ATM and atr expression potentiates hbv replication and contributes to reactivation of hbv infection upon DNA damage

Funding Information: Funding: This work was performed within RSF grant no. 16-15-10426. Funding Information: National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health, Moscow 127994, Russia; [email protected] (S.B.); [email protected] (V.C.) Institute of Immunology, Federal Medical Biological Agency, Moscow 115522, Russia NF Gamaleya Research Center of Epidemiology and Microbiology, Moscow 123098, Russia; [email protected] (E.B.); [email protected] (I.G.); [email protected] (M.I.) Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences, Moscow 108819, Russia Sechenov First Moscow State Medical University, Moscow 119146, Russia; [email protected] Department of Pathology, Riga Stradins University, LV-1007 Riga, Latvia Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-171 76 Stockholm, Sweden Izmerov Research Institute of Occupational Health, Gene Engineering and Biotechnology, Moscow 105275, Russia; [email protected] (I.G.); [email protected] (F.U.); [email protected] (A.N.) Central Research Institute of Epidemiology, Moscow 111123, Russia Correspondence: [email protected] (A.K.); [email protected] (D.K.) Publisher Copyright: © 2019 by the authors.Chronic hepatitis B virus infection (CHB) caused by the hepatitis B virus (HBV) is one of the most common viral infections in the world. Reactivation of HBV infection is a life-threatening condition observed in patients with CHB receiving chemotherapy or other medications. Although HBV reactivation is commonly attributed to immune suppression, other factors have long been suspected to play a role, including intracellular signaling activated in response to DNA damage. We investigated the effects of DNA-damaging factors (doxorubicin and hydrogen peroxide) on HBV reactivation/replication and the consequent DNA-damage response. Dose-dependent activation of HBV replication was observed in response to doxorubicin and hydrogen peroxide which was associated with a marked elevation in the mRNA levels of ataxia-telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR) kinases. Downregulation of ATM or ATR expression by shRNAs substantially reduced the levels of HBV RNAs and DNA. In contrast, transcriptional activation of ATM or ATR using CRISPRa significantly increased HBV replication. We conclude that ATM and ATR are essential for HBV replication. Furthermore, DNA damage leading to the activation of ATM and ATR transcription, results in the reactivation of HBV replication.publishersversionPeer reviewe

HIV-1 Reverse Transcriptase Promotes Tumor Growth and Metastasis Formation via ROS-Dependent Upregulation of Twist

