Expression Of Env Gene Research Articles

Exploring the role of endogenous retroviruses in seasonal reproductive cycles: a case study of the ERV-V envelope gene in mink.

Endogenous retroviruses (ERVs), which originated from exogenous retroviral infections of germline cells millions of years ago and were inherited by subsequent generations as per Mendelian inheritance patterns, predominantly comprise non-protein-coding sequences due to the accumulation of mutations, insertions, deletions, and truncations. Nevertheless, recent studies have revealed that ERVs play a crucial role in diverse biological processes by encoding various proteins. In this study, we successfully identified an ERV envelope (env) gene in a mink species. A phylogenetic tree of mink ERV-V env and reference sequences was constructed using Bayesian methods and maximum-likelihood inference. Phylogenetic analyses indicated a significant degree of sequence conservation and positive selection within the env-surface open reading frame. Additionally, qRT-PCR revealed diverse patterns of mink ERV-V env expression in various tissues. The expression of mink ERV-V env gene in testicular tissue strongly correlated with the seasonal reproductive cycles of minks. Our study suggests that the ERV-V env gene in mink may have been repurposed for host functions.

Characterization of the endogenous retrovirus-derived placenta-specific soluble protein EnvV-Fca from domestic cats.

Endogenous retroviruses (ERVs) are remnants of ancestral viruses in the host genome. The present study identified the expression of a defective retroviral env gene belonging to the ERV group V member Env (EnvV) in Felis catus (EnvV-Fca). EnV-Fca was specifically detected in the placental trophoblast syncytiotrophobic layer and expressed as a secreted protein in cultured cells. Genetic analyses indicated that EnvV2 genes are widely present in vertebrates and are under purifying selection among carnivores, suggesting a potential benefit for the host. This study suggests that birds, bats, and rodents carrying EnvV2 may play significant roles as intermediate vectors in spreading or cross-transmitting viruses among species. Our findings provide valuable insights into the evolution of ERV in vertebrate hosts.

HERV-E ƛ 4-1 activation in peripheral blood mononuclear cells of the recurrent depression patients under the influence of human recombinant IL-1β

IntroductionMental disorders represent complex phenotypes and are the leading causes of global disease burden. Human endogenous retroviruses (HERVs) are ancient retroviral DNA sequences established into germline. Their tight regulation is mainly achieved by epigenetic mechanisms, which can be altered by environmental factors - viral infections, inflammation, leading to HERV activation. The aberrant expression of HERVs associates with neurological diseases and mood disorders. We showed earlier that HERV-E ƛ 4-1 activation is associated with the recurrent depression stage of exacerbation and are accompanied by a pronounced increase in the proinflammatory activity of the peripheral blood mononuclear cells (PBMC).ObjectivesThe purpose of the study was to evaluate the activity of HERV-E ƛ 4-1 on PBMCs of patients with recurrent depression in remission, including under the influence of recombinant human IL-1β.MethodsThe study included 30 patients with an established diagnosis of recurrent depression (F 33.0) aged 26–45 years. PBMC were isolated using the Ficoll density gradient method and further cultured in the presence or absence of 1 mkg/ml of recombinant human IL-1β for 24 hours. HERV-E λ 4 – 1 env gene expression was determined by the PCR. Cells proliferative activity was determined by H3-thymidine incorporation. Cytokines content in culture supernatants was assessed by ELISA.ResultsIt was shown that in all samples of PBMC cultured without IL-1β the HERV-E λ 4 – 1 env expression was not determined. After the PBMC cocultivation with recombinant human IL-1β, HERV-E λ 4 – 1 env gene expression was determined in 86,7% of cases. The HERV-E ƛ 4-1 activation in PBMC after IL-1β influence was accompanied by increased cells proliferative activity and production of IL-1β, IL-6.ConclusionsOur data indicate that the HERV-E λ 4 – 1 env expression in PBMC of recurrent depression patients in the stage of remission induced by the influence of proinflammatory cytokines, such as IL-1β. This mechanism may be one of the possible regulators of HERV-E λ 4 – 1 activation in recurrent depression.Disclosure of InterestNone Declared

Correlation analysis of cancer stem cell marker CD133 and human endogenous retrovirus (HERV)-K env in SKOV3 ovarian cancer cells.

