Papillomavirus E6 Research Articles

Cottontail Rabbit Papillomavirus E1 and E2 Proteins Mutually Influence Their Subcellular Localizations.

The papillomavirus (PV) E2 protein is a nuclear, sequence-specific DNA-binding protein that regulates transcription and nuclear retention of viral genomes. E2 also interacts with the viral E1 protein to replicate the viral genome. E2 residue K111 is highly conserved among PV and has been implicated in contributing to nuclear transport, transcription, and replication. Cottontail rabbit (Sylvilagus floridanus) PV (CRPV or SfPV1) E2 K111R, A, or Q mutations are transcription deficient and localized to the cytoplasm, comparable to other PV types. The addition of a nuclear localization signal (NLS) resulted in nuclear E2 K111 mutant proteins but did not restore transcriptional activation, and this is most likely due to an impaired binding to the cellular Brd4 protein. Surprisingly, coexpression of E1 with E2 K111 mutations resulted in their nuclear localization and, for K111A and R mutations, the activation of an E1/E2-dependent reporter construct. Interestingly, the nuclear localization of E2 K111Q mutant protein was independent from the presence of the conserved bipartite NLS in E1 and the direct interaction between E1 and E2. On the other hand, the cytoplasmic E1 NLS mutation could be targeted to the nucleus by wild-type E2, and this was dependent upon an interaction between E1 and E2. In summary, our studies have uncovered that E1 and E2 control each other's subcellular localization: direct binding of E2 to E1 can direct E1 to the nucleus independently from the E1 NLS, and E1 can direct E2 to the nucleus without an intact NLS or direct binding to E2.IMPORTANCE Papillomaviruses encode the DNA-binding E1 and E2 proteins, which form a complex and are essential for genome replication. Both proteins are targeted to the nucleus via nuclear localization signals. Our studies have uncovered that cytoplasmic mutant E1 or E2 proteins can be localized to the nucleus when E1 or E2 is also present. An interaction between E1 and E2 is necessary to target cytoplasmic E1 mutant proteins to the nucleus, but cytoplasmic E2 mutant proteins can be targeted to the nucleus without a direct interaction, which points to a novel function of E1.

Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis.

Phospholipids regulate numerous cellular functions and their deregulation is known to be associated with cancer development. Here, we show for the first time that expression of the E6 oncoprotein of human papillomavirus type 8 (HPV8) leads to a profound increase in nuclear phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) lipid levels in monolayer cultures, that led to an aberrant phospholipidation of cellular proteins. Elevated PI(4,5)P2 levels in organotypic skin cultures, skin tumors of K14-HPV8-E6 transgenic mice as well as HPV8 positive skin carcinomas highly suggest a decisive role of PI(4,5)P2 in HPV associated squamous-cell-carcinoma development. Furthermore, mass-spectrometric analysis confirmed an increase of PI(4,5)P2, which was characterized by a shift in the distribution of lipid species. PI(4,5)P2 upregulation was independent of E6 interference with MAML1. However, E6 does interfere with the PI(4,5)P2 metabolic pathway by upregulation of phosphatidylinositol-4-phosphate-5-kinase type I and phosphatidylinositol-5-phosphate 4-kinase type II as well as the binding to 5’-phosphatase OCRL and phosphatidylinositol. All of these mechanisms combined may contribute to PI(4,5)P2 elevation in E6 positive cells. The identification of CAND1 and SND1 – two proteins known to be involved in carcinogenic processes – were significantly stronger phospholipidized in the presence of E6. In conclusion we provide evidence that the modulation of the PI(4,5)P2 metabolism is a novel oncogenic mechanism relevant for HPV-induced carcinogenesis.

Synthesis of Proteins Encoded by the Early Genes E2, E6, and E7 of Papillomavirus of Type 16 in the Plant Expression System.

An expression system for the synthesis of early antigenic proteins HPV16 E2, HPV16 E6, and HPV16 E7 of high-risk papillomavirus based on transgenic tomato fruits was developed. It is planned to use the early antigenic proteins synthesized to create a therapeutic vaccine against cervical cancer.

Immunohistochemical detection of E7 human papillomavirus protein in pre‑malignant and malignant lesions of the uterine cervix.

