Pro-Apoptotic Activity and Cell Cycle Arrest of Caulerpa sertularioides against SKLU-1 Cancer Cell in 2D and 3D Cultures.

Cancer is a disease with the highest mortality and morbidity rate worldwide. First-line drugs induce several side effects that drastically reduce the quality of life of people with this disease. Finding molecules to prevent it or generate less aggressiveness or no side effects is significant to counteract this problem. Therefore, this work searched for bioactive compounds of marine macroalgae as an alternative treatment. An 80% ethanol extract of dried Caulerpa sertularioides (CSE) was analyzed by HPLS-MS to identify the chemical components. CSE was utilized through a comparative 2D versus 3D culture model. Cisplatin (Cis) was used as a standard drug. The effects on cell viability, apoptosis, cell cycle, and tumor invasion were evaluated. The IC50 of CSE for the 2D model was 80.28 µg/mL versus 530 µg/mL for the 3D model after 24 h of treatment exposure. These results confirmed that the 3D model is more resistant to treatments and complex than the 2D model. CSE generated a loss of mitochondrial membrane potential, induced apoptosis by extrinsic and intrinsic pathways, upregulated caspases-3 and -7, and significantly decreased tumor invasion of a 3D SKLU-1 lung adenocarcinoma cell line. CSE generates biochemical and morphological changes in the plasma membrane and causes cell cycle arrest at the S and G2/M phases. These findings conclude that C. sertularioides is a potential candidate for alternative treatment against lung cancer. This work reinforced the use of complex models for drug screening and suggested using CSE's primary component, caulerpin, to determine its effect and mechanism of action on SKLU-1 in the future. A multi-approach with molecular and histological analysis and combination with first-line drugs must be included.

Inflammation- and cancer-related microRNAs in rat renal cortex after subchronic exposure to fluoride.

The proximal tubule is a target of subchronic exposure to fluoride (F) in the kidney. Early markers are used to classify kidney damage, stage, and prognosis. MicroRNAs (miRNAs) are small sequences of non-coding single-stranded RNA that regulate gene expression and play an essential role in developing many pathologies, including renal diseases. This study aimed to evaluate the expression of Cytokine-Chemokine molecules (IL-1α/1ß/4/6/10, INF-γ, MIP-1α, MCP-1, RANTES, and TGF ß1/2/3) and inflammation-related miRNAs to evidence the possible renal mechanisms involved in subchronic exposure to F. Total protein and miRNAs were obtained from the renal cortex of male Wistar rats exposed to 0, 15 and 50 mg NaF/L through drinking water during 40 and 80 days. In addition, cytokines-chemokines were analyzed by multiplexing assay, and a panel of 77 sequences of inflammatory-related miRNAs was analyzed by qPCR. The results show that cytokines-chemokines expression was concentration- and time-dependent with F, where the 50 mg NaF/L were the main altered groups. The miRNAs expression resulted in statistically significant differences in thirty-four miRNAs in the 50 mg NaF/L groups at 40 and 80 days. Furthermore, a molecular interaction network analysis was performed. The relevant pathways modified by subchronic exposure to fluoride were related to extracellular matrix-receptor interaction, Mucin type O-glycan biosynthesis, Gap junction, and miRNAs involved with renal cell carcinoma. Thus, F-induced cytokines-chemokines suggest subchronic inflammation; detecting miRNAs related to cancer and proliferation indicates a transition from renal epithelium to pathologic tissue after fluoride exposure.

Fluorescence-Linked Aptamer Assay for SARS-CoV-2 Spike-Protein: A Step-by-Step Performance Analysis in Clinical Samples.