Human endogenous retrovirus (HERV)-K is a type of retrovirus that is present in the human genome, and its expression is usually silenced in healthy tissues. The precise mechanism by which HERV-K env influences cancer stemness is not fully understood, but it has been suggested that HERV-K env may activate various signaling pathways that promote stemness traits in cancer cells. To establish the connection between HERV-K env expression and cancer stemness in ovarian cancer cells, we carried out correlation analyses between HERV-K env and the cancer stem cell (CSC) marker known as the cluster of differentiation 133 (CD133) gene in SKOV3 ovarian cancer cells. To perform correlation analysis between HERV-K env and CSCs, ovarian cancer cells were cultured in a medium designed for cancer stem cell induction. The expression of HERV-K env and CD133 genes was verified using quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Additionally, the expression of stemness-related markers, such as OCT-4 and Nanog, was also confirmed using RT-qPCR. In the stem cell induction medium, the number of tumorsphere-type SKOV3 cells increased, and the expression of CD133 and HERV-K env genes was up-regulated. Additionally, other stemness-related markers like OCT-4 and Nanog also exhibited increased expression when cultured in the cancer stem cell induction medium. However, when HERV-K env knockout (KO) SKOV3 cells were cultured in the same cancer stem cell induction medium, there was a significant decrease in the number of tumorsphere-type cells compared to mock SKOV3 cells subjected to the same conditions. Furthermore, the expression of CD133, Nanog, and OCT-4 did not show a significant increase in HERV-K env KO SKOV3 cells compared to mock SKOV3 cells cultured in the same cancer stem cell induction medium. These findings indicate that the expression of HERV-K env increased in SKOV3 cells when cultured in cancer stem cell induction media, and cancer stem cell induction was inhibited by KO of HERV-K env in SKOV3 cells. These results suggest a strong association between HERV-K env and stemness in SKOV3 ovarian cancer cells.

HERV-K np9 and HERV-R env: Two endogenous retrovirus products with potential Pathogenic roles in pediatric acute lymphoblastic leukemia.

Human endogenous retroviruses (HERVs) are integral components of the human genome, and their reactivation has been implicated in the pathogenesis of some malignancies. External viral co-infections are suspected to play a role in HERV transactivation. This study aimed to investigate the expression of HERV-K np9 elements and HERV-R env gene in pediatric acute lymphoblastic leukemia (ALL) patients. Additionally, we explored potential correlations between HERV expression and common viral infections prevalent in this group of patients. Blood samples were collected from 43 pediatric ALL patients and 48 age- and sex-matched healthy controls. Quantitative real-time PCR (qRT-PCR) was used to assess the expression of HERV-K np9 and HERV-R env, along with herpes simplex virus (HSV), parvovirus B19, and polyomavirus BK. HERV-K np9 and HERV-R env showed significantly higher expression in the peripheral blood of ALL patients compared to healthy controls (p < .001 and p = .003, respectively). HSV positivity was associated with significantly increased HERV-K np9 expression. No significant correlations were observed between other investigated viruses and HERV gene expression. The overexpression of HERV-K np9 and HERV-R env in pediatric ALL patients suggest their potential role in leukemogenesis. Our findings also suggest a possible link between HSV infection and HERV reactivation in this population. Future investigations are needed to understand the precise roles of these genes and viral infections in the development of ALL.

Higher Expression of Human Endogenous Retrovirus-K was Observed in Peripheral B Lymphocytes of Leukemia and Lymphoma Patients.

