High-risk HPV oncoproteins E6 and E7 and their interplay with the innate immune response: Uncovering mechanisms of immune evasion and therapeutic prospects.

Human papillomaviruses (HPVs) are double-stranded DNA (dsDNA) tumor viruses causally associated with 5% of human cancers, comprising both anogenital and upper aerodigestive tract carcinomas. Despite the availability of prophylactic vaccines, HPVs continue to pose a significant global health challenge, primarily due to inadequate vaccine access and coverage. These viruses can establish persistent infections by evading both the intrinsic defenses of infected tissues and the extrinsic defenses provided by professional innate immune cells. Crucial for their evasion strategies is their unique intraepithelial life cycle, which effectively shields them from host detection. Thus, strategies aimed at reactivating the innate immune response within infected or transformed epithelial cells, particularly through the production of type I interferons (IFNs) and lymphocyte-recruiting chemokines, are considered viable solutions to counteract the adverse effects of persistent infections by these oncogenic viruses. This review focuses on the complex interplay between the high-risk HPV oncoproteins E6 and E7 and the innate immune response in epithelial cells and HPV-associated cancers. In particular, it details the molecular mechanisms by which E6 and E7 modulate the innate immune response, highlighting significant progress in our comprehension of these processes. It also examines forward-looking strategies that exploit the innate immune system to ameliorate existing anticancer therapies, thereby providing crucial insights into future therapeutic developments.

A prospective humoral immune monitoring study of kidney transplant recipients receiving three doses of SARS-CoV-2 mRNA vaccine.

Kidney transplant recipients (KTRs), like other solid organ transplant recipients display a suboptimal response to mRNA vaccines, with only about half achieving seroconversion after two doses. However, the effectiveness of a booster dose, particularly in generating neutralizing antibodies (NAbs), remains poorly understood, as most studies have mainly focused on non-neutralizing antibodies. Here, we have longitudinally assessed the humoral response to the SARS-CoV-2 mRNA vaccine in 40 KTRs over a year, examining changes in both anti-spike IgG and NAbs following a booster dose administered about 5 months post-second dose. We found a significant humoral response increase 5 months post-booster, a stark contrast to the attenuated response observed after the second dose. Of note, nearly a quarter of participants did not achieve protective plasma levels even after the booster dose. We also found that the higher estimated glomerular filtration rate (eGFR) correlated with a more robust humoral response postvaccination. Altogether, these findings underscore the effectiveness of the booster dose in enhancing durable humoral immunity in KTRs, as evidenced by the protective level of NAbs found in 65% of the patients 5 months post- booster, especially those with higher eGFR rates.

PAD-mediated citrullination is a novel candidate diagnostic marker and druggable target for HPV-associated cervical cancer.

Citrullination is an emerging post-translational modification catalyzed by peptidyl-arginine deiminases (PADs) that convert peptidyl-arginine into peptidyl-citrulline. In humans, the PAD family consists of five isozymes (PADs 1-4, 6) involved in multiple diseases, including cancer. Given that high-risk (hr) human papillomaviruses (HPVs) are the etiological agents of cervical cancer, in this study, we sought to determine whether PAD-mediated protein citrullination would play a functional role in the HPV-driven transformation of epithelial cells. Here we show that both total protein citrullination and PAD4 expression levels are significantly associated with cervical cancer progression. Specifically, epithelial immunostaining for PAD4 revealed an increasingly higher histoscore from low-grade (CIN1) to high-grade (CIN2, CIN3) cervical intraepithelial neoplasia, and invasive squamous cell carcinoma (SCC) lesions, raising the attractive possibility that PAD4 may be used as tumor staging markers. Furthermore, taking advantage of the epidermoid cervical cancer cell line CaSki, which harbors multiple copies of the integrated HPV16 genome, we show that the expression of E6 and E7 HPV oncoproteins is impaired by treatment with the pharmacological pan-PAD inhibitor BB-Cl-amidine. Consistently, p53 and p21, two targets of HPV oncoproteins, are upregulated by the PAD inhibitor, which undergoes cell growth arrest and apoptosis. Altogether, these findings highlight a novel mechanism by which hrHPVs alter host regulatory pathways involved in cell cycle and survival to gain viral fitness, raising the possibility that PADs may represent an attractive target for developing novel host-targeting antivirals effective in preventing cervical cancer progression.

