only upon complete T-cell depletion or UVB irradiation (22,24,25). While others have detected L1-positive cells in asymptomatically infected skin of C57BL/6 mice (25), we did not detect L1-positive cells in the reproductive tracts of MmuPV1ϩUVϩE2-infected C57BL/6 mice at 4 months postinfection (Fig. S3). Therefore, different genetic backgrounds and tissue sites exhibit variable susceptibility to MmuPV1 infection. Asymptomatic infections may exist in our model below the limit of detection, and longitudinal studies and additional readouts are required to characterize more fully MmuPV1 persistent infections and the contribution of other factors to this process.
We observed a significant effect of UVB radiation on cervicovaginal disease in MmuPV1-infected mice: MmuPV1ϩUVϩE2 mice developed high-grade precancerous lesions and SCC by 4 months postinfection (Fig. 2). We have recently reported that UV radiation (UVR) makes wild-type immunocompetent mice highly susceptible to MmuPV1-induced cutaneous disease by causing systemic immunosuppression (24). It is possible that UV promotes high-grade cervicovaginal disease by a similar mechanism, but one that also requires the action of estrogen. Recent epidemiological studies indicate a correlation between cervical cancer incidence and UV exposure in Caucasian females (49). UV radiation can also affect both skin-associated infections, such as those caused by herpes simplex virus (HSV-1), and others that are systemic and are noncutaneous (reviewed in reference 50). Therefore, the potential role of UV in papillomavirus-mediated cervicovaginal disease is an area that warrants further investigation and is an area that can be addressed using our immunocompetent MmuPV1 infection model.
The work of our lab and others has identified estrogen as a necessary cofactor required for cervical carcinogenesis in HPV16 transgenic mice (29,31,33), and we have demonstrated that this hormone contributes to the onset, maintenance, and malignant progression of HPV16-induced cervical cancer (32). Data presented here support and further strengthen the evidence arising from studies of HPV16 transgenic mice. Estrogen treatment increased the likelihood of establishing a persistent infection and significantly increased the incidence of high-grade precancerous lesions in MmuPV1infected mice at 4 months postinfection (Fig. 2), and 6 months of estrogen treatment was sufficient to promote carcinogenesis in MmuPV1-infected animals (Fig. 4). These data suggest that estrogen not only plays a role in malignant progression, as observed in HPV transgenic mice, but could also potentiate viral infection and persistence. This is the first demonstration of estrogen as a critical cofactor in promoting neoplastic progression in a natural infection model of papillomavirus-mediated cervicovaginal disease. In future studies, we will employ this model to further explore several preexisting areas of estrogen-related research, such as testing whether antiestrogen treatments can prevent and treat cervical cancer in a de novo infection setting, as we have shown previously in HPV16 transgenic mice (51). In HPV16 transgenic mice, we found that the effect of estrogen on cervical carcinogenesis is mediated by estrogen signaling in the stroma (40,52) and that E6 and E7 oncogenes profoundly affect gene expression in the cervical microenvironment (53). Therefore, it will be important to determine if similar changes occur in a natural infection model.
We found reduced expression of viral infection markers in tumors compared to nearby MmuPV1-infected epithelia (Fig. 3). It is possible that MmuPV1 integrates into the host genome, leading to persistent viral oncogene expression as seen with high-risk mucosal HPVs (54). It remains to be determined whether MmuPV1 integrates into the host genome in the cervicovaginal mucosal epithelia in immunocompetent mice, but our RNA-seq analysis of MmuPV1-induced skin papillomas arising in nude mice indicates the presence of virus-host chimeric reads consistent with viral genome integration (unpublished data). Therefore, MmuPV1 infection models open the door to studying papillomavirus viral genome integration in vivo and its association with disease. Some rodent papillomavirus proteins, including MmuPV1, have different expression patterns, structural motifs, and binding partners than HPVs (15,55). Several dependable markers of HPV oncogene function, such as increased suprabasal DNA synthesis and E2F-dependent gene expression, were not markedly affected in MmuPV1-infected cervicovaginal epithelium (Fig. 3C). While some studies have been initiated (55,56), the biochemical properties and molecular functions of MmuPV1 E6 and E7 need to be further explored, as it is still unclear whether and/or how well MmuPV1 viral oncogenes mirror HPV oncogene functions, although both clearly have oncogenic potential.