Human papillomavirus (HPV) E7 protein expression is caused by HPV viral DNA integration into human cellular DNA, and is a prerequisite for the development and progression of cervical cancer. The present study aimed to evaluate the role of E7 protein as a biomarker for identification of transformed cervical epithelial cells during the early stages of cervical cancer. Specific monoclonal antibodies to the E7protein of high‑risk HPVs were generated and characterized for applications in immunocytochemistry and immunohistochemistry using cervical epithelial cells or biopsy tissue slides. The specificity and feasibility for detecting precancerous cells in cervical exfoliated epithelial cells was demonstrated. In addition, antibody staining of cervix biopsies indicated the pathological grades of cervical cancer and precancerous lesions. The results of the present study demonstrated the potential benefit of using E7 protein as a novel and specific clinical diagnostic marker to distinguish transient HPV infections from malignant and pre‑malignant lesions.

Zac1 regulates IL-11 expression in osteoarthritis.

Interleukin (IL)-11, a member of the IL-6 family of cytokines, exerts pleiotropic effects under normal and various disease conditions. We assessed IL-11 expression regulation and the IL-11/IL-6 ratio in osteoarthritis (OA) to better guide clinical therapeutic decision-making. Our findings suggest that Zac1, a zinc finger protein that regulates apoptosis and cell cycle arrest, is a transcription factor regulating IL-11 expression. Zac1 overexpression or knockdown respectively induced or suppressed IL-11 expression in HeLa cells. Zac1 acted synergistically with AP-1, human papillomavirus E2, and hypoxia inducible factor 1 alpha (HIF1α). IL-11 expression under various conditions, including hypoxia or treatment with phorbol 12-myristate 13-acetate or copper sulfate. Recombinant IL-11-induced phosphorylation of signal transducer and activator of transcription 3 at tyrosine 705 was reduced in a dose-dependent manner in HeLa cells. Cross-talk between Zac1, IL-11, p53, and suppressor of cytokine signaling 3 was differentially affected by copper sulfate, digoxin, and caffeine. Finally, aggressive vs. conventional treatment of OA patients was primarily determined by IL-6 levels. However, we suggest that OA patients with higher IL-11 levels may respond well to conventional treatments, even in the presence of high IL-6.

Evaluation of therapeutic potency of human papillomavirus-16 E7 DNA vaccine alone and with interleukin-18 as a genetic adjuvant

AIMS: Despite the existence of effective preventive vaccines for human papillomavirus (HPV), therapeutic vaccines that trigger cell-mediated immune responses are required to treat established infections and malignancies. The aim of this study was to evaluate the therapeutic potency of HPV-16 E7 deoxyribonucleic acid (DNA) vaccine alone and with interleukin (IL)-18. METHODS: In vitro expressions of IL-18 were performed on human embryonic kidney 293 cells and confirmed it by Western blotting methods. DNA vaccine was available from a previous study. A total of 45 female C57BL/6 mice divided into five groups (DNA vaccine, DNA vaccine adjuvanted with IL-18, pcDNA3.1, and phosphate buffer saline) were inoculated with murine tissue culture-1 cell line of HPV related carcinoma, expressing HPV-16 E6/E7 antigens. They were then immunized subcutaneously twice at a seven-day interval. The antitumor and antigen specific-cellular immunity were assessed by lymphocyte proliferation (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide: MTT) assay, lactate dehydrogenase release assay, IL-4 assay and interferon-gamma (IFN-γ) assay. Tumor size was followed for 62 days.RESULTS: MTT assay, which measures the lymphocyte proliferation in response to the specific antigen, increased in the co-administration and the DNA vaccine groups as compared to control and genetic adjuvant groups (p<0.001). The mice immunized with the co-administration generated significantly more IFN-γ and IL-4 than other immunized mice (p<0.001). Reduction of the tumor size in the co-administration and the DNA vaccine groups was significantly more pronounced than in the control and genetic adjuvant groups (p<0.001), but no statistically significant difference between DNA vaccine and co-administration groups (p=0.15) occurred.CONCLUSIONS: IL-18 as a genetic adjuvant and E7 DNA vaccine alone enhanced immune responses in mouse model systems against cervical cancer. However, using of IL-18 as a genetic adjuvant with E7 DNA vaccine had no significant synergistic effect on the immune responses in vivo.

Expression of human papillomavirus oncoproteins E6 and E7 inhibits invadopodia activity but promotes cell migration in HPV-positive head and neck squamous cell carcinoma cells.