Human cytomegalovirus infection triggers a paracrine senescence loop in renal epithelial cells.

Human cytomegalovirus (HCMV) is an opportunistic pathogen causing severe diseases in immunosuppressed individuals. To replicate its double-stranded DNA genome, HCMV induces profound changes in cellular homeostasis that may resemble senescence. However, it remains to be determined whether HCMV-induced senescence contributes to organ-specific pathogenesis. Here, we show a direct cytopathic effect of HCMV on primary renal proximal tubular epithelial cells (RPTECs), a natural setting of HCMV disease. We find that RPTECs are fully permissive for HCMV replication, which endows them with an inflammatory gene signature resembling the senescence-associated secretory phenotype (SASP), as confirmed by the presence of the recently established SenMayo gene set, which is not observed in retina-derived epithelial (ARPE-19) cells. Although HCMV-induced senescence is not cell-type specific, as it can be observed in both RPTECs and human fibroblasts (HFFs), only infected RPTECs show downregulation of LAMINB1 and KI67 mRNAs, and enhanced secretion of IL-6 and IL-8, which are well-established hallmarks of senescence. Finally, HCMV-infected RPTECs have the ability to trigger a senescence/inflammatory loop in an IL-6-dependent manner, leading to the development of a similar senescence/inflammatory phenotype in neighboring uninfected cells. Overall, our findings raise the intriguing possibility that this unique inflammatory loop contributes to HCMV-related pathogenesis in the kidney.

Longitudinal monitoring of SARS-CoV-2 viral load in self-collected saliva from health care workers during breakthrough infections to spare working days.

IMPORTANCE: Real-time quantitative PCR (RT-qPCR) on nasopharyngeal swabs (NPS) has been used as the standard method for detecting and monitoring SARS-CoV-2 infection during the pandemic. However, NPS collection often causes discomfort and poses a higher risk of transmission to health care workers (HCW). Furthermore, RT-qPCR only provides relative quantification and does not allow distinguishing those samples with residual, no longer active infection, whereas droplet digital PCR (ddPCR) allows for precise quantification of viral load, offering greater sensitivity and reproducibility. This study highlights the effectiveness of using self-collected saliva as a convenient and reliable sampling method. By utilizing ddPCR to measure the SARS-CoV-2 viral load in saliva samples, individuals with low or undetectable viral loads can be quickly identified. This approach is particularly advantageous for surveillance programs targeting HCW, as it enables the early identification and release of uninfected personnel, minimizing lost workdays. Additionally, analyzing viral load in saliva samples by ddPCR is valuable in determining virus shedding duration across different SARS-CoV-2 variants, informing transmission and disease control. Finally, testing saliva could overcome the detection of historic cases due to prolonged RNA swabbing past-infection and the unnecessary exclusion of those individuals from the workplace.

SIRT1 is an actionable target to restore p53 function in HPV-associated cancer therapy.

BACKGROUND: Our aim was to evaluate the efficacy and anti-cancer action of a precision medicine approach involving a novel SIRT1-dependent pathway that, when disrupted, leads to the restoration of a functional p53 in human papillomavirus (HPV)-transformed cells. METHODS: The anticancer potential of inhibiting SIRT1 was evaluated by examining the effects of the specific SIRT1 inhibitor EX527 (also known as Selisistat) or genetic silencing, either individually or in conjunction with standard chemotherapeutic agents, on a range of HPV+ cancer cells and a preclinical mouse model of HPV16-induced cancer. RESULTS: We show that SIRT1 inhibition restores a transcriptionally active K382-acetylated p53 in HPV+ but not HPV- cell lines, which in turn promotes G0/G1 cell cycle arrest and inhibits clonogenicity specifically in HPV+ cells. Additionally, EX527 treatment increases the sensitivity of HPV+ cells to sublethal doses of standard genotoxic agents. The enhanced sensitivity to cisplatin as well as p53 restoration were also observed in an in vivo tumorigenicity assay using syngeneic C3.43 cells harbouring an integrated HPV16 genome, injected subcutaneously into C57BL/6J mice. CONCLUSIONS: Our findings uncover an essential role of SIRT1 in HPV-driven oncogenesis, which may have direct translational implications for the treatment of this type of cancer.

