E1 Region Research Articles

A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya.

Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

Sublingual papillomas of cheetahs in southern Africa.

Nine distinct papillomaviruses (Lambdapapillomavirus) have been described in domestic and nondomestic cats, but not in cheetahs. These viruses have been associated with cutaneous papillomas or plaques, bowenoid in situ carcinomas, feline cutaneous squamous cell carcinomas (SCC), feline sarcoids, and oral (often sublingual) papillomas. Fourteen cheetahs from the AfriCat foundation (Namibia) and one from the Ann van Dyk Cheetah center (South Africa) presented with sublingual lesions reminiscent of sublingual papillomas. Two animals were biopsied and the histopathology revealed benign proliferative epithelial lesions with prominent thickening of the overlying squamous epithelium. Throughout the squamous epithelial layers were cells with nuclear enlargement, irregularity of the nuclear membranes and cell contours, focal hyperchromasia of the nuclei, and perinuclear halos, reminiscent of a virus-associated process as seen in papillomavirus infections. Thirteen more cheetahs were sampled and the tissue snap frozen for molecular characterization. Amplification and sequencing of the papillomavirus L1, E6, E7, and E1 gene regions was achieved with modified primers. Maximum likelihood phylogenetic analyses revealed all 15 cheetah papilloma samples were 99.99% genetically similar and closely related to, but genetically distinct from any known felinepapillomaviruses. All cheetahs were FIV and FeLV negative. The results suggest the samples identified in this study can be considered a previously undescribed or novel feline papillomavirus and the authors propose "Acinonyx jubatus papillomavirus type 1" (AjPV-1), within the Lambdapapillomavirus 1 genus (Family: Papillomaviridae).

Deep Sequencing of HPV16 E6 Region Reveals Unique Mutation Pattern of HPV16 and Predicts Cervical Cancer.

ABSTRACTThe genetic diversity of human papillomavirus (HPV) 16 within cervical cells and tissue is usually associated with persistent virus infection and precancerous lesions. To explore the HPV16 mutation patterns contributing to the cervical cancer (CC) progression, a total of 199 DNA samples from HPV16-positive cervical specimens were collected and divided into high‐grade squamous intraepithelial lesion (HSIL) and the non‐HSIL(NHSIL) groups. The HPV16 E6 region (nt 7125-7566) was sequenced using next-generation sequencing. Based on HPV16 E6 amino acid mutation features selected by Lasso algorithm, four machine learning approaches were used to establish HSIL prediction models. The receiver operating characteristic was used to evaluate the model performance in both training and validation cohorts. Western blot was used to detect the degradation of p53 by the E6 variants. Based on the 13 significant mutation features, the logistic regression (LR) model demonstrated the best predictive performance in the training cohort (AUC = 0.944, 95% CI: 0.913–0.976), and also achieved a high discriminative ability in the independent validation cohort (AUC = 0.802, 95% CI: 0.601–1.000). Among these features, the E6 D32E and H85Y variants have higher ability to degrade p53 compared to the E6 wildtype (P < 0.05). In conclusion, our study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting HSIL. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of CC.IMPORTANCE The study provides evidence for the first time that HPV16 E6 sequences contain vital mutation features in predicting the high‐grade squamous intraepithelial lesion and can reduce even more unneeded colposcopies without a loss of sensitivity to detect cervical cancer. Moreover, the D32E and H85Y variants of E6 exhibited a significantly higher ability to degrade p53, which may play a vital role in the development of cervical cancer.