The COVID-19 pandemic has been a main concern over the last two years and has become one of the most important crises in the history of human health. Today, there is still a need for affordable and reliable diagnostic tests for massive disease monitoring. Previously, a set of highly specific DNA-aptamers (C7/C9) binding to the SARS-CoV-2 Spike (S) protein were isolated but its performance in clinical samples remained to be tested. Here, 242 samples were collected through three different methods and subjected to florescence-linked aptamer assays (FLAA) based on C7/C9 aptamers through two readout protocols. Then, a step-by-step statistical approach which included agreement tests, proportion comparisons and binomial and multinomial logistic regressions was used to predict optimal conditions for the novel C7/C9 FLAA test. RTqPCR threshold cycles, symptoms onset and processing time were influential factors on FLAA test results. Naturally occurring mutations on S were also detected and analyzed. Aminoacidic substitutions D614G and T732A appeared relevant for aptamer recognition although further studies are necessary. The methodology presented here is the first step to determine the performance and diagnosis across a range of clinical contexts and it might serve as a base for a complete analysis applicable to other designs of new diagnostic tests.

Non-Functionalized Gold Nanoparticles Inhibit Human Papillomavirus (HPV) Infection.

The spontaneous interaction between human papillomavirus type 16 (HPV16) L1 virus-like particles (VLPs) and non-functionalized gold nanoparticles (nfGNPs) interferes with the nfGNPs' salt-induced aggregation, inhibiting the red-blue color shift in the presence of NaCl. Electron microscopy and competition studies showed that color-shift inhibition is a consequence of direct nfGNP-VLP interaction and, thus, may produce a negative impact on the virus entry cell process. Here, an in vitro infection system based on the HPV16 pseudovirus (PsV) was used to stimulate the natural infection process in vitro. PsVs carry a pseudogenome with a reporter gene, resulting in a fluorescent signal when PsVs infect a cell, allowing quantification of the viral infection process. Aggregation assays showed that nfGNP-treated PsVs also inhibit color shift in the presence of NaCl. High-resolution microscopy confirmed nfGNP-PsV complex formation. In addition, PsVs can interact with silver nanoparticles, suggesting a generalized interaction of metallic nanoparticles with HPV16 capsids. The treatment of PsVs with nfGNPs produced viral infection inhibition at a higher level than heparin, the canonical inhibitor of HPV infection. Thus, nfGNPs can efficiently interfere with the HPV16 cell entry process and may represent a potential active component in prophylactic formulations to reduce the risk of HPV infection.

ZO-2 favors Hippo signaling, and its re-expression in the steatotic liver by AMPK restores junctional sealing.

ZO-2 is a peripheral tight junction (TJ) protein whose silencing in renal epithelia induces cell hypertrophy. Here, we found that in ZO-2 KD MDCK cells, in compensatory renal hypertrophy triggered in rats by a unilateral nephrectomy and in liver steatosis of obese Zucker (OZ) rats, ZO-2 silencing is accompanied by the diminished activity of LATS, a kinase of the Hippo pathway, and the nuclear concentration of YAP, the final effector of this signaling route. ZO-2 appears to function as a scaffold for the Hippo pathway as it associates to LATS1. ZO-2 silencing in hypertrophic tissue is due to a diminished abundance of ZO-2 mRNA, and the Sp1 transcription factor is critical for ZO-2 transcription in renal cells. Treatment of OZ rats with metformin, an activator of AMPK that blocks JNK activity, augments ZO-2 and claudin-1 expression in the liver, reduces the paracellular permeability of hepatocytes, and serum bile acid content. Our results suggest that ZO-2 silencing is a common feature of hypertrophy, and that ZO-2 is a positive regulator of the Hippo pathway that regulates cell size. Moreover, our observations highlight the importance of AMPK, JNK, and ZO-2 as therapeutic targets for blood-bile barrier dysfunction.

Cervical Cancer, Papillomavirus, and miRNA Dysfunction.

Cervical cancer is the leading cause of death by cancer in women from developing countries. Persistent infection with high-risk human papillomavirus (HPV) types 16 and 18 is a major risk factor for cervical carcinogenesis. Nevertheless, only a few women with morphologic expression of HPV infection progress into invasive disease suggesting the involvement of other factors in cervical carcinogenesis. MicroRNAs (miRNAs) are conserved small non-coding RNAs that negatively regulate gene expression including genes involved in fundamental biological processes and human cancer. Dysregulation of miRNAs has been widely reported in cervical cancer. This work focuses on reviewing the miRNAs affected during the HPV infection process, as well relevant miRNAs that contribute to the development and maintenance of malignant cervical tumor cells. Finally, we recapitulate on miRNAs that may be used to distinguish between healthy individuals from patients with precancerous lesions or cervical tumors.