Hematological malignant tumors (HMTs) are serious diseases that threaten human health and life with high mortality. Therefore, it is necessary to develop novel strategies for diagnosis and treatment. Human endogenous retroviruses (HERVs) have recently attracted increasing attention as potential targets for cancer diagnosis and therapy. In this study, we explored the association between HERV-K expression levels and HMTs development. Clinical data and peripheral blood samples were collected from 236 leukemia, 384 lymphoma patients, and 69 healthy controls. Quantitative polymerase chain reaction was used to detect the expression of HERV-K gag, pol, and env genes in peripheral blood mononuclear cells or different cell subpopulations. Differently expressed HERV-K genes were further tested by using deep sequencing method, and further analyzed with gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. B cell- and T cell-related cytokines in patients were also detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the expression levels of the HERV-K gag, pol, and env genes in patients were significantly higher than in healthy controls. There was a correlation between the expression level of HERV-K and the clinicopathological parameters of leukemia patients. HERV-K expression was increased in the B lymphocytes of leukemia and lymphoma patients, but not in the T cells or neutrophils. The GO and KEGG analyses showed that abnormal expression of the HERV-K locus in patients affected immune regulation. The analysis of cytokines proved that the B cell-related cytokines, including interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon-gamma, were significantly decreased in patients, while the T cell-related cytokines, including IL-3, IL-12, and TNF-β, were not significantly changed. In conclusion, HERV-K genes might participate in the occurrence and development of leukemia and lymphoma, and might be biomarkers for the detection or evaluation of leukemia and lymphoma.

Decoding the mystery of MMTV-like virus and its relationship with breast cancer metastasis

BackgroundMMTV causes mammary tumors in mice, and it is associated with invasive and aggressive forms of breast cancer in humans. However, the underlying mechanisms are yet unknown. We aimed to determine the MMTV-like virus (MMTV-LV) association with histological types of breast cancer, nodal involvement, and metastasis. MethodsFirst, 105 breast cancer biopsies and 15 disease-free biopsies were collected. Details of clinicopathological characteristics were retrieved from patients’ records. The status of MMTV-LV was already known for these biopsy samples. Associations of MMTV-LV prevalence with LNM status and metastatic history were determined. Next, quantitative PCR (qPCR) was used to quantify env gene mRNA in biopsies positive for MMTV-LV. Expression of the env gene was compared against different histopathological types of mammary tumor, LNM status, and metastasis by performing Ordinary One Way ANOVA followed by Tukey’s multiple comparisons test. ResultsMMTV-LV prevalence was found to have no significant association with LNM or metastatic history. As compared to normal control, expression of the env gene was significantly higher (>2.8 folds) in invasive samples (P-value: < 0.01). Expression was also higher (3.28 and 2.89 folds) in patient samples with LNM (P-value: 0.0006) or metastatic history (P-value: < 0.0001), respectively. ConclusionWe conclude that MMTV-LV prevalence is not associated with LNM status or breast cancer metastasis; samples with invasive phenotypes, nodal involvement, and metastasis exhibit significantly higher expression of the MMTV-like env gene.

Human endogenous retrovirus HERV-E λ 4-1 in immunopathogenesis of affective disorder

The aim of this work was to study a dependence between the production level of some pro-inflammatory cytokines by peripheral blood mononuclear cells, and activation of human endogenous retrovirus HERV-E 4-1 in the patients with recurrent depression. Patients and methods: the study included 30 patients with an verified diagnosis of recurrent depression (F 33.0) aged 26-45 years, with a disease duration of at least 3 months prior to inclusion into the study. Peripheral blood mononuclear cells were isolated by centrifugation in Ficoll density gradient (1.078 g/cm3). The human endogenous retrovirus HERV-E 4-1 env gene expression was determined by polymerase chain reaction using paired oligonucleotide primers. To assess the cytokine production, peripheral blood mononuclear cells were cultured for 24-72 hours, depending on the experimental conditions. Quantitative determination of spontaneous cytokine production was carried out by a sandwich variant of ELISA method in conditioned media from the cell cultures, according to the manufacturer instructions. Results: our data reveal higher production of IL-1 and IFN in peripheral blood mononuclear cells from those patients with recurrent depression who showed detectable HERV-E 4-1 env expression compared to the patients in whom the HERV-E 4-1 env gene expression was not detected. When studying correlation between HERV-E 4-1 env expression and production of IL-1 and IFN, a positive correlation between the studied parameters was established. Thus, taking into account our earlier data on HERV-E 4-1 immunomodulatory properties, as well as literature data concerning the HERV transcripts found in brains of mentally ill patients, along with increase of IL-1 and IFN production in the patients with recurrent depression and positive HERV-E 4-1 env gene expression, and a positive correlation between the HERV-E 4-1 env gene expression and increased level of cytokines involved in formation of pathological events in the nervous system in the patients with depression, one may conclude that activation of HERV-E 4-1 could participate in immunopathogenesis of recurrent depression by stimulating the synthesis of pro-inflammatory cytokines.