LINC01194 recruits NUMA1 to promote ubiquitination of RYR2 to enhance malignant progression in triple-negative breast cancer

Long intergenic nonprotein coding RNA 1194 (LINC01194) has been reported as an oncogene in several cancer types, but its expression and potential role in triple-negative breast cancer (TNBC) are still unclear. We found that LINC01194 was significantly highly expressed in TNBC based on The Cancer Genome Atlas (TCGA) database. Data from in vitro experiments and in vivo assays demonstrated that LINC01194 promoted TNBC progression. Through bioinformatics prediction, mass spectrometry, and mechanical experiments, we found that LINC01194 could recruit nuclear mitotic apparatus protein 1 (NUMA1) to bind to the untranslated region (3′UTR) of ubiquitin-conjugating enzyme E2 C (UBE2C) 3′ and stabilize UBE2C mRNA. Moreover, we found that UBE2C acted as an ubiquitin ligase to promote the ubiquitination and degradation of ryanodine receptor type 2 (RYR2) that inhibited the progression of TNBC by inhibiting the Wnt/β-catenin signaling pathway. In summary, LINC01194 activate the Wnt/β-catenin signaling pathway and accelerates the malignant progression of TNBC by recruiting NUMA1 to stabilize UBE2C mRNA and thus promotes RYR2 ubiquitination and degradation. These findings might provide a more effective therapeutic strategy for TNBC patients.

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

Multi-omics characterization of productive HPV integration in cervical cancer.

e17508 Background: Cervical cancer (CC), which is caused by high-risk human papillomavirus (HPV), remains a significant public health problem worldwide. HPV integration into the host genome is a critical aetiological event in cervical carcinogenesis and progression and it can be silent or actively transcribed that leads to production of viral-host fusion transcripts. We aim to study the characterization of silent and productive HPV integration in cervical cancer. Methods: Cervical cancer (CC), which is caused by high-risk human papillomavirus (HPV), remains a significant public health problem worldwide. HPV integration into the host genome is a critical aetiological event in cervical carcinogenesis and progression and it can be silent or actively transcribed that leads to production of viral-host fusion transcripts. We aim to study the characterization of silent and productive HPV integration in cervical cancer. Results: Virus capture sequencing revealed 762 virus-host DNA fusion breakpoints and only 142 (18.6%) were actively transcribed into fusion transcripts. The productive HPV integrations were nonrandom, with an increase in breakpoints in the HPV E1 region、 human introns and common fragile sites. They were associated with significant changes in host focal genome structure and gene transcription, but not in protein production in 40 kb region, and produced higher levels of E6 and E7 proteins detected by Western Blot. Clinical, transcriptional, proteomic, phosphoproteomic and single-cell data demonstrated that EMT, proliferation and extracellular matrix associated pathway were enriched in productive HPV integrated tumors and contributed to advanced clinical stage. Conclusions: We demonstrated that productive HPV integration is associated with higher E6/E7 proteins and enhanced tumor aggressiveness and immunoevasion in situ to advanced disease. This study improves our understanding of the effects of HPV fusion transcripts on the biology and pathophysiology of HPV-driven cervical cancer that has the potential to direct new prevention and therapeutic strategies

Genetic diversity of E6, E7 and the long control region in human papillomavirus type 16 variants in Beijing, China

BackgroundHigh-risk human papillomavirus type 16 (HPV16) is a risk factor for cervical cancer. The progression from initial infection to cervical cancer has been linked to properties of the viral sequences. However, the distribution of HPV16 variants among Chinese women has not been extensively addressed and the role of HPV16 variants in the risk of cervical carcinogenesis remains poorly understood. MethodsHPV16 positive cervical exfoliated cell samples were collected from 249 women living in Beijing, China. PCR products from two fragments of E6-E7 and LCR of HPV16 in these samples were sequenced and analyzed. ResultsLineage A was found in the subjects, including A1, A2, A3 and A4 sublineages. Based on the HPV16 reference sequences, 26 nucleotide mutations of A4 sublineage and 39 nucleotide mutations of A1-3 sublineages were found in the E6, E7 and LCR of HPV16 isolates. Point mutations T843C, A7287C and A7872G of A4 sublineage were significantly associated with high-grade cervical lesions. The high-frequency sites in HPV16 LCR located at regions that can bind to multiple transcription factors. ConclusionsThis study contributes to the identification of unique variants in the E6, E7 and LCR of HPV16 isolates infected in Chinese women. Mutations of T843C, A7287C and A7872G in A4 sublineages were significantly associated with high-grade cervical lesions, suggesting that mutations in the E7 and LCR region have potential effects on viral replication and progression of cervical cancer.