Structural insights of the pre-let-7 interaction with LIN28B.

The Let-7:LIN28 regulatory loop is a paradigm in miRNA regulation. LIN28 harbors two RNA binding domains, which interact with well-conserved sequences in pre-let-7 RNAs, the GNGAY and the GGAG motifs. Here, the differential binding between LIN28B and pre-let-7 members was associated with the structural characteristics of the pre-let-7 family mapped by SHAPE, uncovering diverse structural patterns within pre-let-7 members. Pre-let-7 mutants supported a relevant role of the GGAG motif location and the preE-stem stability for the interaction with LIN28B. Based on these results, we propose a core RNA structure for LIN28B interaction.

Effect of naringenin and its combination with cisplatin in cell death, proliferation and invasion of cervical cancer spheroids.

The main treatment alternative for cervical cancer is cisplatin chemotherapy. However, the resistance of tumor cells to cisplatin, in addition to side effects, limits its use. The flavonoid naringenin has shown cytotoxic effects on tumor cells and may be considered as a coadjuvant in the treatment of cervical cancer. In the present study, the effect of naringenin on cell viability, cytotoxicity, proliferation, apoptosis and invasion was evaluated in HeLa spheroid cultures. Naringenin impaired the cell viability as indicated by low ATP levels and caused concentration- and time-dependent cytotoxicity via the loss of cell membrane integrity. Furthermore, it did not activate caspases 3, 7, 8, and 9, suggesting that the cytotoxic effect was by necrotic cell death instead of apoptosis. Additionally, proliferation in the G0/G1 phase of the cell cycle was inhibited. Cell invasion also decreased as time progressed. Later, we determined if naringenin could improve the anti-tumor effect of cisplatin. The combination of naringenin with low concentrations of cisplatin improved the effect of the drug by significantly decreasing cell viability, potentiating the induction of cytotoxicity and decreasing the invasive capacity of the spheroids. Since these effects are regulated by some key proteins, molecular docking results indicated the interaction of naringenin with RIP3 and MLKL, cyclin B and with matrix metalloproteases 2 and 9. The results showed the anti-tumor effect of naringenin on the HeLa spheroids and improved effect of the cisplatin at low concentrations in combination with naringenin, placing flavonoids as a potential adjuvant in the therapy against cervical cancer.

MYPT1 is targeted by miR-145 inhibiting viability, migration and invasion in 2D and 3D HeLa cultures.

The miR-143/145 cluster is down-regulated in cervical tumor cells suggesting a role in tumorigenesis including cytoskeleton remodeling, a key event for tumor progression. The aim of the present work was to determine the role of miR-143/145 in the modulation of the myosin regulator phospho-myosin light chain (pMLC). HeLa monolayer and tridimensional cultures were transfected with miR-143 or miR-145 mimics inhibiting cell viability, proliferation, migration and invasion, mainly through miR-145. MiR-145 transfection increased pMLC levels by targeting the MYPT1 subunit of the regulatory myosin phosphatase. MYPT1 knockdown by siRNAs reproduced miR-145 effects suggesting miR-145 as a tumor suppressor through MYPT1 targeting, leading to a subsequent increase of pMLC levels with implications for cervical cell viability, migration and invasion.

Inflammation-Induced Adverse Pregnancy and Neonatal Outcomes Can Be Improved by the Immunomodulatory Peptide Exendin-4.