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Enhanced Spontaneous Skin Tumorigenesis and Aberrant Inflammatory Response to UVB Exposure in Immunosuppressed Human Papillomavirus Type 8‒Transgenic Mice.

Human papillomaviruses (HPVs) from the beta genus are commensal viruses of the skin usually associated with asymptomatic infection in the general population. However, in individuals with specific genetic backgrounds, such as patients with epidermodysplasia verruciformis, or those with immune defects, such as organ transplant recipients, they are functionally involved in sunlight-induced skin cancer development, mainly keratinocyte carcinoma. Despite their well-established protumorigenic role, the cooperation between ß-HPV infection, impaired host immunosurveillance, and UVB exposure has never been formally shown in animal models. In this study, by crossing skin-specific HPV8-transgenic mice with Rag2-deficient mice, we have generated a preclinical mouse model, named Rag2‒/‒:K14-HPV8. These mice display an unhealthy skin phenotype and spontaneously develop papilloma-like lesions spreading to the entire skin much more rapidly compared with Rag2+/+:K14-HPV8 mice. Exposure to low doses of UVB radiation is sufficient to trigger severe skin inflammation in Rag2‒/‒:K14-HPV8 but not in Rag2+/+:K14-HPV8 mice. Their inflamed skin very much resembled that observed in cutaneous field cancerization in organ transplant recipients, showing high levels of UVB-damaged cells, enhanced production of proinflammatory cytokines, and mast cell recruitment to the dermis. Overall, this immunocompromised HPV8-transgenic mouse model shows that the coexistence of immune defects, ß-HPV, and UVB exposure promotes skin cancer development.

Cancer-Associated Fibroblasts Exert Proangiogenic Activity in Merkel Cell Carcinoma.

The tumor microenvironment is a complex niche enveloping a tumor formed by extracellular matrix, blood vessels, immune cells, and fibroblasts constantly interacting with cancer cells. Although tumor microenvironment is increasingly recognized as a major player in cancer initiation and progression in many tumor types, its involvement in Merkel cell carcinoma (MCC) pathogenesis is currently unknown. In this study, we provide a molecular and functional characterization of cancer-associated fibroblasts (CAFs), the major tumor microenvironment component, in patient-derived xenografts of patients with MCC. We show that subcutaneous coinjection of patient-derived CAFs and human MCC MKL-1 cells into severe combined immunodeficient mice significantly promotes tumor growth and metastasis. These fast-growing xenografts are characterized by areas densely populated with human CAFs, mainly localized around blood vessels. We provide evidence that the growth-promoting activity of MCC-derived CAFs is mediated by the aminopeptidase A/angiotensin II and III/angiotensin II type 1 receptor axis, with the expression of aminopeptidase A in CAFs being a triggering event. Together, our findings point to aminopeptidase A as a potential marker for MCC prognostic stratification and as a candidate for therapeutic intervention.

Subversion of Host Innate Immunity by Human Papillomavirus Oncoproteins.