BackgroundThe rapid increase in the incidence of head and neck squamous cell carcinoma (HNSCC) is caused by high‐risk human papillomavirus (HPV) infections. The HPV oncogenes E6 and E7 promote carcinogenesis by disrupting signaling pathways that control survival and proliferation. Although these cancers are often diagnosed with metastases, the mechanisms that regulate their dissemination are unknown.AimsThe aim of this study was to determine whether the HPV‐16 E6 and E7 oncogenes affected the invasive and migratory properties of HNSCC cells which promote their spread and metastasis.Methods and resultsInvasiveness was determined using invadopodia assays which allow for quantitation of extracellular matrix (ECM) degradation by invadopodia which are proteolytic membrane protrusions that facilitate invasion. Using cell lines and genetic manipulations, we found that HPV inhibited invadopodia activity in aggressive cell lines which was mediated by the E6 and E7 oncogenes. Given these findings, we also tested whether HPV caused differences in the migratory ability of HNSCC cells using Transwell assays. In contrast to our invadopodia results, we found no correlation between HPV status and cell migration; however, blocking the expression of the E6 and E7 oncoproteins in a HPV‐positive (HPV+) HNSCC cell line resulted in decreased migration.ConclusionsOur data suggest that the E6 and E7 oncoproteins are negative regulators of invadopodia activity but may promote migration in HPV+ HNSCC cells. Despite the need for ECM proteolysis to penetrate most tissues, the unique structure of the head and neck tissues in which these cancers arise may facilitate the spread of migratory cancer cells without significant proteolytic ability.

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens

ABSTRACTCervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins E6 and E7. Our group pioneered a recombinant viral vector system based on Semliki Forest virus (SFV) for vaccination against cervical cancer. The most striking benefit of this alphavirus vector-based vaccine platform is its high potency. DNA vaccines on the other hand, have a major advantage with respect to ease of production. In this study, the benefits associated with both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA vaccine did not prevent tumor outgrowth, immunization with a 200-fold lower equimolar dose of DREP (0.05 µg of DREP) resulted in approximately 85% of tumor-free mice. To overcome the safety concern of potential malignant transformation at the vaccination site, we evaluated the anti-tumor effect of a DREP vaccine encoding a shuffled version of E7 (DREP-E7sh). DREP-E7sh delayed tumor growth yet not to the same extent as DREP-E6,7. In addition, inclusion of a helper cassette and an ER targeting signal (sigHelp) did not significantly further enhance the suppression of tumor outgrowth in the long term, albeit exhibiting better tumor control early after immunization. Collectively, this study points towards the clinical evaluation of DREP encoding HPV antigens as a potent immunotherapy for patients with HPV16 (pre)-malignancies.

Interleukin‑17A and heparanase promote angiogenesis and cell proliferation and invasion in cervical cancer.

Interleukin‑17A (IL‑17A) is a CD4 T-cell-derived pro-inflammatory cytokine that is involved in human cervical tumorigenesis. Heparanase (HPSE) is an endo-glycosidase expressed in mammals, which has been confirmed to be associated with cervical cancer invasion. In the present study, it was hypothesized that IL‑17A and HPSE are key proteins promoting tumor angiogenesis and cell proliferation and invasion in cervical cancer. The expression of IL‑17A and HPSE in cervical cancer tissues was detected by immunohistochemical staining. In addition, the expression of IL‑17A and HPSE was down- and upregulated via RNAi and human recombinant proteins, and MTT and Transwell assays were performed to examine cervical cancer cell proliferation and invasion, respectively. Flow cytometry analysis was also performed to detect cell cycle distribution, and the levels of target mRNA and protein were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. IL‑17A and HPSE were highly expressed in cervical cancer tissues, and microvessel density was notably higher in the IL‑17A-positive group. IL‑17A and/or HPSE recombinant protein promoted the proliferation and invasion of cervical cancer cells, increased the proportion of cells in the G2/M phase, and enhanced the mRNA and protein expression of human papillomavirus E6, P53, vascular endothelial growth factor and CD31, whereas downregulation of IL‑17A and/or HPSE exerted the opposite effects. Furthermore, downregulation of IL‑17A and/or HPSE was found to inhibit the expression of nuclear factor (NF)-κB P65. In summary, IL‑17A and HPSE may promote tumor angiogenesis and cell proliferation and invasion in cervical cancer, possibly via the NF-κB signaling pathway. These findings may lead to the identification of new diagnostic markers and therapeutic targets.

The SMC5/6 Complex Interacts with the Papillomavirus E2 Protein and Influences Maintenance of Viral Episomal DNA.

