Human papillomavirus (HPV) epidemiological data from Pakistan remain limited, particularly in unscreened and unvaccinated populations. We performed a crosssectional study in Karachi, Pakistan, to determine overall type-and age-specific HPV prevalence between May 2022 and November 2023. A total of 3119 women were invited to participate; 497 women gave consent, provided cervical samples and were included in the final analysis. HPV detection and genotyping were done using the highly sensitive INNO-LiPA HPV Genotyping Extra II line probe assay. Overall HPV prevalence was 16.7%. High-risk (HR) HPV types (counted once per genotype within IARC groups 1, 2A and 2B) were detected in 11.8% of women, and low-risk or unclassified types in 5.0%. Single HR-HPV infections occurred in 8.4% and multiple HR-HPV infections in 3.4%. Fourteen HR genotypes were detected as single infections, with HPV31 and HPV53 (1.2% each) being most prevalent, followed by HPV68 (1.0%), HPV16 (0.8%), and HPV33/HPV39/HPV51 (0.6% each). Less frequent types included HPV82 and HPV18 (0.4% each), and HPV35/ HPV70/HPV73 (0.2% each). The most prevalent low-risk type was HPV6 (1.2%). HPV prevalence peaked in women aged 25-34 years (21.4%) and remained detectable in women above 54 years (12.5%). These data indicate a substantial and genetically diverse HR-HPV burden in Karachi. They support organized HPVbased cervical cancer screening using high-performance assays that detect the full panel of carcinogenic HPV types, with adequate triage and follow-up. They also support rapid scale-up and monitoring of HPV vaccination in Pakistan, including assessment of broader-valency vaccination strategies where feasible, to reduce cervical cancer, pre-cancerous lesions and other HPV-related cancers.

Flaviviruses pose a major threat to global health and can cause severe disease in animals and humans. Among them, tick-borne encephalitis virus (TBEV) and related members of the tick-borne encephalitis (TBE) serocomplex are transmitted primarily by ticks and can lead to neurological disease, including encephalitis and death. Despite many years of research on the TBE-serocomplex, no specific antiviral treatment has been developed. Langat virus (LGTV), another member of this serocomplex typically causes asymptomatic infections in humans, but may cause severe neurological disease in rare cases. Moreover, experimentally LGTV-infected mice and monkeys show neurological symptoms. While viruses of the TBE-serocomplex are canonically transmitted by ticks, tick-independent transmission of LGTV has been reported in mice, suggesting a broader transmission potential. Notably, the full host range, vector competence and global distribution of LGTV remain poorly defined, and climate change-driven expansion of tick vectors may enable the emergence of this virus in new regions. In this review, we summarise our current knowledge of LGTV, focussing on vector competence, transmission routes, tissue and host tropism, pathogenicity and animal models. Furthermore, we discuss the potential application of LGTV as a vaccine candidate for TBEV. By highlighting the unique features of LGTV and identifying current research gaps, we aim to inspire interest in this understudied virus and its value as a tool to uncover fundamental aspects of tick-borne flavivirus biology and strategies for vaccine development.

Torque teno virus (TTV) DNAemia is a surrogate marker of immune function in renal transplant recipients (RTRs), usually peaking around 3 months after transplantation. Post-transplantation diabetes mellitus (PTDM), a condition with impaired immune response, is common after renal transplantation, but its relationship with TTV replication remains unclear. In this retrospective study, TTV-DNA loads were analysed in 303 plasma samples from 93 RTRs collected shortly after transplantation and at 3, 6, and 12 months. Clinical and laboratory parameters, including glycaemic status, were assessed. 17 patients (18.3