Preterm birth is the leading cause of neonatal morbidity and mortality worldwide. Inflammation is causally linked to preterm birth; therefore, finding an intervention that dampens maternal and fetal inflammatory responses may provide a new strategy to prevent adverse pregnancy and neonatal outcomes. Using animal models of systemic maternal inflammation [intraperitoneal injection of lipopolysaccharide (LPS)] and fetal inflammation (intra-amniotic administration of LPS), we found that (1) systemic inflammation induced adverse pregnancy and neonatal outcomes by causing a severe maternal cytokine storm and a mild fetal cytokine response; (2) fetal inflammation induced adverse pregnancy and neonatal outcomes by causing a mild maternal cytokine response and a severe fetal cytokine storm; (3) exendin-4 (Ex4) treatment of dams with systemic inflammation or fetal inflammation improved adverse pregnancy outcomes by modestly reducing the rate of preterm birth; (4) Ex4 treatment of dams with systemic, but not local, inflammation considerably improved neonatal outcomes, and such neonates continued to thrive; (5) systemic inflammation facilitated the diffusion of Ex4 through the uterus and the maternal-fetal interface; (6) neonates born to Ex4-treated dams with systemic inflammation displayed a similar cytokine profile to healthy control neonates; and (7) treatment with Ex4 had immunomodulatory effects by inducing an M2 macrophage polarization and increasing anti-inflammatory neutrophils, as well as suppressing the expansion of CD8+ regulatory T cells, in neonates born to dams with systemic inflammation. Collectively, these results provide evidence that dampening maternal systemic inflammation through novel interventions, such as Ex4, can improve the quality of life for neonates born to women with this clinical condition.

Inhibition of Human Papillomavirus Type 16 Infection Using an RNA Aptamer.

Human papillomavirus type 16 (HPV16) DNA has been found in ∼50% of cervical tumors worldwide. HPV infection starts with the binding of the virus capsid to heparan sulfate (HS) receptors exposed on the surface of epithelial basal layer keratinocytes. Previously, our group isolated a high-affinity RNA aptamer (Sc5c3) specific for HPV16 L1 virus-like particles (VLPs). In this study, we report the inhibition of HPV16 infection by Sc5c3 in a pseudovirus (PsVs) model. 293TT cells were infected by HPV16 PsVs containing the yellow fluorescent protein (YFP) as reporter gene. Incubation of HPV16 PsVs with Sc5c3 before infection resulted in a dose-dependent decrease in YFP fluorescence, suggesting infection inhibition. Aptamer degradation by RNase A restored PsVs infectivity, supporting the previous observation that Sc5c3 aptamer can inhibit infection. VLP mutants with removed HS binding sites were used in binding assays to elucidate the Sc5c3 blocking mechanism; however, no binding difference was observed between wild-type and mutant VLPs, suggesting that pseudoinfection inhibition relies on mechanisms additional to electrostatic HS binding site interaction. A DNA/RNA Sc5c3 version also inhibited HPV PsVs infection, suggesting that a modified, nuclease-resistant Sc5c3 may be used to inhibit HPV16 infection in vivo.

Let-7 miRNA Precursors Co-express with LIN28B in Cervical Cells.

BACKGROUND: The let-7 microRNAs (miRNAs) are frequently dysregulated in carcinogenic processes, including cervical cancer. LIN28 proteins regulate let-7 biogenesis by binding to conserved sequences within the pre-miRNA structure. Nevertheless, recent research has shown that some let-7 miRNAs may escape LIN28 regulation. OBJECTIVE: Correlate pre-let-7 miRNAs and LIN28B levels in cervical cell lines with different malignancy and HPV content. METHODS: Pre-let-7 levels were determined by RTqPCR. LIN28B and other let-7 targets were analyzed by immunoblot. In silico tools were used to correlate let-7 and LIN28B expression and to analyze prelet- 7 sequences and structures. RESULTS: Lin28B protein was detected in all tested cell lines although it was more expressed in tumor cell lines. High levels of pre-let-7c/f-1 and pre-miR-98 were present in almost all cell lines regardless malignancy and LIN28B expression. Pre-let-7g/i were mainly expressed in tumor cell lines, pre-let-7e and pre-let-7-a3 were absent in all cell lines and pre-let-7a-2 showed indistinct expression. LIN28B showed positive correlation with pre-let-7i/g/f-1 and pre-miR-98 in tumor cell lines, suggesting escape from regulation. Sequence alignment and analysis of pre-let-7 miRNAs showed distinctive structural features within the preE region that may influence the ideal pre-let-7 structuring for LIN28B interaction. Short preE-stems were present in pre-let-7 that may escape LIN28B regulation, but long preEstems were mostly associated with high-level pre-let-7 miRNAs. CONCLUSION: The observed differences of pre-let-7 levels in cervical cell lines may be the result of alternative preE structuring affecting interaction with LIN28B thus resulting in differential let-7 regulation.