Adenovirus Vector With ADP Gene Induces Cytopathic Effects in HEK293 Cells Without Significant Elevation of Virus Titers.

Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome. We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined. The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers. The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells.

Determination of Human Papillomavirus Type 18 Lineage of E6: A Population Study from Iran.

The epidemiological studies in Iran on HPV18 nucleotide changes are rare. This type of virus is prevalent in the Iranian population. Therefore, in the present study, we aimed to identify the genetic variability in HPV18 in the E6 region to evaluate the prevalence of lineage distribution and sublineages in a sample population in Iran. Overall, 60 HPV18 confirmed cases were investigated between 2019 and 2021. The specimens were collected, and molecular genotyping was done using the Linear Array HPV Genotyping Test. DNA extraction was performed by a viral DNA/RNA kit. The HPV E6 gene was amplified by using type-specific primers designed according to the HPV18 genome prototype sequence. The sequencing of the E6 region was successfully done on 43 samples which were then compared to the reference sequence. The most frequent sublineage of HPV18 in this study was A4 (69.7%), followed by A1 (18.6%) and A3 (11.6%). Neither A2 nor A5 sublineage was not detected in this study. The related nucleotide acid changes according to the main references were as follows: A3: T104C/T232G/T485C/C549A, A4: T104C/T485C/C549A. The predominance of A lineage with the high frequency of A4 sublineage was found in the present research. The importance of sublineages in susceptibility to a progressive form of infection requires to be more investigated among the different population.

Occurrence and molecular characterization of human pegivirus-1 (HPgV-1) viremia in healthy volunteer blood donors from Northern Portugal.

Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family and the Pegivirus genus. Despite having been discovered 25 years ago, there is still much to know regarding HPgV-1 clinical impact, as this virus is currently not associated with any pathology. Yet, HPgV-1 prevalence and molecular characterization are still unknown in many countries, including Portugal. To fill in this knowledge gap, this study aimed to determine the occurrence and molecular characterization of HPgV-1 in a group of healthy blood donors from the north of Portugal. Blood samples from 465 Portuguese blood donors were collected from a major Hospital Center in the north of Portugal. RNA was extracted and an initial nested RT-PCR was performed targeting the conserved 5'-untranslated region region of the HPgV-1 genome. A second nested RT-PCR targeting the E2 region was performed for genotyping. Only one sample tested positive for HPgV-1 RNA, resulting in a prevalence of approximately 0.22%. Phylogenetic analyses confirmed the characterization as genotype 2, the most prevalent in Europe.

Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector.

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.

Intra-host analysis of hepaciviral glycoprotein evolution reveals signatures associated with viral persistence and clearance.

Even 30 years after the discovery of the hepatitis C virus (HCV) in humans there is still no vaccine available. Reasons for this include the high mutation rate of HCV, which allows the virus to escape immune recognition and the absence of an immunocompetent animal model for vaccine development. Phylogenetically distinct hepaciviruses (genus Hepacivirus, family Flaviviridae) have been isolated from diverse species, each with a narrow host range: the equine hepacivirus (EqHV) is the closest known relative of HCV. In this study, we used amplicon-based deep-sequencing to investigate the viral intra-host population composition of the genomic regions encoding the surface glycoproteins E1 and E2. Patterns of E1E2 substitutional evolution were compared in longitudinally sampled EqHV-positive sera of naturally and experimentally infected horses and HCV-positive patients. Intra-host virus diversity was higher in chronically than in acutely infected horses, a pattern which was similar in the HCV-infected patients. However, overall glycoprotein variability was higher in HCV compared to EqHV. Additionally, selection pressure in HCV populations was higher, especially within the N-terminal region of E2, corresponding to the hypervariable region 1 (HVR1) in HCV. An alignment of glycoprotein sequences from diverse hepaciviruses identified the HVR1 as a unique characteristic of HCV: hepaciviruses from non-human species lack this region. Together, these data indicate that EqHV infection of horses could represent a powerful surrogate animal model to gain insights into hepaciviral evolution and HCVs HVR1-mediated immune evasion strategy.