Persistent infections with high-risk human papillomaviruses (HPV) can result in different malignancies. Productive replication of HPV is normally restricted to suprabasal keratinocytes that have entered terminal differentiation and is characterized by vegetative genome amplification, activation of the late promoter, and expression of the viral late E4 protein. Cells undergoing productive replication remain in a prolonged G2 phase and exit the cell cycle without division. The viral E8^E2 protein binds to NCoR/SMRT co-repressor complexes to repress viral transcription and replication in undifferentiated keratinocytes, but the biological rationale for this repression has remained unclear. Recent studies have revealed that Mus musculus PV1 E8^E2 prevents late viral E4 expression in undifferentiated cells to enable tumor formation in vivo. Here, we demonstrate that loss of E8^E2 function in high-risk HPV31 leads to inappropriate activation of genome amplification in undifferentiated keratinocytes, resulting in expression of E4 protein and cell cycle perturbation which explains why HPV31 E8^E2 mutant genomes fail to be maintained as episomes and instead are always found integrated in surviving cell lines. Interestingly, this is independent from E4 expression suggesting that vegetative genome amplification is sufficient to prevent cell division. Remarkably, depletion of NCoR/SMRT complexes in cell lines maintaining HPV31 episomes phenocopies E8^E2 inactivation and induces genome amplification and E4 expression. Notably, most E4-positive cells generated by E8^E2 inactivation or NCoR/SMRT depletion retain basal-like characteristics, indicating that genome amplification and E4 expression can be uncoupled from differentiation when E8^E2 repression is relieved. Interestingly, differentiation diminishes the effects of NCoR/SMRT depletion, suggesting that E8^E2 activity is likely inactivated during differentiation to permit productive replication. Collectively, these findings identify E8^E2 as a critical gatekeeper preventing premature genome amplification and E4 expression in basal keratinocytes and suggest that targeting the E8^E2-NCoR/SMRT interaction may represent a novel antiviral strategy.

Dengue virus (DENV) poses a critical health threat causing millions of cases every year. The absence of proof-reading activity of the RNA-dependent RNA polymerases (RdRp) leads to significant mutations in its genome. This may result in genetic diversity among the genotypes which contribute to severe complications during the infection. Targeting these mutations may help to understand the viral transmission and identify a potential treatment. An aggregate of 152 dengue positive serum samples were collected from different tertiary care hospitals in Karachi. RNA isolation, cDNA synthesis as well as amplification of the C-prM region was performed, followed by the Sanger sequencing. The resultant sequences of each serotype were aligned with their respective genome sequences and phylogenetic trees based on genotypes were constructed using Maximum Likelihood (ML) method with 1000 bootstraps replicates. The sequences were then converted to the amino acid sequence and genetic variations in comparison to the reference genomes were evaluated. The results showed that there was a higher number of positive cases in males (58%) than in females (42%). The most affected age group was 21-30. The study suggested that the dominant serotype in the population of Karachi was DENV-1 (78.33%), followed by DENV- 2 (15%) and DENV-3 (6.66%). Overall, the acquired data provide the information about genetic variation in the C-prM region of dengue virus in Karachi, Pakistan.

Epidermodysplasia verruciformis (EV) is an autosomal recessive disorder characterized by an extraordinary susceptibility to infections with human papillomaviruses (HPV), mainly from the genus beta. EV patients carry biallelic loss-of-function mutations in TMC6 encoding Transmembrane channel-like protein 6 or EV protein 1 (EVER1), TMC8 encoding Transmembrane channel-like protein 8 or EV protein 2 (EVER2), or CIB1 encoding Calcium and integrin-binding protein 1. TMC6, TMC8 and CIB1 form a protein complex in the endoplasmic reticulum which has been hypothesized to act as a restriction factor for beta-HPV in keratinocytes. SiRNA-mediated knock-down of CIB1, TMC6, or TMC8 greatly reduces transcript and protein levels, but does not activate beta-HPV8 gene expression in normal keratinocytes. TMC6 and TMC8 transcript levels are much lower in normal and HPV-positive human keratinocytes than in CD4+ T-cells suggesting that the levels are too low for anti-viral activity. However, neither the activation of DNA sensing pathways by HPV genomes, supernatants from activated immune cells, nor activation of pathways important for the viral life cycle such as the DNA damage response or keratinocyte differentiation induce high levels of TMC6 or TMC8 in normal keratinocytes. This is consistent with findings that TMC6 and TMC8 do not restrict Mus musculus PV1 expression in keratinocytes. Taken together, we find no evidence for restriction factor activity of EV susceptibility genes for beta-HPV8 or conditions to induce high levels of TMC6 and TMC8 in keratinocytes. Thus, it is plausible that the EV phenotype in humans may be associated with an immune deficiency involving immune cells.