The growth of human papillomavirus (HPV)-transformed cells depends on the ability of the viral oncoproteins E6 and E7, especially those from high-risk HPV16/18, to manipulate the signaling pathways involved in cell proliferation, cell death, and innate immunity. Emerging evidence indicates that E6/E7 inhibition reactivates the host innate immune response, reversing what until then was an unresponsive cellular state suitable for viral persistence and tumorigenesis. Given that the disruption of distinct mechanisms of immune evasion is an attractive strategy for cancer therapy, the race is on to gain a better understanding of E6/E7-induced immune escape and cancer progression. Here, we review recent literature on the interplay between E6/E7 and the innate immune signaling pathways cGAS/STING/TBK1, RIG-I/MAVS/TBK1, and Toll-like receptors (TLRs). The overall emerging picture is that E6 and E7 have evolved broad-spectrum mechanisms allowing for the simultaneous depletion of multiple rather than single innate immunity effectors. The cGAS/STING/TBK1 pathway appears to be the most heavily impacted, whereas the RIG-I/MAVS/TBK1, still partially functional in HPV-transformed cells, can be activated by the powerful RIG-I agonist M8, triggering the massive production of type I and III interferons (IFNs), which potentiates chemotherapy-mediated cell killing. Overall, the identification of novel therapeutic targets to restore the innate immune response in HPV-transformed cells could transform the way HPV-associated cancers are treated.

Human Papillomavirus E7 Oncoprotein Subverts Host Innate Immunity via SUV39H1-Mediated Epigenetic Silencing of Immune Sensor Genes.

Subversion of innate immunity by oncoviruses, such as human papillomavirus (HPV), favors carcinogenesis because the mechanism(s) of viral immune evasion can also hamper cancer immunosurveillance. Previously, we demonstrated that high-risk (hr) HPVs trigger simultaneous epigenetic silencing of multiple effectors of innate immunity to promote viral persistence. Here, we expand on those observations and show that the HPV E7 oncoprotein upregulates the H3K9-specific methyltransferase, whose action shuts down the host innate immune response. Specifically, we demonstrate that SUV39H1 contributes to chromatin repression at the promoter regions of the viral nucleic acid sensors RIG-I and cGAS and the adaptor molecule STING in HPV-transformed cells. Inhibition of SUV39H1 leads to transcriptional activation of these genes, especially RIG-I, followed by increased beta interferon (IFN-ß) and IFN-λ1 production after poly(dA·dT) or RIG-I agonist M8 transfection. Collectively, our findings provide new evidence that the E7 oncoprotein plays a central role in dampening host innate immunity and raise the possibility that targeting the downstream effector SUV39H1 or the RIG-I pathway is a viable strategy to treat viral and neoplastic disease.IMPORTANCE High-risk HPVs are major viral human carcinogens responsible for approximately 5% of all human cancers. The growth of HPV-transformed cells depends on the ability of viral oncoproteins to manipulate a variety of cellular circuits, including those involved in innate immunity. Here, we show that one of these strategies relies on E7-mediated transcriptional activation of the chromatin repressor SUV39H1, which then promotes epigenetic silencing of RIG-I, cGAS, and STING genes, thereby shutting down interferon secretion in HPV-transformed cells. Pharmacological or genetic inhibition of SUV39H1 restored the innate response in HPV-transformed cells, mostly through activation of RIG-I signaling. We also show that IFN production upon transfection of poly(dA·dT) or the RIG-I agonist M8 predominantly occurs through RIG-I signaling. Altogether, the reversible nature of the modifications associated with E7-mediated SUV39H1 upregulation provides a rationale for the design of novel anticancer and antiviral therapies targeting these molecules.

PYHIN genes as potential biomarkers for prognosis of human papillomavirus-positive or -negative head and neck squamous cell carcinomas.