HPV E1 qPCR, a Low-Cost Alternative Assay to Roche Diagnostic Linear Array is Effective in Identifying Women at Risk for Developing Cervical Cancer.

ObjectiveSince a quantitative polymerase chain reaction (qPCR) assay targeting the E1 region of HPV genome is cost-effective/simple to perform, we evaluated the agreement between the Roche Diagnostics Linear Array (RDLA) genotyping test and qPCR-based E1 assay to detect HR-HPV genotypes that are included or not included in HPV vaccines and compared their accuracy to detect CIN 2+.MethodsStudy population included 257 African American (AA) and 266 Caucasian American (CA) diagnosed with intraepithelial neoplasia (CIN) grades ≤CIN 1 or ≥CIN 2 (CIN 2+) and tested for HPV by the RDLA and E1 assay. The concordance was determined using Gwet’s AC1. The calculated positive predictive value (PPV) and negative predictive value (NPV) of the two assays were used to determine their suitability to detect CIN lesions.ResultsOverall, the E1 assay showed substantial agreement with the RDLA assay to detect any HR-HPV genotype and the agreement was higher in women diagnosed with CIN 2+ than ≤CIN 1. The concordance was largely higher in Cas than in Aas. The NPV and PPV values to detect CIN lesions were similar between the two assays.ConclusionUtilization of the HPV E1 assay as a tool for CC screening could be a cost-effective approach that applies to both vaccinated and unvaccinated populations.

Isolation and Genomic Analysis of Single Circulating Tumor Cell Using Human Telomerase Reverse Transcriptase and Desmoglein-2.

As epithelial cells in the circulation are considered to originate from the tumor, the epithelial cell adhesion molecule has been commonly used as a standard marker for circulating tumor cells (CTCs) isolation. However, it seems to disappear after the epithelial-mesenchymal transition that most cancer cells undergo for intravasation. Thus, more advanced techniques for CTC detection are needed to better understand the clinical significance of CTCs. A cancer cell-specifically-infecting or replicating virus that codes a fluorescent monitor gene can be a solution to efficiently detect CTCs. Thus, the authors designed an adenovirus to bind to desmoglein-2, which is highly expressed in most cancer cells. A cancer-specific human telomerase reverse transcriptase promoter is inserted to control a viral E1 region. The adenovirus is utilized to compare the number of CTCs from renal cell carcinoma and prostate cancer patients before and after surgery. The isolated two or three CTCs are eligible for whole genome sequencing. The genomic analysis proves the difference of variants between primary tumors and CTCs. Taken together, it is a fast and exact serial method for CTC isolation and the enriched genome sequencing may be used to determine the prognosis and as a point-of-care system for patients with cancer.

Development of Nucleic Acid Lateral Flow Immunoassay for Rapid and Accurate Detection of Chikungunya Virus in Indonesia.

Chikungunya fever is an arboviral disease caused by the Chikungunya virus (CHIKV). The disease has similar clinical manifestations with other acute febrile illnesses which complicates differential diagnosis in low-resource settings. We aimed to develop a rapid test for CHIKV detection based on the nucleic acid lateral flow immunoassay technology. The system consists of a primer set that recognizes the E1 region of the CHIKV genome and test strips in an enclosed cassette which are used to detect amplicons labeled with FITC/biotin. Amplification of the viral genome was done using open-source PCR, a low-cost open-source thermal cycler. Assay performance was evaluated using a panel of RNA isolated from patients' blood with confirmed CHIKV (n = 8) and dengue virus (n = 20) infection. The open-source PCR-NALFIA platform had a limit of detection of 10 RNA copies/ml. The assay had a sensitivity and specificity of 100% (95% CI: 67.56% - 100%) and 100% (95% CI: 83.89% - 100%), respectively, compared to reference standards of any positive virus culture on C6/36 cell lines and/or qRT-PCR. Further evaluation of its performance using a larger sample size may provide important data to extend its usefulness, especially its utilization in the peripheral healthcare facilities with scarce resources and outbreak situations.