High-risk human papillomaviruses (HPV), particularly HPV16, are major causes of anogenital and oropharyngeal cancers. The HPV late promoter, P670 in the case of HPV16, is activated upon host cell differentiation and drives the expression of viral capsid proteins. While differentiation-specific host transcription factors have been implicated in regulating this promoter, the mechanism remains incompletely understood. HPV E2 proteins activate transcription by interacting with the host protein BRD4 (Bromodomain-containing protein 4). A biotin proximity ligation screen identified several novel E2 interactors, of which many overlap with the BRD4 interactome, suggesting BRD4 mediates a large fraction of these interactions. One such interactor, ZC3H4 (Zinc finger CCCH domain-containing protein 4), is known to restrict the expression of long non-coding RNAs, including enhancer and promoter upstream antisense RNAs (uaRNAs). E2 recruits ZC3H4 in a BRD4-dependent manner to specifically activate the P670 promoter in reporter assays. Supporting this, E2 and ZC3H4 co-localize in cells with high P670 activity. ZC3H4 is upregulated during differentiation, and its knockdown in differentiated HPV16- or HPV31-positive cells reduces late viral transcripts in an E2-BRD4-dependent manner. Interestingly, knockdown of ZC3H4 does not increase viral uaRNAs, suggesting that ZC3H4 does not enhance HPV late transcription by regulating viral antisense transcription. IMPORTANCE:High-risk human papillomaviruses (HPVs), particularly HPV16, can cause anogenital and oropharyngeal cancers. HPV16 relies on the differentiation-dependent activation of its late promoter, P670, to produce capsid proteins. While host transcription factors contribute to this regulation, the mechanisms remain incompletely defined. Our findings reveal that the viral E2 protein collaborates with the host protein BRD4-a critical transcriptional regulator-to recruit other cellular partners, such as ZC3H4. Normally, ZC3H4 suppresses non-coding RNAs in cells, but HPV16 repurposes it via BRD4 to activate P670. This interaction intensifies in differentiated cells, where ZC3H4 levels rise, and disrupting ZC3H4 specifically blocks late viral gene expression without affecting antisense viral transcription. This highlights a unique, differentiation-dependent strategy HPV16 uses to hijack host machinery for its replication.

Human papillomaviruses (HPV) from the genus beta have been implicated in the development of cutaneous squamous cell cancer in epidermodysplasia verruciformis and organ transplant patients. In contrast to alpha-high-risk HPV, which cause ano-genital and oropharyngeal cancers, beta-HPV replication is not well understood. The beta-HPV49 transcriptome was analyzed by RNA sequencing using stable keratinocyte cell lines maintaining high levels of extrachromosomally replicating E8- genomes, which can be established due to a lack of the viral E8^E2 repressor protein. This analysis indicated the presence of four transcription start sites, two polyadenylation signals, and splice donor (SD) and acceptor sites consistent with the conserved gene expression patterns of animal and human PV. Surprisingly, a novel SD in the E6 oncogene (SD217) was identified resembling the SD in E6 of carcinogenic alpha-HPV. Mutation of SD217 enhanced E6 protein expression but had no influence on the growth of keratinocytes transduced with retroviral HPV49 E6 and E7 expression vectors. Inactivation of SD217 in the context of the HPV49 wild-type genome did not enable immortalization and prevented immortalization in the context of the E8- genome. The analysis of SD217 mutant genomes revealed a strong down-regulation of SD217 usage, but only weak effects on other viral transcripts. This suggests that SD217 does not contribute to immortalization by modulating viral gene expression. Usage of SD217 is increased in immortalized E8- cell lines compared with transiently transfected cells, which may indicate that long-term extrachromosomal maintenance requires reduced E6 protein levels.IMPORTANCEHigh-risk (hr) human papillomaviruses (HPV) from the genus alpha cause ano-genital and oropharyngeal cancers, whereas beta-HPV have been implicated to cause skin cancer in epidermodysplasia verruciformis and organ transplant patients. In contrast to alpha hr-HPV, the replication cycle of beta-HPV is not very well understood. Transcriptional profiling of beta-HPV49 by RNA sequencing reveals transcription start sites and splice sites conserved among HPV. Surprisingly, a splice donor site in the E6 oncogene (SD217), previously only described for hr-HPV, was identified that controls E6 oncoprotein levels and is required for immortalization of keratinocytes by the HPV49 genome.

