Probes for INS

Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

A murine papillomavirus, MmuPV1, infects both cutaneous and mucosal epithelia of laboratory mice and can be used to model high-risk human papillomavirus (HPV) infection and HPV-associated disease. We have shown that estrogen exacerbates papillomavirus-induced cervical disease in HPV-transgenic mice. We have also previously identified stress keratin 17 (K17) as a host factor that supports MmuPV1-induced cutaneous disease. Here, we sought to test the role of estrogen and K17 in MmuPV1 infection and associated disease in the female reproductive tract. We experimentally infected wild-type and K17 knockout (K17KO) mice with MmuPV1 in the female reproductive tract in the presence or absence of exogenous estrogen for 6 mon. We observed that a significantly higher percentage of K17KO mice cleared the virus as opposed to wild-type mice. In estrogen-treated wild-type mice, the MmuPV1 viral copy number was significantly higher compared to untreated mice by as early as 2 wk postinfection, suggesting that estrogen may help facilitate MmuPV1 infection and/or establishment. Consistent with this, viral clearance was not observed in either wild-type or K17KO mice when treated with estrogen. Furthermore, neoplastic disease progression and cervical carcinogenesis were supported by the presence of K17 and exacerbated by estrogen treatment. Subsequent analyses indicated that estrogen treatment induces a systemic immunosuppressive state in MmuPV1-infected animals and that both estrogen and K17 modulate the local intratumoral immune microenvironment within MmuPV1-induced neoplastic lesions. Collectively, these findings suggest that estrogen and K17 act at multiple stages of papillomavirus-induced disease at least in part via immunomodulatory mechanisms.

The artemisinin family of compounds is cytopathic in certain cancer cell lines that are positive for human papillomaviruses (HPV) and can potentially drive the regression of dysplastic lesions. We evaluated the efficacy of topical dihydroartemisinin (DHA) on cervical dysplasia and anal dysplasia in two papillomavirus mouse models: K14E6/E7 transgenic mice, which express HPV16 oncogenes; and immunodeficient NOD/SCID gamma (NSG) mice infected with Mus musculus papillomavirus (MmuPV1). Mice started treatment with DHA at 25 weeks of age (K14E6/E7) or 20 weeks post infection (MmuPV1-infected), when the majority of mice are known to have papillomavirus-induced low- to high-grade dysplasia. Mice were treated with or without topical DHA at the cervix or anus and with or without topical treatment with the chemical carcinogen 7,12 dimethylbenz(a)anthracene (DMBA) at the anus of in transgenic mice to induce neoplastic progression. Mice were monitored for overt tumor growth, and tissue was harvested after 20 weeks of treatment and scored for severity of histological disease. For MmuPV1-infected mice, anogenital lavages were taken to monitor for viral clearance. Tissues were also evaluated for viral gene expression at the RNA and/or protein levels. Treatment with topical DHA did not reduce dysplasia in the anogenital tract in either papillomavirus-induced mouse model and did not prevent progression to anal cancer in the DMBA-treated K14E6/E7 mice.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.

Select protease inhibitors (PI) have been found to be effective in decreasing human papillomavirus oncoprotein expression. This study evaluated whether the topical PI, Saquinavir (SQV), promotes viral clearance in an infectious mouse model with Mus musculus papillomavirus 1 (MmuPV1). NOD scid gamma (NSG) mice were anally infected with ∼4 × 108 viral genome equivalents of MmuPV1 and 120 days post-infection (when majority have high-grade anal dysplasia), began topical treatments: control (mock), 7,12-dimethylbenz(a)anthracene (DMBA) only, once weekly to promote carcinogenesis, 1% SQV only, daily (Monday - Friday), and SQV + DMBA. Viral MmuPV1 load was analyzed from anal lavages pre and post-treatment. Anal tissue was harvested, processed, and evaluated for drug absorption, grade of anal disease, and anal viral RNA. Results suggest that topical SQV promotes decreased viral shedding in female mice treated with SQV.

Penile Squamous Cell Carcinoma (PSCC) is associated with high-risk human papillomavirus (HR-HPV). The immunohistochemical (IHC) test for p16INK4a (p16) is highly correlated with HR-HPV expression in other SCCs. To investigate whether the expression of p16 IHC or HR-HPV is associated with survival in PSCC, we conducted a single institution analysis of 143 patients with a diagnosis of PSCC and, available tissue were tested for p16 IHC staining patterns, histological subtype, tumor grade, and lymphovascular invasion (LVI) by an experienced pathologist. HR-HPV status using the Cobas PCR Assay or the RNAScope high-risk HPV in situ hybridization kit were also assessed. Patient characteristics were summarized using descriptive statistics of clinico-pathologic variables. Kaplan-Meier was used to estimate median overall survival (OS), cancer specific survival (CSS) and correlated with HPV, p16, and other study variables. Patients with p16+ tumors had a significantly longer median CSS in comparison to the p16- group (p = 0.004), with respective 5-year CSS probability of 88% (95% CI; 0.84, 1) versus 58% (95% CI; 0.55, 0.76; p = 0.004). HPV status did not predict survival outcomes. Multivariable analysis with respect to OS and CSS, showed that p16+ status was associated with a lower risk of death (HR = 0.36, 95%CI; 0.20-0.67, p = 0.001), and improved CSS (HR = 0.20, 95% CI; 0.07-0.54, p = 0.002) after adjusting for covariates. In conclusion, tumor p16 status via IHC was an easy to perform independent prognostic factor for OS and CSS that correlates with HR-HPV expression.

Although both the 2014 and 2020 World Health Organization (WHO) criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma (ASC), in practice, ASC diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphologic, and clinical features and outcomes associated with ASCs, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed ASCs (including glassy cell carcinoma and related lesions) to confirm an ASC diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as ASCs, 34 retained their ASC diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or ASCs), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy adenocarcinomas were reclassified as poorly differentiated HPV-associated carcinomas based on morphology and immunophenotype. There were no significant immunophenotypic differences between ASCs and pure invasive stratified mucin-producing carcinomas with regard to HPV and other markers including p16 expression. Although limited by a small sample size, survival outcomes seemed to be similar between all groups. ASCs should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The 2 putative glassy cell carcinomas studied did not meet our criteria for ASC and categorizing them as such should be reconsidered.