MARK1 is a Novel Target for miR-125a-5p: Implications for Cell Migration in Cervical Tumor Cells.

BACKGROUND: Aberrant miRNA expression is associated with the development of several diseases including cervical cancer. Dysregulation of miR-125a-5p is present in a plethora of tumors, but its role in cervical cancer is not well understood. OBJECTIVE: The aim was to analyze the expression profile of miR-125a-5p in tumor and immortal cell lines with further target prediction, validation and function analysis. METHODS: MiR-125a-5p expression was determined by real-time RT-PCR from nine cervical cell lines. In silico tools were used to find target transcripts with an miR-125-5p complementary site within the 3'UTR region. Further target selection was based on gene ontology annotation and ΔG analysis. Target validation was performed by transfection of synthetic miR-125a-5p mimics and luciferase assays. Functional evaluation of miR-125a-5p on migration was performed by transwell migration assays. RESULTS: Differential miR-125a-5p expression was observed between immortal and tumor cells regardless of the human papillomavirus (HPV) content. Thermodynamic and ontological analyses showed Microtubule-Affinity-Regulating Kinase1 (MARK1) as a putative target for miR-125a-5p. An inverse correlation was observed among miR-125a-5p expression and MARK1 protein levels in tumor but not in immortal cells. Luciferase assays showed direct miR-125a-5p regulation over MARK1 through recognition of a predicted target site within the 3'-UTR. HeLa and C-33A cervical tumor cells enhanced migration after transfection with miR-125a-5p mimics and stimulation of cell migration was reproduced by siRNA-mediated inhibition of MARK1. CONCLUSION: The results showed MARK1 as a novel functional target for miR-125a-5p with implications on cell migration of tumor cervical cancer cells.

Effect of HPV16 L1 virus-like particles on the aggregation of non-functionalized gold nanoparticles.

Colorimetric assays based on gold nanoparticles (GNPs) are of considerable interest for diagnostics because of their simplicity and low-cost. Nevertheless, a deep understanding of the interaction between the GNPs and the intended molecular target is critical for the development of reliable detection technologies. The present report describes the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and non-functionalized GNPs (nfGNPs) resulting in the inhibition of nfGNPs salt-induced aggregation and the stabilization of purified VLPs. Ionic-competition experiments suggested that the nature of nfGNPs-VLPs interaction is non-covalent. Adsorption of an RNA aptamer on nfGNPs surface showed an additive aggregation-inhibitory effect. The use of mutant VLPs confirmed that the interaction nfGNPs-VLPs is not mediated by the opposing superficial electrostatic charges, suggesting that non-electrostatic forces participate in the arrangement of nfGNPs on the VLPs surface. Competition experiments using increasing ethanol concentrations on nfGNPs-VLPs complexes suggested hydrophobic interactions as the main stabilizing force. Therefore, the nfGNPs-VLPs interaction described here should facilitate the development of adsorption assays based on nfGNPs for HPV detection and cervical cancer prevention.

Targeting of the HPV-16 E7 protein by RNA aptamers.

The expression of high-risk human papillomavirus E6 and E7 proteins in most cervical tumors raised a considerable interest in the diagnostic and therapeutic applications of functional oligonucleotides (i.e., DNAzymes, ribozymes, and aptamers) directed against HPV targets. Aptamers are short single-stranded oligonucleotides that specifically recognize a wide variety of molecular targets, including HPV proteins. Here, we describe a protocol for the successful isolation of RNA aptamers directed at the recombinant HPV-16 E7 protein through the application of the SELEX method. Once the nucleic acid sequence of a functional aptamer is determined, large amounts of the oligonucleotide can be produced and modified at low cost and high efficiency. The remarkable affinity and specificity of aptamers for their targets make these molecules the next-generation tool for diagnostics and therapeutics of cervical cancer.