The COVID-19 pandemic caused immense mortality and morbidity reporting 704,753,890 cases worldwide. The repercussions of this pandemic are still being felt in the form of newly evolving variants and infections. The pandemic has pointed towards the need for the development of new and effective agents against SARS-CoV-2 infection. Sulfonohydrazides are a class of compounds with a wide range of therapeutic potential. The present study aims to identify the anti-SARS-CoV-2 potential of Sulfonohydrazide compounds. Twenty-five Sulfonohydrazides derivatives were evaluated for anti-viral potential via plaque reduction assay (PRA) and cytopathic effect (CPE) analysis in-vitro. Treatment point assay was employed for the strategic evaluation of antiviral compound at the particular stages of the SARS-CoV-2 life cycle. Gene expression analysis was also carried out, which was supported by immunofluorescence assays targeting the N and S proteins of SARS-CoV-2, alongside fold-change analysis, to identify a robust and multifaceted approach for the understanding of viral dynamics. Moreover, ligand-inhibitor interactions were assessed by in- silico studies. Compound 24 (4(E)-4-methyl-N'-(2,3,4-trihydroxybenzylidene)benzenesulfonohydrazide) was identified as the most potent molecule that inhibited SARS-CoV-2 infection (92.85 ± 3.57%) via PRA. The time point assay revealed that the effect of the compound might be at the entry point, which might be due to the down-regulation of the Spike (S) and Angiotensin-converting enzyme 2 (ACE-2) genes by the compound. The gene expression analysis of ORF1a/b by qRT-PCR indicated reduction in viral load after compound treatment, as indicated by a higher cycle threshold (Ct) value. Moreover, the compound 24 also downregulated the expression of S, RdRp, and ACE-2. Furthermore, the interaction of compound 24 with S, RdRp, and ACE-2 was predicted via molecular docking, which validated the interaction and possible anti-SARS-CoV-2 effect. Additionally, immunofluorescence staining analysis of spike and nucleocapsid proteins also showed downregulation in SARS-CoV-2 infected cells. Overall, the acquired data suggested that Sulfonohydrazide derivative 24 inhibits SARS-CoV-2 entry and replication.

Identification of previous SARS-CoV-2 infection typically relies on serology, yet T-cells play a key role in the adaptive immune response against SARS-CoV-2. Here, we investigated in parallel the SARS-CoV-2-specific as well as endemic human coronavirus-specific humoral and cross-reactive cellular responses in children and adults. We analyzed clinical data and blood samples from a family cohort of 96 children and 144 adults at 3-4 and 11-12 months after their first contact with SARS-CoV-2. Humoral response was assessed by a multiplex immunoassay with high sensitivity and specificity (MULTICOV-AB). Cellular responses were analyzed by IFN-γ ELISPOT using four different established epitope compositions (ECs) to discriminate between SARS-CoV-2 specific and HCoV cross-reactive T-cell responses. While the majority of adults had a combined serological and T-cell response, relatively more children had a T-cell response alone rather than a combined response. The magnitude of the T-cell response correlated with symptoms and the humoral response. In addition, SARS-CoV-2 infection significantly boosted the endemic coronavirus-specific cellular response. Overall, our data suggest discordant humoral and cellular responses, reflecting either abortive infection, cellular sensitization with rapid viral clearance or rapid antibody waning or a combination of these phenomena. Restricting epidemiologic analysis to SARS-CoV-2 serological data may underestimate rates of infection with or at least exposure to SARS-CoV-2 in children.