The aim of the present study is to determine the expression levels of PYHIN (IFI16 and AIM2) and APOBEC3 (A3A, A3B, A3C, A3D, A3F, A3G, and A3H) gene family members in a cohort of patients with head and neck squamous cell carcinoma (HNSCC) and assess their potential correlation with human papillomavirus (HPV) infection status, clinical characteristics, and survival. For this purpose, 34 HNSCC tissue specimens along with healthy surrounding mucosa were collected from patients surgically treated for HNSCC. Nucleic acids were isolated to assess the presence of HPV and the expression levels of selected molecular markers. Survival analysis was carried out using the Kaplan-Meier method. In HPV-negative (HPV-) HNSCCs, we detected low mRNA expression levels of IFI16, A3A, and A3B, whereas these genes were upregulated of 2-100 folds in HPV-positive (HPV+) tumors (p < 0.05). Interestingly, AIM2 gene expression levels were predominantly unchanged in HPV+ HNSCCs compared to their HPV- counterparts, in which AIM2 was predominantly upregulated (10% vs. 50% of patients). In HPV- tumors, upregulation of TP53, NOTCH1, PD-L1, and IFI16 correlated with lower occurrence of nodal metastases. On the other hand, the expression of APOBEC family members did not correlate with clinical characteristics. Regarding survival, patients with upregulated A3F gene expression had a worse prognosis, while patients without changes in A3H expression had a lower survival rate. In conclusion, our findings indicate that the innate immune sensors IFI16 and AIM2 and some APOBEC family members could be potentially used as biomarkers for disease outcome in HNSCC patients regardless of HPV presence.

HPV18 Persistence Impairs Basal and DNA Ligand-Mediated IFN-ß and IFN-λ1 Production through Transcriptional Repression of Multiple Downstream Effectors of Pattern Recognition Receptor Signaling.

Although it is clear that high-risk human papillomaviruses (HPVs) can selectively infect keratinocytes and persist in the host, it still remains to be unequivocally determined whether they can escape antiviral innate immunity by interfering with pattern recognition receptor (PRR) signaling. In this study, we have assessed the innate immune response in monolayer and organotypic raft cultures of NIKS cells harboring multiple copies of episomal HPV18 (NIKSmcHPV18), which fully recapitulates the persistent state of infection. We show for the first time, to our knowledge, that NIKSmcHPV18, as well as HeLa cells (a cervical carcinoma-derived cell line harboring integrated HPV18 DNA), display marked downregulation of several PRRs, as well as other PRR downstream effectors, such as the adaptor protein stimulator of IFN genes and the transcription factors IRF1 and 7. Importantly, we provide evidence that downregulation of stimulator of IFN genes, cyclic GMP-AMP synthase, and retinoic acid-inducible gene I mRNA levels occurs at the transcriptional level through a novel epigenetic silencing mechanism, as documented by the accumulation of repressive heterochromatin markers seen at the promoter region of these genes. Furthermore, stimulation of NIKSmcHPV18 cells with salmon sperm DNA or poly(deoxyadenylic-deoxythymidylic) acid, two potent inducers of PRR signaling, only partially restored PRR protein expression. Accordingly, the production of IFN-ß and IFN-λ1 was significantly reduced in comparison with the parental NIKS cells, indicating that HPV18 exerts its immunosuppressive activity through downregulation of PRR signaling. Altogether, our findings indicate that high-risk human papillomaviruses have evolved broad-spectrum mechanisms that allow simultaneous depletion of multiple effectors of the innate immunity network, thereby creating an unreactive cellular milieu suitable for viral persistence.

The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters.

UNLABELLED: The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses. IMPORTANCE: Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.

The Extracellular IFI16 Protein Propagates Inflammation in Endothelial Cells Via p38 MAPK and NF-κB p65 Activation.

The nuclear interferon-inducible-16 (IFI16) protein acts as DNA sensor in inflammasome signaling and as viral restriction factor. Following Herpesvirus infection or UV-B treatment, IFI16 delocalizes from the nucleus to the cytoplasm and is eventually released into the extracellular milieu. Recently, our group has demonstrated the occurrence of IFI16 in sera of systemic-autoimmune patients that hampers biological activity of endothelia through high-affinity membrane binding. As a continuation, we studied the activity of endotoxin-free recombinant IFI16 (rIFI16) protein on primary endothelial cells. rIFI16 caused dose/time-dependent upregulation of IL-6, IL-8, CCL2, CCL5, CCL20, ICAM1, VCAM1, and TLR4, while secretion of IL-6 and IL-8 was amplified with lipopolysaccharide synergy. Overall, cytokine secretion was completely inhibited in MyD88-silenced cells and partially by TLR4-neutralizing antibodies. By screening downstream signaling pathways, we found that IFI16 activates p38, p44/42 MAP kinases, and NF-kB. In particular, activation of p38 is an early event required for subsequent p44/42 MAP kinases activity and cytokine induction indicating a key role of this kinase in IFI16 signaling. Altogether, our data conclude that extracellular IFI16 protein alone or by synergy with lipopolysaccharide acts like Damage-associated molecular patterns propagating "Danger Signal" through MyD88-dependent TLR-pathway.