The papillomavirus E2 protein executes numerous essential functions related to viral transcription, replication of viral DNA, and viral genome maintenance. Because E2 lacks enzymatic activity, many of these functions are mediated by interactions with host cellular proteins. Unbiased proteomics approaches have successfully identified a number of E2-host protein interactions. We have extended such studies and have identified and validated the cellular proteins structural maintenance of chromosome 5 (SMC5) and SMC6 as interactors of the viral E2 protein. These two proteins make up the core components of the SMC5/6 complex. The SMC5/6 complex is a member of the conserved structural maintenance of chromosomes (SMC) family of proteins, which are essential for genome maintenance. We have examined the role of SMC5/6 in various E2 functions. Our data suggest that SMC6 is not required for E2-mediated transcriptional activation, E1/E2-mediated transient replication, or differentiation-dependent amplification of viral DNA. Our data, however, suggest a role for SMC5/6 in viral genome maintenance.IMPORTANCE The high-risk human papillomaviruses (HPVs) are the etiological cause of cervical cancer and the most common sexually transmitted infection. While the majority of infections may be asymptomatic or cause only benign lesions, persistent infection with the oncogenic high-risk HPV types may lead to serious diseases, such as cervical cancer, anogenital carcinoma, or head and neck oropharyngeal squamous cell carcinoma. The identification of virus-host protein interactions provides insights into the mechanisms of viral DNA persistence, viral genome replication, and cellular transformation. Elucidating the mechanism of early events in the virus replication cycle as well as of integration of viral DNA into host chromatin may present novel antiviral strategies and targets for counteracting persistent infection. The E2 protein is an important viral regulatory protein whose functions are mediated through interactions with host cell proteins. Here we explore the interaction of E2 with SMC5/6 and the functional consequences.

Human Oncoviruses and p53 Tumor Suppressor Pathway Deregulation at the Origin of Human Cancers.

Viral oncogenesis is a multistep process largely depending on the complex interplay between viruses and host factors. The oncoviruses are capable of subverting the cell signaling machinery and metabolic pathways and exploit them for infection, replication, and persistence. Several viral oncoproteins are able to functionally inactivate the tumor suppressor p53, causing deregulated expression of many genes orchestrated by p53, such as those involved in apoptosis, DNA stability, and cell proliferation. The Epstein–Barr virus (EBV) BZLF1, the high-risk human papillomavirus (HPV) E6, and the hepatitis C virus (HCV) NS5 proteins have shown to directly bind to and degrade p53. The hepatitis B virus (HBV) HBx and the human T cell lymphotropic virus-1 (HTLV-1) Tax proteins inhibit p53 activity through the modulation of p300/CBP nuclear factors, while the Kaposi’s sarcoma herpesvirus (HHV8) LANA, vIRF-1 and vIRF-3 proteins have been shown to destabilize the oncosuppressor, causing a decrease in its levels in the infected cells. The large T antigen of the Merkel cell polyomavirus (MCPyV) does not bind to p53 but significantly reduces p53-dependent transcription. This review describes the main molecular mechanisms involved in the interaction between viral oncoproteins and p53-related pathways as well as in the development of therapeutic strategies targeting such interactions.

Chaperone-assisted selective autophagy in healthy and papillomavirus-associated neoplastic urothelium of cattle

Chaperone-assisted selective autophagy (CASA) is a newly-described selective tension-induced macroautophagy pathway mediated by Bag3 that is believed to be essential for mechanotransduction in skeletal muscle and to be an important regulator of the immune system. We investigated CASA machinery both in healthy and in fifteen papillomavirus-associated neoplastic bovine urothelium. The components of CASA complex, that comprises the molecular chaperones HspA8/Hsc70 and Hsp8B/Hsp22 and the cochaperones Bag3 and STUB1/CHIP, were studied by molecular, microscopic and submicroscopic investigations. CASA complex was found to be constitutively expressed in healthy bovine urothelium; its expression increased in urothelial cancers of cattle, namely thirteen papillary carcinomas and two papillary urothelial neoplasm of low malignant potential (PUNLMPs). We suggest that basal levels of CASA are important in the healthy urothelium which interfaces with the community of urinary microbiota thus representing an important epithelial cell-autonomous mechanism of antibacterial defense. Co-immunoprecipitation studies using an antibody against bovine papillomavirus E5 protein revealed that the oncoprotein co-localized with CASA complex in urothelial cancer cells. This suggests that infection by BPV E5 could influence cell behaviour by interfering with basal autophagy processes although this study did not conclusively show that this interaction increased the expression of CASA proteins. In neoplastic urothelium, CASA could be involved in regulating fundamental cellular processes such adhesion, migration, and proliferation and so might influence the biological behaviour of urothelial tumors in cattle.