Funding Information: https://orcid.org/0000-0002-6160-2203 Bayurova Ekaterina [email protected] 1 2 Jansons Juris [email protected] 3 4 Skrastina Dace [email protected] 3 4 https://orcid.org/0000-0002-4980-9754 Smirnova Olga [email protected] 5 Mezale Dzeina [email protected] 3 Kostyusheva Anastasia [email protected] 6 Kostyushev Dmitry [email protected] 6 Petkov Stefan [email protected] 7 Podschwadt Philip [email protected] 7 https://orcid.org/0000-0003-0365-570X Valuev-Elliston Vladimir [email protected] 5 Sasinovich Sviataslau [email protected] 7 https://orcid.org/0000-0003-2278-4451 Korolev Sergey [email protected] 8 Warholm Per [email protected] 9 https://orcid.org/0000-0002-2260-6551 Latanova Anastasia [email protected] 1 5 https://orcid.org/0000-0003-2183-0858 Starodubova Elizaveta [email protected] 1 5 https://orcid.org/0000-0001-8506-2339 Tukhvatulin Amir [email protected] 1 Latyshev Oleg [email protected] 1 Selimov Renat [email protected] 10 Metalnikov Pavel [email protected] 10 Komarov Alexander [email protected] 10 https://orcid.org/0000-0002-3673-4714 Ivanova Olga [email protected] 5 Gorodnicheva Tatiana [email protected] 11 https://orcid.org/0000-0002-7443-6961 Kochetkov Sergey [email protected] 5 Gottikh Marina [email protected] 8 Strumfa Ilze [email protected] 3 https://orcid.org/0000-0002-5659-9679 Ivanov Alexander [email protected] 5 Gordeychuk Ilya [email protected] 1 2 12 https://orcid.org/0000-0001-9382-2254 Isaguliants Maria [email protected] 1 2 3 7 García-Rivas Gerardo 1 NF Gamaleya Research Center of Epidemiology and Microbiology Moscow Russia 2 Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products of Russian Academy of Sciences Moscow Russia chumakovs.ru 3 Department of Pathology Riga Stradins University Riga Latvia rsu.lv 4 Latvian Biomedical Research and Study Centre Riga Latvia lu.lv 5 Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia ras.ru 6 National Medical Research Center for Tuberculosis and Infectious Diseases Moscow Russia 7 Department of Microbiology Tumor and Cell Biology Karolinska Institutet Stockholm Sweden ki.se 8 Chemistry Department and Belozersky Institute of Physico-Chemical Biology Lomonosov Moscow State University Moscow Russia msu.ru 9 Science for Life Laboratory Stockholm University Stockholm Sweden su.se 10 Russian State Center for Quality and Standardization of Veterinary Drugs and Feed (VGNKI) Moscow Russia 11 Evrogen Moscow Russia 12 Sechenov First Moscow State Medical University Moscow Russia mma.ru 2019 2 12 2019 2019 08 05 2019 01 11 2019 05 11 2019 2 12 2019 2019 Copyright © 2019 Ekaterina Bayurova et al. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of “non-AIDS-defining” malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist . These properties, particularly, the expression of Twist , correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist . No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression. Ministry of Science and Higher Education of the Russian Federation 075-15-2019-1660 Latvian Science Council LZP-2018/2-0308 EU VACTRAIN Russian Foundation for Basic Research 17-00-00085 17_04_00583 17_54_30002 Publisher Copyright: © 2019 Ekaterina Bayurova et al.HIV-induced immune suppression results in the high prevalence of HIV/AIDS-associated malignancies including Kaposi sarcoma, non-Hodgkin lymphoma, and cervical cancer. HIV-infected people are also at an increased risk of "non-AIDS-defining" malignancies not directly linked to immune suppression but associated with viral infections. Their incidence is increasing despite successful antiretroviral therapy. The mechanism behind this phenomenon remains unclear. Here, we obtained daughter clones of murine mammary gland adenocarcinoma 4T1luc2 cells expressing consensus reverse transcriptase of HIV-1 subtype A FSU_A strain (RT_A) with and without primary mutations of drug resistance. In in vitro tests, mutations of resistance to nucleoside inhibitors K65R/M184V reduced the polymerase, and to nonnucleoside inhibitors K103N/G190S, the RNase H activities of RT_A. Expression of these RT_A variants in 4T1luc2 cells led to increased production of the reactive oxygen species (ROS), lipid peroxidation, enhanced cell motility in the wound healing assay, and upregulation of expression of Vimentin and Twist. These properties, particularly, the expression of Twist, correlated with the levels of expression RT_A and/or the production of ROS. When implanted into syngeneic BALB/C mice, 4T1luc2 cells expressing nonmutated RT_A demonstrated enhanced rate of tumor growth and increased metastatic activity, dependent on the level of expression of RT_A and Twist. No enhancement was observed for the clones expressing mutated RT_A variants. Plausible mechanisms are discussed involving differential interactions of mutated and nonmutated RTs with its cellular partners involved in the regulation of ROS. This study establishes links between the expression of HIV-1 RT, production of ROS, induction of EMT, and enhanced propagation of RT-expressing tumor cells. Such scenario can be proposed as one of the mechanisms of HIV-induced/enhanced carcinogenesis not associated with immune suppression.publishersversionPeer reviewe

Dead Cas Systems: Types, Principles, and Applications

The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions

Clinical Implications of Hepatitis B Virus RNA and Covalently Closed Circular DNA in Monitoring Patients with Chronic Hepatitis B Today with a Gaze into the Future: The Field Is Unprepared for a Sterilizing Cure

Chronic hepatitis B virus (HBV) infection has long remained a critical global health issue. Covalently closed circular DNA (cccDNA) is a persistent form of the HBV genome that maintains HBV chronicity. Decades of extensive research resulted in the two therapeutic options currently available: nucleot(s)ide analogs and interferon (IFN) therapy. A plethora of reliable markers to monitor HBV patients has been established, including the recently discovered encapsidated pregenomic RNA in serum, which can be used to determine treatment end-points and to predict the susceptibility of patients to IFN. Additionally, HBV RNA splice variants and cccDNA and its epigenetic modifications are associated with the clinical course and risks of hepatocellular carcinoma (HCC) and liver fibrosis. However, new antivirals, including CRISPR/Cas9, APOBEC-mediated degradation of cccDNA, and T-cell therapies aim at completely eliminating HBV, and it is clear that the diagnostic arsenal for defining the long-awaited sterilizing cure is missing. In this review, we discuss the currently available tools for detecting and measuring HBV RNAs and cccDNA, as well as the state-of-the-art in clinical implications of these markers, and debate needs and goals within the context of the sterilizing cure that is soon to come