Adenovirus Biology, Recombinant Adenovirus, and Adenovirus Usage in Gene Therapy.

Gene therapy is currently in the public spotlight. Several gene therapy products, including oncolytic virus (OV), which predominantly replicates in and kills cancer cells, and COVID-19 vaccines have recently been commercialized. Recombinant adenoviruses, including replication-defective adenoviral vector and conditionally replicating adenovirus (CRA; oncolytic adenovirus), have been extensively studied and used in clinical trials for cancer and vaccines. Here, we review the biology of wild-type adenoviruses, the methodological principle for constructing recombinant adenoviruses, therapeutic applications of recombinant adenoviruses, and new technologies in pluripotent stem cell (PSC)-based regenerative medicine. Moreover, this article describes the technology platform for efficient construction of diverse “CRAs that can specifically target tumors with multiple factors” (m-CRAs). This technology allows for modification of four parts in the adenoviral E1 region and the subsequent insertion of a therapeutic gene and promoter to enhance cancer-specific viral replication (i.e., safety) as well as therapeutic effects. The screening study using the m-CRA technology successfully identified survivin-responsive m-CRA (Surv.m-CRA) as among the best m-CRAs, and clinical trials of Surv.m-CRA are underway for patients with cancer. This article also describes new recombinant adenovirus-based technologies for solving issues in PSC-based regenerative medicine.

A FLI1-KLF6 Axis Regulates Aging in Human Hematopoietic Stem and Progenitor Cells and Normalization of KLF6 Levels in Aged Cells Leads to Their Rejuvenation