In summary, we have developed a novel preclinical model of papillomavirusinduced cervicovaginal disease by infecting the female reproductive tracts of wild-type mice with MmuPV1. By establishing our model in immunocompetent mice, this system provides a physiologically relevant platform that more accurately models several aspects of papillomavirus-induced disease in the female reproductive tract that can be used to advance several areas of basic and translational research in the papillomavirus field.

MATERIALS AND METHODS
Animals. The following mice were included in this study: 6-to 8-week-old immunodeficient athymic FoxN1 nu mice (Hsd:Athymic Nude-FoxN1 nu ; Envigo, Somerset, NJ) and immunocompetent, wild-type FVB/N mice (Taconic Biosciences, Albany, NY) and C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). All animal experiments were performed in full compliance with standards outlined in the Guide for the Care and Use of Laboratory Animals (57)  Tissue procurement, processing, and histopathological analysis. Reproductive tracts were harvested, fixed in 4% paraformaldehyde, and paraffin embedded. Serial sections (5 m) were cut, and every 10th section was stained with H&E and evaluated by histopathological analysis and scored for worst disease by a trained pathologist in the Department of Pathology and Laboratory Medicine (University of Wisconsin School of Medicine and Public Health). The scoring system is described in detail in Results. Images of H&E-stained cervical tumors and epithelia were captured using a Zeiss AxioImager M2 microscope and AxioVision software version 4.8.2 (Jena, Germany). Full-slide scans of H&E-stained slides were performed using an Aperio ScanScope XT slide scanner using a 20ϫ/0.75 Plan Apo objective.
MmuPV1 infection of female reproductive tracts. Mice were infected with MmuPV1 virus stock generated by isolating virions from papillomas from infected FoxN1 nu/nu mice as described previously (24). The female reproductive tract infection strategy was adapted from methods described previously (34,35). Briefly, mice were injected subcutaneously with 3 mg medroxyprogesterone acetate (Amphastar Pharmaceuticals, Rancho Cucamonga, CA) 4 days prior to MmuPV1 infection to induce diestrus. On the day of the infection, mice were pretreated vaginally with 50 l Conceptrol (Options; catalog no. 247149) containing 4% nonoxynol-9 to induce chemical injury to the cervicovaginal epithelium (34). At 4 h after treatment with Conceptrol, 10 8 VGE MmuPV1 virions suspended in 25 l 4% carboxyl methylcellulose (Sigma; catalog no. C4888) were delivered intravaginally. All treatments were performed while mice were anesthetized with 5% isoflurane.
Estrogen treatment. Treatment with exogenous estrogen was performed as described previously (29,32). Briefly, female mice were anesthetized with 5% isoflurane, and a continuous-release estrogen (E2) tablet (17␤-estradiol; 0.05 mg/60 days; Innovative Research of America, Sarasota, FL) was inserted subcutaneously in the shoulder fat pads of the dorsal skin. For those mice receiving estrogen, treatment began 5 days following MmuPV1 infection. A new tablet was inserted every 2 months as needed.
UVB radiation. Animals were exposed to a single dose of UVB at 1000 mJ/cm 2 as described previously (24,58). UVB was administered using a custom-designed research irradiation unit (Daavlin, Bryan, OH) with lamps controlled using Daavlin flex control integrating dosimeters.
FISH for MmuPV1 DNA. MmuPV1 DNA fluorescent in situ hybridization (FISH) was performed as described previously (58,59). This protocol has been adapted from a DNA FISH protocol used to detect Epstein-Barr virus (EBV) DNA in monolayer cells and is described in detail at https://mcardle.wisc.edu/ sugden/protocols.html.
RNA in situ hybridization. MmuPV1 viral transcripts were detected using RNAscope 2.5 HD Assay-Brown (Advanced Cell Diagnostics, Newark, CA) according to the manufacturer's instructions (61) with probes specific for MmuPV1 E1^E4 (catalog no. 473281). Tissue sections were treated following protease treatment and prior to probe hybridization with 20 units of DNase I (Thermo Fisher Scientific; catalog no. EN0521) for 30 min at 40°C. Slides were counterstained with hematoxylin before mounting and coverslipping.
Fluorescence image acquisition. High-resolution wide-field fluorescent images were acquired using a Leica SP8 3X STED microscope (61) by means of a 20ϫ lens objective (specifications: HC PL APO 20ϫ/0.75 CS2, dry) and the LAS-X suite (version 2.0.1). All other images were captured using a Zeiss AxioImager M2 microscope and AxioVision software version 4.8.2 (Jena, Germany).