Introduction:During gestation the immune system undergoes dramatic remodelling to protect the maternal-fetal dyad from infections whilst also preventing fetal rejection. We investigated how SARS-CoV-2 modifies the immune landscape during infection and in recovered pregnant women. Methods:We immunophenotyped our two independent geographical cohorts using a 14-colour flow cytometry panel (surface and intracellular staining). We estimated cytokines and SARS-CoV-2 IgG antibodies in validation cohort using a multiplexd flow cytometry panel. Single-cell RNA sequencing (scRNA-seq) was performed using a Chromium Single Cell 3' Gel Bead Chip and Library Kit from 10x Genomics (Drop-seq method). Furthermore, we estimated the cytotoxic functions of natural killer (NK) cells by flow cytometry using surface and intracellular staining. Results:Using two independent geographical cohorts, we identified that NK cells had a sustained reduction during active infection and after recovery. Further, scRNA-seq data revealed that infection with SARS-CoV-2 rewired the gene expression profile of NK, monocytes, CD4+, CD8+ effector T cells and antibody producing B cells in convalescent pregnant women. Several gene pathways associated with cytotoxic function, interferon signalling type I & II, and pro- and anti-inflammatory functions in NK and CD8+ cytotoxic T cells were attenuated in recovered pregnant patients compared with healthy pregnancies. We validated our scRNA-seq of NK cells from convalescent pregnant women and confirmed that NK cells had diminished levels of cytotoxic proteins; perforin, CD122 and granzyme B. Discussion:Overall, our study uncovers that SARS-CoV-2 infection deranges the adaptive immune response in pregnant women even after recovery and may contribute to post-COVID19 sequalae of symptoms.

We performed a population-based study in densely populated areas of the largest city of Pakistan (Karachi) to determine the overall type- and age-specific prevalence of human papillomavirus (HPV) in the setting of an unscreened and non-vaccinated female population between May 2022 and November 2023. Women (n=3,119) were invited to participate in the study from whom a total of 497 women gave consent and provided cervical samples. HPV positivity was determined and specific HPV genotypes were identified using the INNO LiPa Extra II-line probe assay. Total HPV positivity among all age groups was 16.7%. High risk HPV types (groups 1 and 2) were found in 11.9% whereas low risk types and unclassified types were 4.8% in all samples. Fourteen HR types were detected as single infections (8.4%), highest prevalent types are HPV-31 and HPV-53 (each 1.2%) followed by HPV-68 (1.0%), HPV-16 (0.8%), HPV-33, -39 and -51 (each 0.6%). While HPV-35, HPV-52, HPV-70, HPV-73 and HPV-82 were the least prevalent types. The most prevalent (1.2%) low-risk HPV type detected was HPV-6 of all samples. The highest HPV prevalence (21.3%) was observed in subjects aged 25–34 years (n=220), whereas in the age group above 54 years (n=24) we detected HPV in 12.5% of samples tested. Single HR HPV infections were observed in 8.4%, while multiple HR infections were detected in 4.02% of the 497 women tested. Extrapolating this data to the total female population of Pakistan allows to estimate that about 20 million women are HPV positive. This rough estimation forms a strong basis of an organized cervical cancer screening program using high precision HPV tests for early detection of HPV infections and related diseases including cervical cancer. In addition, this also establish the need of implementation of immunization program with the recently licensed nonavalent human papillomavirus vaccine. This can significantly reduce the future morbidity and mortality from cervical and oral cancer, pre-cancerous lesions and other HPV-related cancers in the female and male population of Pakistan. The current study, therefore, provide a credible basis of further research, and follow-up action on HPV-related disease burden.

Background In Denmark, a girls-only human papillomavirus (HPV) vaccination program was initiated in 2008-2009. The study aim was to assess the HPV prevalence and type distribution in younger men prior to HPV vaccination in men.Methods The study population was younger men who attended information days regarding military service. At random days (2019-2020), 280 men were included. We collected questionnaire data regarding risk factors for HPV infection and a penile swab for HPV testing. We compared results in this study with those from a previous study of young men (2006-2007).Results The majority of participants (94%) were 18-20 years old. The median number of lifetime sexual partners was 4. Altogether, 130 men (46.4%) were HPV positive. No infections with HPV types 6, 11, 16, 18, 31, and 45 were detected. The most frequent type was HPV-51 (detected in 11.1%). Comparison showed that the odds of high-risk HPV type infection were higher in 2019-2020 (prevalence odds ratio [POR], 1.7 [95% confidence interval {CI}, 1.1-2.7]) compared with 2006-2007. In contrast, the odds were lower (POR, 0.3 [95% CI, .1-.6]) for HPV types targeted by the 9-valent HPV vaccine.Conclusions The multicohort girls-only vaccination program has to a large degree protected young men against the HPV types included in the licensed vaccines. This does not speak against gender-neutral vaccination as the HPV prevalence is still high, although consisting largely of less carcinogenic HPV types. The multicohort girls-only vaccination program in Denmark has to a large degree protected young men against the HPV types included in licensed vaccines. However, this does not speak against gender-neutral vaccination as, for example, male sexual minority populations are not profiting from this protection.