Aging causes a gradual decline in hematopoietic stem cell (HSC) function, which increases the risk for hematological malignancies. While much has been done in murine models, human HSC aging impairment is less understood. We recently showed that Krüppel-like transcription factor 6 (KLF6) is among the top downregulated genes during human HSC aging, which correlates with H3K27ac loss at several upstream putative enhancers. Moreover, loss of KLF6 in human CD34 + cells resulted in impaired in vitro differentiation, increased colony-forming potential and a transcriptional profile similar to that of aged CD34 + CD38 - cells. We hypothesized that age-acquired deregulation of KLF6 may be a key player in age-related HSC dysfunction and sought to fully characterize this. Thus, we isolated CD34 + cells from young (<32 y.o) and aged (>65 y.o.) healthy donors and performed CRISPR-Cas9 genome editing and transcriptional activation of KLF6, respectively, followed by epigenetic and transcriptional reprogramming, in vivo hematopoietic reconstitution, and analysis of DNA damage, apoptosis, and reactive oxygen species (ROS) levels.KLF6 knock-out (KO) and non-targeting control (NTC) cells from young healthy donors were engrafted into immunodeficient NSGS mice. Hematopoietic reconstitution analysis showed that KLF6 KO cells led to increased myeloid and reduced lymphoid reconstitution in peripheral blood (PB; p<1.62 -7) and an increase in immunophenotypically defined HSC and CD34 + CD38 - progenitor fractions in the bone marrow (BM; p=0.02, and p=0.04, respectively). H3K27ac analysis of KLF6 KO cells revealed a loss of 3,390 ChIP-seq peaks (FDR < 0.05) and 285 peaks gained. Functional annotation using ChIP-Enrich showed that H3K27ac loss associates with myeloid homeostasis, erythroid differentiation and oxidative stress (FDR < 0.05). Three putative enhancer (E) regions upstream of the KLF6 locus showed loss of H3K27ac with aging. Depletion of the E1 but not E2 or E3 regions phenocopied in vitro and in vivo findings of KLF6 KO. Transcription factor (TF) ChIP-seq data analysis revealed FLI1, ERG, and RUNX1 binding overlapping the E1 region. Knockdown of FLI1 but not ERG or RUNX1 led to an increase in KLF6. Notably, FLI1 mRNA levels, but not ERG or RUNX1, are increased during normal aging.We next performed in vitro KLF6 activation in aged CD34 + (KLF6a) cells using a dCas9-VP64 system to test if we could rejuvenate these cells. KLF6a cells exhibited a decrease in their in vitro myeloid differentiation potential, compared to aged NTC CD34 + cells (p<0.0041), and behaved instead similar to young controls. ChIP-seq analysis of KLF6a showed marked decrease of H3K4me1 (n=3,273 peaks) with relatively few regions with increased H3K4me1 (n=602) (FDR < 0.05). In contrast, we observed an increase in H3K27ac (n=3,361 peaks) with only 71 peaks lost compared to aged NTC (FDR < 0.05). Regions that gained H3K27ac in KLF6a were associated with platelet activation, cell junction and adhesion. In vivo analysis of KLF6a cells injected into NSGS mice revealed a significant reduction in the PB myeloid fraction compared to NTC (p<1.2-8), with a concomitant expansion in the lymphoid compartment (p<4.4 -11). BM composition analysis at week 16 showed a decrease in the HSC fraction in KLF6a cells (p=0.0029) as well as a reduction in CD34 +CD38 -, CD34 +CD38 + and MEPs (p=0.036, p<0.0001 and p=0.041, respectively). We next examined the impact of KLF6 modulation on DNA damage and observed that young human KLF6 KO cells had a significant increase in gH2AX and 53BP1 (p<0.0001, for both) whereas KLF6a in aged CD34 + cells exhibited reduced gH2AX and 53BP1 foci in comparison to aged NTC (p<0.0001, for both). In addition, apoptotic levels in KLF6 KO cells were higher than in NTC cells (p=0.006) whereas aged KLF6a cells showed a reduction in the incidence of apoptotic cells compared to NTC (p=0.019). Finally, ROS analysis in young KLF6 KO showed increased levels of total and mitochondrial ROS compared to NTC (p=0.0008 and p<0.0001, respectively) whereas both ROS fractions were reduced in KLF6a cells (p=0.0002 and p<0.0001, respectively).In summary, these results show that the FLI1-KLF6 axis plays a key role in regulating HSPC aging and that KLF6 is required for normal HSPC function and differentiation. In addition, normalization of KLF6 levels in aged HSPCs resulted in reprogramming and rejuvenation HSPCs, confirming the central role of this TF in aging HSPC biology. DisclosuresNo relevant conflicts of interest to declare.

Assessment of New E2 Protein Domain Interaction with PKR Protein to Control IFN Signaling

Introduction: Controversy exists regarding the impact of Phosphorylation Homology Domain (PePHD) of Hepatitis C Virus (HCV) E2 protein on the interruption of the antiviral signaling pathway. A mechanism by which the virus evades the antiviral effect of interferon (IFN) alpha involves protein kinase (PKR) eukaryotic Initiation Factor 2 alpha (eIF2a) PePHD. By binding to PKR, PePHD inhibits its activity and, therefore, causes the virus to evade the antiviral activity of IFN. This study aimed to clarify the inconsistency of different conclusions reached in previous studies using reliable bioinformatics tools. Methods: Fifty-eight Iranian patients infected with HCV genotypes 1a and 3a and 58 healthy control individuals were examined. Plasma viral RNA was used to amplify and sequence the HCV E2 gene; also, HCV viral load, genotyping, IL-28B genotyping, alanine Aminotransferase (ALT), and aspartate aminotransferase (AST) test were determined. Bioinformatics tools determined the physicochemical properties, B-cell epitopes, post-modification changes, and secondary/tertiary structures, and also evaluated the interactions between E2 and PePHD regions. Results: The results showed a new domain that is responsible for binding to PKR protein and is important to the intrigued antiviral response. Physicochemical features and post-modifications were defined, showing that E2 is highly phosphorylated and there were numerous possible disulfide bonds. Secondary and tertiary structures for E2 protein were constructed. No significant relationship between ALT and AST and treatment failure was detected. Conclusions: Docking analysis showed a new domain in E2 protein that can be involved in the interaction between PKR and E2 protein. This finding may justify our results revealing the non-significant relationship between mutations in PePHD and treatment failure.