Differential expression of HER2, STAT3, SOX2, IFI16 and cell cycle markers during HPV-related head and neck carcinogenesis.

The aim of this study was to analyze protein and gene expression of HER2 in 224 head and neck precancerous and malignant lesions by immunohistochemistry and FISH analysis. In parallel, expression of pStat3, Sox2, IFI16 and p16, Ki67 was evaluated. Immunohistochemical analysis was assessed on formalin-fixed paraffin-embedded (FFPE) tissue specimens. A combined method for HPV detection consisting of p16 immunostaining and two PCR probes was applied. HER2 gene status was evaluated by FISH analysis. HPV DNA was detected in 24% of cases with predominant HPV16 genotype. HPV-positive lesions had higher HER2, pStat3 and within carcinoma group, and higher IFI16 expression compared to the HPV-negative group (Fig. 1A-B-C). A strong positive correlation between Sox2 and proliferative activity was observed, whereas IFI16 expression displayed a negative relationship with Sox2 and Ki67 activity. The most striking result was higher pStat3 expression in HPV-positive lesions and its strong positive correlation with IFI16 expression. The presence of HPV may induce upregulation of HER2/neu, pStat3 and IFI16. High levels and a strong positive correlation between pStat3 and IFI16 suggest their synergistic pro-apoptotic effects in HPV-positive lesions.

Innate nuclear sensor IFI16 translocates into the cytoplasm during the early stage of in vitro human cytomegalovirus infection and is entrapped in the egressing virions during the late stage.

UNLABELLED: Intrinsic immune mechanisms mediated by constitutively expressed proteins termed "restriction factors" provide frontline antiviral defense. We recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. We show here that at an early time point during the in vitro infection of low-passage-number human embryonic lung fibroblasts, IFI16 binds to HCMV DNA. However, during a later phase following infection, IFI16 is mislocalized to the cytoplasmic virus assembly complex (AC), where it colocalizes with viral structural proteins. Indeed, upon its binding to pUL97, IFI16 undergoes phosphorylation and relocalizes to the cytoplasm of HCMV-infected cells. ESCRT (endosomal sorting complex required for transport) machinery regulates the translocation of IFI16 into the virus AC by sorting and trafficking IFI16 into multivesicular bodies (MVB), as demonstrated by the interaction of IFI16 with two MVB markers: Vps4 and TGN46. Finally, IFI16 becomes incorporated into the newly assembled virions as demonstrated by Western blotting of purified virions and electron microscopy. Together, these results suggest that HCMV has evolved mechanisms to mislocalize and hijack IFI16, trapping it within mature virions. However, the significance of this IFI16 trapping following nuclear mislocalization remains to be established. IMPORTANCE: Intracellular viral DNA sensors and restriction factors are critical components of host defense, which alarm and sensitize immune system against intruding pathogens. We have recently demonstrated that the DNA sensor IFI16 restricts human cytomegalovirus (HCMV) replication by downregulating viral early and late but not immediate-early mRNAs and their protein expression. However, viruses are known to evolve numerous strategies to cope and counteract such restriction factors and neutralize the first line of host defense mechanisms. Our findings describe that during early stages of infection, IFI16 successfully recognizes HCMV DNA. However, in late stages HCMV mislocalizes IFI16 into the cytoplasmic viral assembly complex and finally entraps the protein into mature virions. We clarify here the mechanisms HCMV relies to overcome intracellular viral restriction, which provides new insights about the relevance of DNA sensors during HCMV infection.