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

Inhibition of human papillomavirus expression using DNAzymes.

Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion.

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.

Design and function of triplex hairpin ribozymes.

Triplex ribozymes allow for the individual activity of multiple trans-acting ribozymes producing higher target cleavage relative to tandem-expressed RZs. A triplex expression system based on a single hairpin ribozyme for the multiple expression (multiplex) vectors can be engineered to target RNAs with single or multiple antisense-accessible sites. System construction relies on triplex expression modules consisting of hairpin ribozyme cassettes flanked by ribozymes lacking catalytic domains. Multiplex vectors can be generated with single or multiple specificity by tandem cloning of triplex expression modules. Triplex ribozymes are initially tested in vitro using cis- and trans-cleavage assays against radioactive-labeled targets. In addition, triplex ribozymes are tested for cis and trans cleavage in vivo by transfection in cultured cells followed by ribonuclease protection assays (RPAs) and RT-PCR. The use of triplex configurations with multiplex ribozymes will provide the basis for the development of future RZ-based therapies and technologies.

Regulación genética de los papilomavirus humanos genitales / Genetic regulation of human genital papillomavirus

Los papilomavirus humano (PVH) infectan epitelios estratificados queratinizados con una alta especificidad y están asociados con la aparición y persistencia de neoplasias benignas y malignas. Los elementos que dirigen la expresión genética de estos virus se localizan en una región no codificadora conocida como región larga de control (RLC). Al inicio del ciclo viral, una combinación particular de factores celulares que interactúan con la RLC promueven la transcripción temprana de los oncogenes virales E6 y E7. Estos favorecen la división celular interrumpiendo los mecanismos regulatorios celulares: E6 se une a la proteína supresora de tumor p35 y E7 se une a p105RB. La continuidad en la transcripción temprana conlleva al aumento gradual de las proteínas virales E1 y E2. La proteína E2 impide la transcripción temprana y confiere especificidad de unión a E1, la cual promueve la replicación viral. El cese paulatino de la transcripción de los oncogenes virales a través de la represión por E2, libre la regulación del crecimiento celular mediada por p53 y p105RB, permitiendo que la diferenciación celular progrese. Es entonces cuando el promotor tardío funciona para la producción de las proteínas de la cápside viral L1 y L2, permitiendo la maduración de viriones en los estratos superiores del epitelio. La disrupción del gen E2 durante un evento de integración del genoma viral, impide la progreción del ciclo vira y la entrada del programa de diferenciación de la célula epitelial, sosteniendo el estado transformado producido por E6 y E7

Human papillomavirus (HPV) specifically infect stratified epithelial cells, causing benign and malignant neoplasia. Several elements directing this virus' genetic expression are present in a non-coding region called LCR. HPV infection starts in the basal cells of stratified epithelia, where a particular combination of cellular factors interacting with the LCR starts the transcription of the viral E6 and E7 oncogenes. The E6 and E7 genes alter the cell cycle because they interact and inactivate tumor suppressor proteins: E6 binds and degrades protein p53 and E7 associates with p105RB. E1 and E2 are the next synthesized proteins. E2 blocks the early transcription and permits E1 specific binding to the viral origin of replication located within the lcr, initiating the viral genome replication. Following the course of viral infection, the E2-induced E6 and E7 down-regulation releases p53 and p105RB proteins, and the differentiation process can continue. Then, a putative late promoter can activate the capsid genes L1 and L2. At this step, mature virions can be detected in the upper layers of the epithelium. Disruption in E2 gene transcription is usually associated to genital malignant neoplasia. In the absence of E2, E6 and E7 remain constitutively expressed, sustaining the immortality of the infected cell and blocking the epithelial differentiation program.