title: Oxidative stress induced by HIV-1 reverse transcriptase modulates the enzyme's performance in gene immunization.
abstract: HIV-1 infection induces chronic oxidative stress. The resultant neurotoxicity has been associated with Tat protein. Here, we for the first time describe the induction of oxidative stress by another HIV-1 protein, reverse transcriptase (RT). 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. 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
  quinone oxidoreductase (Nqo1) and heme oxygenase 1 (HO-1), indicating the induction of oxidative stress response. 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. 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. 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 capacity of RT genes to induce the oxidative stress and stress response was then correlated with their immunogenic performance. For this, RT genes were administered into BALB/c mice by intradermal injections followed by electroporation. 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. 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. 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. Thus, we have revealed a direct link between the propensity of the microbial proteins to induce oxidative stress and their immunogenicity.
authors.affiliation: Microbiology, Tumor, and Cell Biology Center; Karolinska Institutet; Stockholm, Sweden; DI Ivanovsky Institute of Virology; Moscow, Russia.
authors.forename: Maria
authors.initials: M
authors.lastname: Isaguliants
authors.affiliation: Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Smirnova
authors.affiliation: Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Ivanov
authors.affiliation: Microbiology, Tumor, and Cell Biology Center; Karolinska Institutet; Stockholm, Sweden.
authors.forename: Athina
authors.initials: A
authors.lastname: Kilpelainen
authors.affiliation: Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Kuzmenko
authors.affiliation: Microbiology, Tumor, and Cell Biology Center; Karolinska Institutet; Stockholm, Sweden.
authors.forename: Stefan
authors.initials: S
authors.lastname: Petkov
authors.affiliation: Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Anastasia
authors.initials: A
authors.lastname: Latanova
authors.affiliation: DI Ivanovsky Institute of Virology; Moscow, Russia; Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Olga
authors.initials: O
authors.lastname: Krotova
authors.affiliation: Microbiology, Tumor, and Cell Biology Center; Karolinska Institutet; Stockholm, Sweden.
authors.forename: Gunnel
authors.initials: G
authors.lastname: Engström
authors.affiliation: Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Vadim
authors.initials: V
authors.lastname: Karpov
authors.affiliation: Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Kochetkov
authors.affiliation: Microbiology, Tumor, and Cell Biology Center; Karolinska Institutet; Stockholm, Sweden.
authors.forename: Britta
authors.initials: B
authors.lastname: Wahren
authors.affiliation: Microbiology, Tumor, and Cell Biology Center; Karolinska Institutet; Stockholm, Sweden; Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow, Russia.
authors.forename: Elizaveta
authors.initials: E
authors.lastname: Starodubova
mesh_terms: D016915:AIDS Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002460:Cell Line; D020869:Gene Expression Profiling; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007114:Immunization / Q000379:methods*; D007371:Interferon-gamma / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D019444:Vaccines, DNA / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*
chemical_list: D016915:AIDS Vaccines; D017382:Reactive Oxygen Species; D019444:Vaccines, DNA; D007371:Interferon-gamma; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase
keywords: DNA immunization; HIV-1 reverse transcriptase; oxidative stress; reactive oxygen species; transcription factor Nrf2