Effect of human endogenous retrovirus-K env gene knockout on proliferation of ovarian cancer cells.

Among various human endogenous retroviruses (HERVs), the HERV-K (HML-2) group has been reported to be highly related to cancer. In pancreatic cancer cells, shRNA-mediated downregulation of HERV-K env RNA decreases cell proliferation and tumor growth through the RAS-ERK-RSK pathway; in colorectal cancer, CRISPR-Cas9 knockout (KO) of the HERV-K env gene affects tumorigenic characteristics through the nupr-1 gene. The effect of HERV-K env KO has not been studied in ovarian cancer cell lines. In this study, we analyzed the tumorigenic characteristics of ovarian cancer cell lines, including cell proliferation, migration, and invasion, and the expression patterns of related proteins after CRISPR-Cas9 KO of the HERV-K env gene. The HERV-K env gene KO was achieved using the CRISPR-Cas9 system in ovarian cancer cell lines SKOV3 and OVCAR3. Tumorigenic characteristics including cell proliferation, migration, and invasion were analyzed, and related protein expression was investigated by western blot analysis. The expression of the HERV-K env gene in KO cells was significantly reduced at RNA and protein levels, and tumorigenic characteristics including cell proliferation, migration, and invasion were significantly reduced. In HERV-K env KO SKOV3 cells, the expression of the RB protein was significantly up-regulated and the cyclin B1 protein level was significantly reduced. In contrast, in HERV-K env KO OVCAR3 cells, the level of phospho-RB protein was significantly reduced, but other protein levels were not changed. The results of this study showed that HERV-K env gene KO affects cell proliferation, invasion, and migration of ovarian cells through RB and Cyclin B1 proteins, but the specific regulation pattern can differ by cell line.

Effects of prolonged exposure to ELF-EMF on HERVs expression in human melanoma cells.

The Human endogenous retroviruses (HERVs) are ancient remnants of exogenous retroviral infections. Their abnormal activation is associated with several diseases, such as cancer and autoimmunity. Epigenetic and environmental factors are probably playing essential roles in the expression of these elements. This study aimed to examine the 96-hour effects of ELF-EMF on HERV-H, K, and W expression in human melanoma cells. SK-MEL-37 cells (the human skin malignant melanoma) were continuously exposed to ELF-EMF (50 Hz) at 1.5 and 3 mT intensity for 96 hours. Following mRNA extraction, the expression level of HERV-H, K, and W was assessed by qPCR. According to our results, exposure to ELF-EMF intensities for 96 hours could significantly downregulate HERV-H, K, and W env gene expression (P<0.001). Our obtained data suggest that low intensity and long-term exposure to ELF-EMF may pave using this type of radiation as a novel therapeutic approach by neutralizing the HERVs upregulated expression in melanoma cells.

High Expression of HERV-K (HML-2) Might Stimulate Interferon in COVID-19 Patients.

