HCV core can aggravate this effect, leading to early apoptosis of co-expressing cells, preventing the induction of immune response. The effect(s) could be due to interference of the ectopically expressed xenogeneic rat TERT with biogenesis of mRNA, ribosomes and protein translation in murine cells, affecting the expression of immunogens. TERT with deleted N-terminal nucleoli localization signal and mutations abrogating telomerase activity still suppressed the IFN-β driven Luc expression, while the inactivated reverse transcriptase domain of TERT and its analogue, enzymatically active HIV-1 reverse transcriptase, exerted only weak suppressive effects, implying that suppression relied on the presence of the full-length/nearly full-length TERT, but not its enzymatic activity. Panel of mutant TERT variants was created and tested for their expression effects. A loss of bioluminescence signal after co-delivery of TERT and Luc DNA into mice indicated that TERT affects the in vivo expression of luciferase directed by the immediate early cytomegalovirus and interferon-β promoters. However, DNA-immunization with Core152opt and TERT mix resulted in abrogation of immune response against both components. Additionally, DNA-immunization with TERT enhanced cellular immune response against luciferase encoded by a co-delivered plasmid (Luc DNA). Core152opt and TERT DNA were highly immunogenic in BALB/c mice, inducing IFN-γ/IL-2/TNF-α response of CD4+ and CD8+ T cells. Their synthetic genes were expression-optimized, and HCV core was truncated after aa 152 (Core152opt) to delete the domain interfering with immunogenicity. To meet this need we developed a two-component DNA vaccine based on the highly conserved core protein of HCV to target HCV-infected cells, and a renowned tumor-associated antigen telomerase reverse transcriptase (TERT) based on the rat TERT, to target malignant cells. A possible remedy could be a multi-component immunotherapeutic vaccine targeting both HCV-infected and malignant cells, but also those not infected with HCV. Direct acting antivirals eliminate HCV, unless it is drug resistant, and partially reverse liver disease, but they cannot cure HCV-related cancer. Thus, we have revealed a direct link between the propensity of the microbial proteins to induce oxidative stress and their immunogenicity.Ĭhronic HCV infection and associated liver cancer impose a heavy burden on the healthcare system. Poor gene immunogenicity was also associated with a high (per unit of protein) transcription of antioxidant response element (ARE) dependent phase II detoxifying enzyme genes, specifically HO-1. RT variants generating high total ROS levels induced significantly stronger IFN-γ responses than the variants inducing lower total ROS, while high levels of ROS normalized per unit of protein in expressing cell were associated with a weak IFN-γ response. Splenocytes of immunized mice were stimulated with the RT-derived and control antigens and antigen-specific proliferation was assessed by IFN-γ/IL-2 Fluorospot. For this, RT genes were administered into BALB/c mice by intradermal injections followed by electroporation. The capacity of RT genes to induce the oxidative stress and stress response was then correlated with their immunogenic performance. However, the viral RT genes induced higher levels of ROS production and higher levels of HO-1 mRNA than the synthetic genes per unit of protein in the expressing cell. The total ROS production induced by RT genes of the viral origin was found to be lower than that induced by the synthetic/expression-optimized or chimeric RT genes. The capacity to induce oxidative stress and stress response appeared to be an intrinsic property of a vast variety of RTs: enzymatically active and inactivated, bearing mutations of drug resistance, following different routes of processing and presentation, expressed from viral or synthetic expression-optimized genes. Quinone oxidoreductase (Nqo1) and heme oxygenase 1 (HO-1), indicating the induction of oxidative stress response. Quantitative RT-PCR demonstrated that expression of RT in HEK293 cells induced a 10- to 15-fold increased transcription of the phase II detoxifying enzymes human Expression of HIV-1 RT in human embryonic kidney cells generated potent production of the reactive oxygen species (ROS), detected by the fluorescence-based probes. Here, we for the first time describe the induction of oxidative stress by another HIV-1 protein, reverse transcriptase (RT). The resultant neurotoxicity has been associated with Tat protein. HIV-1 infection induces chronic oxidative stress.
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