Papillomavirus E2 protein is regulated by specific fibroblast growth factor receptors

The papillomavirus (PV) E2 protein activates transcription and replication by recruiting cellular proteins and the E1 DNA helicase to their binding sites in the viral genome. We recently demonstrated that phosphorylation of tyrosine 102 in the bovine papillomavirus (BPV-1) E2 protein restricts these activities and that fibroblast growth factor receptor-3 (FGFR3) tyrosine kinase binds PV E2. Expression of FGFR3 decreased viral replication with both wild-type and the phenylalanine substitution at position 102, inferring that another kinase targets Y102. Here we tested FGFR- 1, −2 and −4 for association with PV E2 proteins. FGFR2 but not FGFR1 or FGFR4 co-immunoprecipitated with BPV-1 E2. We found that FGFR2 suppressed replication but did not depend on phosphorylation of BPV-1 Y102. HPV-16 and −31 E2 interacted with FGFR1, −2, and −4. These results imply that the expression and activity of FGF receptors in epithelial cells can regulate the function of E2 in viral replication.

Regression of epithelial cancers in humans following t-cell receptor gene therapy targeting human papillomavirus-16 E7.

3043Background: Adoptive T-cell therapy with gene-engineered T cells is an emerging cancer treatment strategy. Study of this approach in epithelial cancers, the most common types of malignancies, h...

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute.

CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

Generation of the Fluorescent HPV16 E7 Protein for Detection of Delivery In vitro.

Immunotherapies targeting the human papillomavirus (HPV) oncogenic proteins, E6 and E7, are effective to treat HPV-associated cervical malignancies. The main objective of this study was to generate the fluorescent HPV16 E7 protein for detection of delivery in vitro. Two types of the fusion E7-GFP proteins (i.e., with or without linker) were expressed in different E. coli strains. Then, the efficiency of GFP and E7-GFP transfection was compared with FITC-antibody protein control using TurboFect reagent in the HEK-293T cell line. Our data indicated that both E7-GFP fusion proteins were efficiently produced in M15 E. coli strain, but not in BL21 or Rosetta strains. The E7-GFP fusion showed a clear band of ~ 50 kDa in SDS-PAGE. Moreover, the E7-GFP protein maintained the fluorescent properties only when there was a distance between E7 and GFP genes, suggesting a promising potential to use GFP fusion protein in generating soluble form of protein. This fluorescent property was stable and could be detected in vitro. Moreover, the HEK-293T cells transfected by GFP/TurboFect and E7- GFP/TurboFect complexes demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the GFP fluorescence was stable even at 24 h post-transfection. Briefly, the E7-GFP fusion protein with linker can be useful for the development of protein vaccines against HPV16 infections and detection in vivo.

Association of antibody to E2 protein of human papillomavirus and p16INK4A with progression of HPV-infected cervical lesions.

Human papillomavirus (HPV) E2 and L1 proteins are expressed in cervical cells during the lytic stage of infection. Overexpression of p16INK4A is a biomarker of HPV-associated cervical neoplasia. This study investigated antibodies to HPV16 E2, HPV16 L1, and p16INK4A in sera from women with no squamous intraepithelial lesion (No-SIL) of the cervix, low-grade SIL, high-grade SIL, and cervical squamous cell carcinoma (SCC). HPV DNA was detected by polymerase chain reaction. Anti-E2, -L1, and -p16INK4A antibodies in sera were determined by western blot. Among 116 samples, 69 (60%) were HPV DNA-positive. Percentages seropositive for anti-E2, -L1, and -p16INK4A antibodies were 39.6, 22.4, and 23.3%, respectively. Anti-E2 antibody was significantly correlated with HPV DNA-positive cases. Eighty-seven women (75%) were regarded as infected with HPV, having at least one positive result from HPV DNA, L1, or E2 antibody. Antibody to p16INK4A was associated with HPV infection (odds = 5.444, 95% CI 1.203-24.629, P = 0.028) and precancerous cervical lesions (odds = 5.132, 95% CI 1.604-16.415, P = 0.006). Interestingly, the concurrent detection of anti-E2 and -p16INK4A antibodies was significantly associated with HPV infection (odds = 1.382, 95% CI 1.228-1.555, P = 0.044). These antibodies might be good candidate biomarkers for monitoring HPV-associated cervical lesion development to cancer.

Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems.

In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry. The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p < 0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~ 83.94 and ~ 77.01% for HEK-293T), and cancer cells (~ 71.69 and ~ 67.19% for TC-1, and ~ 73.86 and ~ 67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~ 60-80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~ 63.66%) compared to TurboFect (~ 32.95%); however, the efficiency level of hPP10 was only ~ 17.51 and ~ 16.36% in TC-1 and A549 cells. Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

A compact, low-cost, quantitative and multiplexed fluorescence detection platform for point-of-care applications

An effective method of combating infectious diseases is the deployment of hand-held devices at the point-of-care (POC) for screening or self-monitoring applications. There is a need for very sensitive, low-cost and quantitative diagnostic devices. In this study, we present a low-cost, multiplexed fluorescence detection platform that has a high sensitivity and wide dynamic range. Our system features inexpensive 3 × 3 mm interference filters with a high stopband rejection, sharp transition edges, and greater than 90% transmission in the passband. In addition to the filters, we improve signal-to-noise ratio by leveraging time for accuracy using a charge-integration-based readout. The fluorescence sensing platform provides a sensitivity to photon flux of ∼1×104photons/mm2sec and has the potential for 2–3 orders of magnitude improvement in sensitivity over standard colorimetric detection that uses colored latex microspheres. We also detail the design, development, and characterization of our low-cost fluorescence detection platform and demonstrate 100% and 97.96% reduction in crosstalk probability and filter cost, respectively. This is achieved by reducing filter dimensions and ensuring appropriate channel isolation in a 2 × 2 array configuration. Practical considerations with low-cost interference filter system design, analysis, and system performance are also discussed. The performance of our platform is compared to that of a standard laboratory array scanner. We also demonstrate the detection of antibodies to human papillomavirus (HPV16) E7 protein, as a potential biomarker for early cervical cancer detection in human plasma.

The Human Papillomavirus E6 Oncoprotein Targets USP15 and TRIM25 To Suppress RIG-I-Mediated Innate Immune Signaling.

Retinoic acid-inducible gene I (RIG-I) is a key pattern recognition receptor that senses viral RNA and interacts with the mitochondrial adaptor MAVS, triggering a signaling cascade that results in the production of type I interferons (IFNs). This signaling axis is initiated by K63-linked ubiquitination of RIG-I mediated by the E3 ubiquitin ligase TRIM25, which promotes the interaction of RIG-I with MAVS. USP15 was recently identified as an upstream regulator of TRIM25, stabilizing the enzyme through removal of degradative K48-linked polyubiquitin, ultimately promoting RIG-I-dependent cytokine responses. Here, we show that the E6 oncoprotein of human papillomavirus type 16 (HPV16) as well as of other HPV types form a complex with TRIM25 and USP15 in human cells. In the presence of E6, the K48-linked ubiquitination of TRIM25 was markedly increased, and in line with this, TRIM25 degradation was enhanced. Our results further showed that E6 inhibited the TRIM25-mediated K63-linked ubiquitination of RIG-I and its CARD-dependent interaction with MAVS. HPV16 E6, but not E7, suppressed the RIG-I-mediated induction of IFN-β, chemokines, and IFN-stimulated genes (ISGs). Finally, CRISPR-Cas9 gene targeting in human keratinocytes showed that the TRIM25-RIG-I-MAVS triad is important for eliciting an antiviral immune response to HPV16 infection. Our study thus identifies a novel immune escape mechanism that is conserved among different HPV strains and further indicates that the RIG-I signaling pathway plays an important role in the innate immune response to HPV infection.IMPORTANCE Persistent infection and tumorigenesis by HPVs are known to require viral manipulation of a variety of cellular processes, including those involved in innate immune responses. Here, we show that the HPV E6 oncoprotein antagonizes the activation of the cytoplasmic innate immune sensor RIG-I by targeting its upstream regulatory enzymes TRIM25 and USP15. We further show that the RIG-I signaling cascade is important for an antiviral innate immune response to HPV16 infection, providing evidence that RIG-I, whose role in sensing RNA virus infections has been well characterized, also plays a crucial role in the antiviral host response to small DNA viruses of the Papillomaviridae family.