Background. Interferon is a marker of host antiviral immunity, which is disordered in COVID-19 patients. ERV can affect the secretion of interferon through the cGAS-STING pathway. In this study, we explored whether IFN-I and HERV-K (HML-2) were activated in COVID-19 patients and whether there was an interaction between them. Methods. We collected blood samples from COVID-19 patients and healthy controls. We first detected the expression of HERV-K (HML-2) gag, env, and pol genes and IFN-I-related genes between patients and healthy people by qPCR, synchronously detected VERO cells infected with SARS-CoV-2. Then, the chromosome distributions of highly expressed HERV-K (HML-2) gag, env, and pol genes were mapped by the next-generation sequencing results, and GO analysis was performed on the related genes. Results. We found that the HERV-K (HML-2) gag, env, and pol genes were highly expressed in COVID-19 patients and VERO cells infected with SARS-CoV-2. The interferon-related genes IFNB1, ISG15, and IFIT1 were also activated in COVID-19 patients, and GO analysis showed that HERV-K (HML-2) can regulate the secretion of interferon. Conclusions. The high expression of HERV-K (HML-2) might activate the increase of interferon in COVID-19 patients, proving that HERV-K does not only play a negative role in the human body.

Epstein-Barr Virus Load Correlates with Multiple Sclerosis-Associated Retrovirus Envelope Expression.

pHERV-W ENV and syncytin-1, the envelope proteins of the human endogenous retrovirus W family (HERV-W), have been proposed as etiological factors for MS development. In addition, herpesviruses, such as the Epstein-Barr virus (EBV) and the human herpesvirus 6A/B (HHV-6A/B), have been also strongly associated with the disease. This work aims to study the possible link between viral loads and antibody titers against EBV and HHV-6A/B and the pHERV-W ENV/syncytin-1 protein/gene expression. For this purpose, we conducted a 12-month longitudinal study involving 98 RRMS patients. Peripheral blood samples were obtained from each patient. Serum antibody titers against EBV and HHV-6A/B were determined by ELISA, while viral loads were analyzed using qPCR. HLA MS-related alleles were also genotyped. pHERV-W ENV/syncytin-1 protein and gene expression levels in immune cells were assessed by flow cytometry and qPCR, respectively. We found that the 12-month variation of the pHERV-W ENV gene expression levels positively correlated with the variation of the EBV viral load, especially in those patients with high baseline EBV loads. Therefore, these results could support previous studies pointing to the transactivation of pHERV-W ENV by EBV. However, further studies are needed to better understand this possible relationship.

Construction of a recombinant avipoxvirus expressing the env gene of Zika virus as a novel putative preventive vaccine

BackgroundZika virus (ZIKV) has been declared a public health emergency that requires development of an effective vaccine, as it might represent an international threat.MethodsHere, two novel DNA-based (pVAXzenv) and fowlpox-based (FPzenv) recombinant putative vaccine candidates were constructed that contained the cPrME genes of ZIKV. The env gene inserted into the fowlpox vector was verified for correct transgene expression by Western blotting and by immunofluorescence in different cell lines. The production of virus-like particles as a result of env gene expression was also demonstrated by electron microscopy. BALB/c mice were immunosuppressed with dexamethasone and immunized following a prime–boost strategy in a heterologous protocol where pVAXzenv was followed by FPzenv, to evaluate the immunogenicity of the Env protein. The mice underwent a challenge with an epidemic ZIKV after the last boost.ResultsThese data show that the ZIKV Env protein was correctly expressed in both normal human lung fibroblasts (MRC-5 cells) and green monkey kidney (Vero) cells infected with FPzenv, and that the transgene expression lasted for more than 2 weeks. After mucosal administration of FPzenv, the immunized mice showed specific and significantly higher humoral responses compared to the control mice. However, virus neutralizing antibodies were not detected using plaque reduction assays.ConclusionsAlthough BALB/c mice appear to be an adequate model for ZIKV infection, as it mimics the natural mild infection in human beings, inadequate immune suppression seemed to occur by dexamethasone and different immune suppression strategies should be applied before challenge to reveal any protection of the mice.

