Lnc-ITM2C-1 and GPR55 are Proviral Host Factors for Hepatitis C Virus Dissertation for the degree Doctor rerum naturalium (Dr. rer. nat.) submitted by Pan Hu, M. Sc. Institute of Biochemistry Faculty of Medicine Justus Liebig University Gießen Gießen, October 2019 This work was accomplished from May 2015 to October 2019 under the supervision of Prof. Dr. Michael Niepmann in the Institute of Biochemistry, Faculty of Medicine, Justus Liebig University Gießen. This research was funded by a Land Hessen LOEWE grant (Medical RNomics). Supervisors: Prof. Dr. Albrecht Bindereif Institute of Biochemistry Faculty of Biology and Chemistry Justus Liebig University Gießen Prof. Dr. Michael Niepmann Institute of Biochemistry Faculty of Medicine Justus Liebig University Gießen Summary Summary Multiple host factors are known to play important roles in Hepatitis C Virus (HCV) replication, in immune responses induced by HCV infection, or in processes that facilitate virus escape from immune clearance, while yet only few studies examined the contribution of long non-coding RNAs (lncRNAs/lncRs). Using microarrays, we identified lncRNAs with altered expression levels in HCV replicating Huh-7.5 hepatoma cells. Of these, lncR 8/Lnc-ITM2C-1 was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) to be upregulated early after HCV infection. Nucleus/cytoplasm fractionation showed a preferential nuclear localization of lncR 8. Expression of lncR 8 in Huh-7.5 could be largely repressed by GapmeRs (GmRs). After suppressing the expression of lncR 8, HCV RNA and protein were downregulated, confirming a positive correlation between lncR 8 expression and HCV replication. LncR 8 knockdown in Huh-7.5 cells reduced mRNA expression level of the neighboring gene G protein-coupled receptor 55 (GPR55) at early times, and leads to increased levels of several interferon stimulated genes (ISG) including interferon stimulated gene 15 (ISG15), MX dynamin like GTPase 1 (Mx1) and interferon induced transmembrane protein 1 (IFITM1). Importantly, the effect of lncR 8 on ISGs and GPR55 precedes its effect on HCV replication. Furthermore, knockdown of GPR55 mRNA induces ISG expression, providing a possible link between lncR 8 and ISGs. We conclude that HCV induces lncR 8 expression, while lncR 8 indirectly favors HCV replication by stimulating expression of its neighboring gene GPR55, which in turn downregulates expression of ISGs. The latter fact is also consistent with a pro-inflammatory role of GPR55. These events may contribute to the failure of cells to eliminate ongoing HCV infection. Content Content 1. Introduction ............................................................................................. 4 1.1 Hepatitis C Virus Infection ...................................................................................... 4 1.2 Hepatitis C Virus Genotypes ..................................................................................... 5 1.3 Hepatitis C Virus Life Cycle ...................................................................................... 7 1.3.1 Viral Translation and Replication ............................................................... 8 1.3.1.1 Viral Translation ...................................................................................... 8 1.3.1.2 Viral Replication ...................................................................................... 9 1.3.1.3 Host Factors Regulate Translation and Replication ................... 10 1.3.2 Viral Assembly and Secretion .................................................................... 10 1.4 The Antiviral Response Against HCV .................................................................. 11 1.4.1 Innate Immune Response ............................................................................. 11 1.4.1.1 Cellular Sensors ...................................................................................... 13 1.4.1.2 IFN Signaling .......................................................................................... 13 1.4.1.3 ISGs ............................................................................................................ 15 1.4.2 Adaptive Immune Response ....................................................................... 16 1.4.3 HCV Evasion from Immune System ....................................................... 16 1.4.3.1 Viral Proteins Contribute to Immune Escape ............................... 17 1.4.3.2 Cellular Sensors Contribute to Immune Escape .......................... 17 1.4.3.3 ISGs Contribute to Immune Escape ................................................. 17 1.4.3.4 Exosomes Contribute to Immune Escape ...................................... 18 1.5 Long non-coding RNA (lncRNA) .......................................................................... 18 1.5.1 HCV or the Antiviral Response Induced LncRNAs ........................... 20 1.5.2 Function of LncRNAs ................................................................................... 21 1.5.2.1 Antiviral LncRNA ................................................................................. 21 1.5.2.2 Proviral LncRNA ................................................................................... 22 1.6 Animal Model and Cell Culture System for HCV Research ......................... 23 1.7 Aim of Work ................................................................................................................. 24 2. Results .................................................................................................... 25 2.1 Identification of LncRNAs Deregulated by HCV Replication ................ 25 2.2 HCV Replication Increases the Expression of Four LncRNAs .................... 28 2.3 Low Protein Coding Potential and Subcellular Localization ........................ 28 2.4 LncR 8 Favors HCV Viral Replication and Infection ..................................... 30 2.5 LncR 8 may be a non-polyadenylated RNA ....................................................... 34 1 Content 2.6 LncR 8 is a Short-Term Cis-Acting Regulator of Its Neighbor GPR55 .... 35 2.7 LncR 8 is a Negative regulator of the Antiviral Response ............................. 38 2.8 GPR55 Negatively Regulates ISGs ....................................................................... 43 2.9 LncR 8 is Induced by poly(I:C) .............................................................................. 43 2.10 LncR 8 is Downregulated by JAK/STAT Pathway ....................................... 46 3. Discussion .............................................................................................. 48 3.1 Identification of LncRNAs Deregulated by HCV Replication ..................... 48 3.2 HCV Replication Increases the Expression of Four LncRNAs .................... 49 3.3 LncR 8 may Regulate GPR55 by Cis-regulation .............................................. 50 3.4 Immune Responses Induced after HCV Infection ............................................ 50 3.5 LncR 8 is a Negative Regulator of the Antiviral Response ........................... 51 3.6 GPR55 Negatively Regulates ISGs ....................................................................... 52 3.7 LncR 8 can be Induced by Poly(I:C) ..................................................................... 54 3.8 LncR 8 is Downregulated by JAK/STAT Pathway .......................................... 54 4. Materials and Methods ......................................................................... 56 4.1 Materials ......................................................................................................................... 56 4.1.1 Bacterial Strains, Cell Lines and plasmid ............................................... 56 4.1.2 Materials for Bacterial Growth and Cell Culture ................................. 56 4.1.3 Chemicals and Reagents .............................................................................. 56 4.1.4 Enzymes ............................................................................................................ 57 4.1.5 Antibodies ......................................................................................................... 58 4.1.6 Kits ...................................................................................................................... 58 4.1.7 Oligonucleotides and primers ..................................................................... 59 4.1.7.1 miR122 duplex ........................................................................................ 59 4.1.7.2 Anti-miR122 LNA ................................................................................ 59 TM 4.1.7.3 LNA LongRNA GmR Oligos ....................................................... 59 4.1.7.4 Primers ...................................................................................................... 60 4.1.8 Buffers and Solutions .................................................................................... 63 4.1.8.1 Bacterial Growth and Cell Culture Solutions ............................... 63 4.1.8.2 General Use Buffer ................................................................................ 64 4.1.8.3 Western Blot and Immunofluorescence Buffers .......................... 65 4.1.9 Consumables .................................................................................................... 66 4.1.10 Laboratory Equipment ................................................................................ 66 4.2 Methods .........................................................................................................................