Designing DNA probe from HPV 18 and 58 in the E6 region for sensing element in the development of genosensor-based gold nanoparticles.

The E6 region has higher protuberant probability annealing than consensus probe focusing on another region in the human papillomavirus (HPV) genome in terms of detection and screening method. Here, we designed the first multiple virus single-stranded deoxyribonucleic acid (ssDNA) for multiple detections in an early phase of screening for cervical cancer in the E6 region and became a fundamental evolution of detection electrochemical HPV biosensor. Gene profiling of the virus ssDNA sequences has been carried by high-end bioinformatics tools such as GenBank, Basic Local Alignment Searching Tools (BLAST), and Clustal OMEGA in a row. The output from bioinformatics tools resulted in 100% of similarities between our virus ssDNA probe and HPV complete genome in the databases. The cross-validation between HPV genome and our designed virus ssDNA provided high specificity and selectivity during screening methods compared with Pap smear. The DNA probe for HPV 18, 5' COOH-GAT CCA GAA GGT ACA GAC GGG GAG GGC ACG 3', while 5'COOH-GGG CGC TGT GCA GTG TGT TGG AGA CCC CGA3' as DNA probe for HPV 58 designed with 66.77% guanine (G) and cytosine (C) content for both. Our virus ssDNA probe for the HPV biosensor promises high sensitivity, specificity, selectivity, repeatability, low fluid consumption, and will be useful in mini-size diagnostic devices for cervical cancer detection.

Effect of E2 and long control region polymorphisms on disease severity in human papillomavirus type 11 mediated mucosal disease: Protein modelling and functional analysis

Interaction of the long control region (LCR) and the E2 protein of HPV11s was studied by in silico modelling and in vitro functional analysis. Genomes of HPV11s from fifteen (six known and nine novel) patients (two solitary papillomas, eleven respiratory papillomatoses of different severity, one condyloma acuminatum and one cervical atypia) were sequenced; E2 polymorphisms were analysed in silico by protein modelling. E2 and LCR variants were cloned into pcDNA3.1+ expression vector and into pALuc reporter vector, respectively, transfected to HEp2 cells alone or in different combinations and the luciferase activity was measured. In the E2, the ubiquitous polymorphism K308R caused stronger binding between the dimers but did not alter DNA binding; E2s with this polymorphism were significantly less efficient than the reference in promoting LCR activity. The unique polymorphism Q86K changed the negative surface charge of E2 (Q86) to positive (K86). The unique polymorphisms S245F and N247T in the hinge region disrupt a probable phosphorylation site in a RXXS motif targeted by protein kinase A and B, but do not affect directly the amino acids critical to nuclear transport. Both unique patterns partly restored the LCR activating potential disrupted by K308R. A unique E2/E4 ORF with a 58-bp deletion leading to a frameshift and an early stop codon resulted in a practically nonfunctional E2, and was associated with a papillomatosis with dysplasia. When testing existing LCR-E2 combinations, LCR with intrinsically lower enhancer capacity was only marginally activated by its E2 (R308 and the deletion mutant), and did not significantly exceed the activity of the reference LCR without E2. Combined with more potent LCRs associated with more severe disease, the activity was significantly higher, but still significantly lower than LCRs with reference E2. In summary, LCR-E2 interaction determined by their polymorphisms may explain, at least partly, differences in disease severity.