Human endogenous retroviruses env gene expression and long terminal repeat methylation in colorectal cancer patients

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p < 0.05).The HERV env gene was expressed in the EVs at of 54% (-H), 38% (-K), 31% (-R) patients. Association was not found between HERV env expression and LTR methylation, but significant higher expression of HERV-P and -R env was found in tumor tissues arising from the right colon. Our findings do not demonstrate significant overexpression of the studied HERV in colorectal cancer, but their association with tumor localization and specificity of the changes in DNA methylation of retroelements are shown. HERV sequences were packaged in the EVs and might be transferred from one cell to another.

THE COMPARATIVE STUDY OF HUMAN ENDOGENOUS RETROVIRUS HERV-E λ 4-1 EXPRESSION IN BLOOD IMMUNE CELLS OF MULTIPLE SCLEROSIS PATIENTS

In order to investigate the function of human endogenous retrovirus HERV-E λ 4-1 in multiple sclerosis pathogenesis, the comparative research of frequency of this retrovirus expression and mRNA level on different types of blood immune cells of progredient course multiple sclerosis patients have been conducted with using of the reverse - transcriptase polymerase chain reaction method. Peripheral blood mononuclear cells were isolated on Ficoll density gradient centrifugations. Monocytes were separated by the adhesion to plastic Petri dishes. For the estimation of the mitogen-induced HERV-E λ 4-1 expression, blood mononuclear cells were incubated with adding of phytogemagglutinin or pokeweed mitogen during 72 hours in CO2 incubator at 37 °C and 5% CO2. Results of HERV-E λ 4-1 env gene expression estimation demonstrate that both monocytes and lymphocytes express HERV-E λ 4-1. The level of the HERV-E λ 4-1 env mRNA was higher in lymphocytes than in monocytes. The main source of HERV-E λ 4-1 in progredient course MS patients between blood immune cells are lymphocytes, especially B lymphocytes.

Expression of human endogenous retroviruses in pemphigus vulgaris patients.

Pemphigus is a severe, potentially life-threatening autoimmune blistering mucocutaneous disease which establishes with autoreactive IgG antibodies that target cellular adhesions, precisely extracellular domains of keratinocyte proteins. Several genetic and environmental elements are believed to contribute to the pathogenesis of the disease. The extent to whichthe initiation and progress of this autoimmune blistering disease may be influenced by the expression of human endogenous retroviruses (HERVs) remains to be elucidated. In this study, we evaluated the expression of HERV groups (K, W, and H) in pemphigus vulgaris (PV) patients in comparison to controls. Peripheral blood samples were collected from 24 PV patients and the corresponding age- and sex-matched healthy controls to extract total RNA for evaluation of HERV-K (HML-2), HERV-W, and HERV-H, env gene expression profile by qPCR. The mRNA expression level of HERV-K, HERV-W, and HERV-H were significantly upregulated in PV patients in comparison to healthy controls (P < 0.0001). The difference in expression of studied HERVs groups between men and women was no significant (P > 0.05). Although rituximab taking patients had a decreased expression level of studied HERVs, the results were not significant (P > 0.05). According to our obtained data, HERVs expression could be measured as a possible diagnostic tool for detection of PV and monitoring of the treatment.

A New Insight on Activation of Human Endogenous Retroviruses (HERVs) in Malignant Melanoma upon Exposure to CuSO4.

Human endogenous retroviruses (HERVs) are semi-conserved subtypes of long-terminal repeats containing retrotransposons that constitute approximately 8% of the genome. Under pathological conditions, the expression of HERVs is also affected by epigenetic modifications. The extent to which the activation of human endogenous retroviruses can be influenced upon exposure to copper remains to be evaluated. Thus, the present study was designed to evaluate the effects of CuSO4 administration on the transcriptional activity of three HERV families (H, K, and W) in human malignant melanoma cells. For this purpose, following the determination of less cytotoxic concentrations of copper sulfate, the human skin malignant melanoma SK-MEL-37 cells were treated with 25, 50, and 75μM CuSO4 for 96h. Then, mRNA expression of env gene of HERV-H, HERV-K, and HERV-W was evaluated by qPCR. According to the results, 96-h treatment of SK-MEL-37 cells with 75μM CuSO4 could significantly downregulate HERV-H evn expression (P< 0.05). Moreover, exposure of 25μM copper significantly upregulated the expression of HERV-K env (P< 0.05). Regarding HERV-W env, the expression level increased significantly in all treated concentrations (P< 0.05). It seems that the expression change was decreased in both HERV-W and HERV-K by increasing doses. The study results demonstrated that copper exposure to melanoma cells might promote tumor growth by inducing HERVs and/or control tumor development by decreasing the activation of HERVs in defined levels of copper. According to the findings of this study, copper might exert a binary effect on malignant melanoma.