Abstract Human adenoviruses (HAdVs) are a diverse group of viruses associated with respiratory infections in humans worldwide. However, there is a lack of research on the genetic diversity and epidemiology of HAdVs in Pakistan. This study characterized HAdVs in pediatric patients with respiratory tract infections in Karachi, Pakistan, between 2022 and 2023. We analyzed 762 nasopharyngeal samples of children ≤ 5 years. DNA extraction, followed by PCR targeting E2B and hexon genes, was carried out. Data analysis was performed on SPSS 25.0, and phylogenetic analysis of hexon gene was performed on MEGA 11. HAdV was detected in 7.34% (56/762) of patients round the year, but at a significantly higher rate during the winter season. Age was insignificantly associated with HAdV incidence (p = 0.662), but more than 62.5% (35/56) of positive cases were younger than 10 months. The circulating HAdVs were identified as six different types from species B (78.57%) and C (21.42%), with the majority of isolates found to be like B3. HAdV was found to be co-infected with bocavirus (5.4%) and measles (7.14%). These findings revealed a high frequency and genetic diversity of respiratory HAdVs in Karachi, Pakistan. We conclude that periodic and continuous surveillance of adenoviruses and other respiratory pathogens is necessary to improve the prognosis and management of respiratory diseases, thereby reducing the child mortality rate in Pakistan.

BACKGROUND:Reliable and fast detection and quantification of human cytomegalovirus (CMV) DNA in various diagnostic specimens is essential for care of immunocompromised or congenitally infected individuals. OBJECTIVES:To evaluate the analytical and clinical performance of the Panther Aptima® CMV (Hologic) quantitative real-time transcription mediated amplification (TMA) assay. STUDY DESIGN:Performance of the TMA assay run on the Hologic Panther Fusion was analysed for 32 proficiency testing samples and 21 quantitative reproducibility panel samples; additionally, we compared results of TMA assay and routine quantitative real-time PCR assays ("PCR-A"= Biomérieux CMV R-gene® or "PCR-B"= Laboratory-developed CMV-PCR) in 518 diagnostic specimens (254 plasma, 120 EDTA whole blood, 43 urine, 45 amniotic fluid and 56 breast milk) at two university hospital laboratories. RESULTS:All proficiency panel samples were correctly identified and quantified by the TMA assay; replicate testing of the reproducibility panel samples showed good reproducibility within and between the two laboratories. Sensitivity in plasma and WB was higher for the TMA assay detecting low-level CMV-DNAemia in samples tested negative by routine PCR. Quantitative CMV-DNAemia values correlated well between TMA and real-time PCR. Similarly, urine, AF and BM specimens showed a high rate of concordant results (91%, 98% and 98%, respectively) among TMA and PCR with good correlation of quantitative values. CONCLUSION:The performance of the Aptima® CMV TMA assay for viral blood load testing compared well to established real-time PCRs. In addition, it can be useful for diagnostics in urine, amniotic fluid and breast milk specimens.

Productive replication of human papillomaviruses (HPV) only takes place in differentiating keratinocytes. The HPV16 E8^E2 protein acts as a repressor of viral gene expression and genome replication and HPV16 E8^E2 knock-out (E8-) genomes display enhanced viral late protein expression in differentiated cells. Global transcriptome analysis of differentiated HPV16 wild-type and E8-cell lines revealed a small number of differentially expressed genes which are not related to cell cycle, DNA metabolism or keratinocyte differentiation. The analysis of selected genes suggested that deregulation requires cell differentiation and positively correlated with the expression of viral late, not early transcripts. Consistent with this, the additional knock-out of the viral E4 and E5 genes, which are known to enhance productive replication, attenuated the deregulation of these host cell genes. In summary, these data reveal that productive HPV16 replication modulates host cell transcription.