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na+,K+-ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 107 infectious particles·ml-1). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.

MiR-34b-5p Suppresses Melanoma Differentiation-Associated Gene 5 (MDA5) Signaling Pathway to Promote Avian Leukosis Virus Subgroup J (ALV-J)-Infected Cells Proliferaction and ALV-J Replication.

Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus that has a similar replication cycle to multiple viruses and therefore can be used as a model system for viral entry into host cells. However, there are few reports on the genes or microRNAs (miRNAs) that are responsible for the replication of ALV-J. Our previous miRNA and RNA sequencing data showed that the expression of miR-34b-5p was significantly upregulated in ALV-J-infected chicken spleens compared to non-infected chicken spleens, but melanoma differentiation-associated gene 5 (MDA5) had the opposite expression pattern. In this study, a dual-luciferase reporter assay showed that MDA5 is a direct target of miR-34b-5p. In vitro, overexpression of miR-34b-5p accelerated the proliferation of ALV-J-infected cells by inducing the progression from G2 to S phase and it promoted cell migration. Ectopic expression of MDA5 inhibited ALV-J-infected cell proliferation, the cell cycle and cell migration, and knockdown of MDA5 promoted proliferation, the cell cycle and migration. In addition, during ALV-J infections, MDA5 can detect virus invasion and it triggers the MDA5 signaling pathway. MDA5 overexpression can activate the MDA5 signaling pathway, and thus it can inhibit the mRNA and protein expression of the ALV-J env gene and it can suppress virion secretion. In contrast, in response to the knockdown of MDA5 by small interfering RNA (siRNA) or an miR-34b-5p mimic, genes in the MDA5 signaling pathway were significantly downregulated (P < 0.05), but the mRNA and protein expression of ALV-J env and the sample-to-positive ratio of virion in the supernatants were increased. This indicates that miR-34b-5p is able to trigger the MDA5 signaling pathway and affect ALV-J infections. Together, these results suggest that miR-34b-5p targets MDA5 to accelerate the proliferation and migration of ALV-J-infected cells, and it promotes ALV-J replication, via the MDA5 signaling pathway.

Biochemical and Structural Characterization of Recombinant Human Endogenous Retrovirus‐R

Human endogenous retroviruses (HERVs) comprise approximately 8% of the human genome, and the majority of HERVs exist in truncated form with multiple stop codons, insertions, and deletions. Because HERVs contribute to genome instability via mutation events such as rearrangement and retrotransposition, it has been proposed that they may play a role in human pathology. Although the role of the env gene expression of full‐length endogenous retrovirus (HERV)‐R has been investigated in various human tissues and cancer cells, there have been no studies of soluble HERV‐R protein or structural characterizations of the protein expressed in Escherichia coli to date. Full‐length HERV‐R is composed of a surface (SU) domain and a transmembrane (TM) domain. In this study, we successfully expressed and purified the TM region of HERV‐R with high solubility, and then modeled the three‐dimensional structure of the HERV‐R TM and SU domains by homology modeling based on the structure of the Ebola virus membrane fusion subunit and the feline leukemia virus receptor‐binding domain. Furthermore, we found that the SU domain was bound to RNA. Our results will enable the production of recombinant HERV‐R protein in large quantities and facilitate future functional and structural analyses of HERV‐R.