High-risk human papillomaviruses (PV) account for approximately 600,000 new cancers per year. The early protein E8^E2 is a conserved repressor of PV replication, whereas E4 is a late protein that arrests cells in G2 and collapses keratin filaments to facilitate virion release. While inactivation of the Mus musculus PV1 (MmuPV1) E8 start codon (E8-) increases viral gene expression, surprisingly, it prevents wart formation in FoxN1nu/nu mice. To understand this surprising phenotype, the impact of additional E8^E2 mutations was characterized in tissue culture and mice. MmuPV1 and HPV E8^E2 similarly interact with cellular NCoR/SMRT-HDAC3 co-repressor complexes. Disruption of the splice donor sequence used to generate the E8^E2 transcript or E8^E2 mutants (mt) with impaired binding to NCoR/SMRT-HDAC3 activates MmuPV1 transcription in murine keratinocytes. These MmuPV1 E8^E2 mt genomes also fail to induce warts in mice. The phenotype of E8^E2 mt genomes in undifferentiated cells resembles productive PV replication in differentiated keratinocytes. Consistent with this, E8^E2 mt genomes induced aberrant E4 expression in undifferentiated keratinocytes. In line with observations for HPV, MmuPV1 E4-positive cells displayed a shift to the G2 phase of the cell cycle. In summary, we propose that in order to enable both expansion of infected cells and wart formation in vivo, MmuPV1 E8^E2 inhibits E4 protein expression in the basal keratinocytes that would otherwise undergo E4-mediated cell cycle arrest. IMPORTANCE Human papillomaviruses (PVs) initiate productive replication, which is characterized by genome amplification and expression of E4 protein strictly within suprabasal, differentiated keratinocytes. Mus musculus PV1 mutants that disrupt splicing of the E8^E2 transcript or abolish the interaction of E8^E2 with cellular NCoR/SMRT-HDAC3 co-repressor complexes display increased gene expression in tissue culture but are unable to form warts in vivo. This confirms that the repressor activity of E8^E2 is required for tumor formation and genetically defines a conserved E8 interaction domain. E8^E2 prevents expression of E4 protein in basal-like, undifferentiated keratinocytes and thereby their arrest in G2 phase. Since binding of E8^E2 to NCoR/SMRT-HDAC3 co-repressor is required to enable expansion of infected cells in the basal layer and wart formation in vivo, this interaction represents a novel, conserved, and potentially druggable target.

Human papillomaviruses (HPVs) are DNA tumor viruses that infect mucosal and cutaneous epithelial cells of more than 20 vertebrates. High-risk HPV causes about 5% of human cancers worldwide, and the viral proteins E6 and E7 promote carcinogenesis by interacting with tumor suppressors and interfering with many cellular pathways. As a consequence, they immortalize cells more efficiently in concert than individually. So far, the networks of E6 and E7 with their respective cellular targets have been studied extensively but independently. However, we hypothesized that E6 and E7 might also interact directly with each other in a novel interaction affecting HPV-related carcinogenesis. Here, we report a direct interaction between E6 and E7 proteins from carcinogenic HPV types 16 and 31. We demonstrated this interaction via cellular assays using two orthogonal methods: coimmunoprecipitation and flow cytometry-based FRET assays. Analytical ultracentrifugation of the recombinant proteins revealed that the stoichiometry of the E6/E7 complex involves two E7 molecules and two E6 molecules. In addition, fluorescence polarization showed that (I) E6 binds to E7 with a similar affinity for HPV16 and HPV31 (in the same micromolar range) and (II) that the binding interface involves the unstructured N-terminal region of E7. The direct interaction of these highly conserved papillomaviral oncoproteins may provide a new perspective for studying HPV-associated carcinogenesis and the overall viral life cycle.