1. Major Credit Seminar Adil Anamul Haq PhD Scholar Division of Virology, IVRI Mukteswar Welcome Role of miRNA in host-virus interaction: current insights and future perspectives

2. Introduction Biogenesis Viral miRNAs Cellular miRNAs Applications Summary Future Perspectives Contents 3. What are miRNAs? Non-coding RNAs ~22nts in length Post-transcriptionally regulate gene expression Regulate ~60% protein coding genes (Friedman et al., 2009) Encoded both by hosts and viruses Important effects in Signaling pathway Metabolism Apoptosis Cell proliferation Regeneration Stem cell division Oncogenesis Nervous system control Developmental timing Hematopoietic cell fate Regulation of Immune system (Libri et al., 2008) miRNA mRNA 4. Victor R. Ambros Historical Background 1993: lin-4 in C. elegans (Lee et al., 1993) Thought to be an oddity of nematodes No other miRNAs found for next 7 years !!! Second miRNA let-7 in C. elegans (Reinhart et al.,2000) Let-7 found 100% conserved in genomes of mice and humans (Pasquinelli et al., 2000) Three landmark papers in Science 1. Lagos-Quintana et al., 2001; 2. Lau et al., 2001 3. Lee and Ambros, 2001 C. elegans 5. Historical Background Sbastien Pfeffer First reported by Thomas Tuschl and colleagues (Pfeffer et al., 2004) First v-miRNA seen in EBV Most not conserved between viruses Presently > 250 viral miRNAs are known (Cullen et al., 2013) 6. Canonical miRNA biogenesis pathways (Sonkoly and Pivarcsi, 2009) 7. Non-canonical miRNA biogenesis pathways Two pathways: Drosha- independent MHV68 BLV Dicer- independent miR-451 (Libri et al., 2013) 8. miRNAs mRNA Cleavage Translational Repression Fate of mRNA Boss et al., 2011 9. Seed Sequence (Zhao and Srivastava, 2007) 6-7 nt signature region at 5 end (Bartel, 2009) Hexamer = 2-7 nt Heptamer = 2-8 nt Directs miRISC to target 3 UTR of mRNA Plant miRNAs differ in that they are entirely complementary to their target genes 10. miRNA in host-viral Interaction 11. Which viruses encode miRNA ? Herpesviruses code for ~ 90% of known miRNAs (Cullen, 2013) Herpesviridae Polyomaviridae Ascoviridae Baculoviridae Iridoviridae Adenoviridae Most recently Retroviridae Based on the requirements of nuclear machinery and RNA cleavage for miRNA processing, it is no surprise that cytoplasmic replicating DNA viruses and RNA viruses have not been found to express miRNAs (Boss et al., 2011) 12. Family/Sub-family Species Host Pre-miR Hairpins Mature miRs -Herpesviruses Herpes Simplex Virus-1 Human 16 25 Herpes Simplex Virus-2 Human 18 24 Herpes B virus Human 3 3 Herpesvirus of turkey Turkey 17 28 Bovine herpesvirus 1 Bovine 10 12 Pseudorabies virus Swine 13 13 Mareks disease virus -1 Chicken 14 26 Mareks disease virus -2 Chicken 18 36 -Herpesviruses Human cytomegalovirus Human 11 17 Mouse Cytomegalovirus Murine 18 28 Human herpesvirus 6B Human 4 8 -Herpesviruses Kaposis sarcoma associated herpes virus Human 12 25 Epstein-Barr Virus Human 25 44 Rhesus Lymphocryptovirus Simian 36 50 Rhesus Monkey Rhadinovirus Simian 15 25 Mouse Gamma Herpesvirus 68 Murine 15 28 Herpesvirus Saimiri strain A11 Simian 3 6 List of Viral encoded miRNAs 13. Family/Sub- family Species Host Pre-miR Hairpins Mature miRs Polyomaviruses Simian Virus 40 Simian 1 2 BK Polyomavirus Human 1 2 JC Polyomavirus Human 1 2 Mouse polyomavirus Mouse 1 2 Merkel cell polyomavirus Human 1 2 SA12 Simian 1 2 RETROVIRUS Bovine Leukemia virus Bovine 5 8 Iridoviridae Singapore Grouper Iridovirus Fish 14 15 Ascoviridae Heliothis virescens ascovirus Insect 1 1 Baculoviridae Bombyx mori nucleopolyhedrosis virus Insect 4 4 Adenoviridae Human adenoviruses types 2 and 5 (others likely) Human 2 3 Unclassified Bandicoot papillomatosis carcinomatosis virus type 1 Bandicoots (marsupial) 1 1 Bandicoot papillomatosis carcinomatosis virus type 2 Bandicoots (marsupial) 1 1 (Kinkaid & Sullivan, 2012) 14. Location of miRNA genes within herpesvirus genomes Boss et al., 2009 15. Strategies for determining viral-encoded miRNA function Bottom-up Approach Top-down Approach Grundhoff and Sullivan, 2011 16. Functions of viral encoded miRNAs Grouped into two classes: Analogs of host miRNAs Specific to viruses Regulation of latency/persistency Prevention of apoptosis Alteration of cell cycle Viral immune evasion Transformation Autoregulation of virus gene expression (Grundhoff & Sullivan, 2011) Gottwein & Collen, 2008 17. Regulation of latency/persistency The role of virus-encoded miRNAs in promoting KSHV latency (Grundhoff & Sullivan, 2011) KSHV miR-K12-5p Rbl2 Increase in DNA methylation First reported evidence that viral miRNAs can directly impact the epigenetic status of herpesvirus genomes during latency (Lu et al., 2010) 18. Evading the Immune Response Direct Regulation Autoregulation KSHV miRK12-3 KSHV miRK12-7 LIP Secretion IL-6 & IL-10 (Quin et al., 2010) (Boss & Renne, 2011) 19. Autoregulation miRS1- 5p miR-S1-3p T- Antigen CTL Response (Sullivan et al., 2005) JCV BKV muPyV This study provided the first evidence that viral miRNAs can inhibit CTL recognition by directly targeting viral antigens (Boss et al., 2011) T- Antigen SV40: miRNAs encoded antisense to the T-antigen that mediate its transcript cleavage during infection (Sullivan et al., 2005) 20. Evading the Immune Response HCMV miR- Ul112-1 KSHV miR-K12-7 MICB NK Cell & CTL Response EBV miR-BART2- 5p NKG2D-R (Stern-Ginossar et al., 2007; Thomas et al., 2008) HCMV-, EBV-, and KSHV-encoded miRNAs target the MICB gene by completely different sequences raises a very interesting question about the co-evolution of viral miRNAs and their corresponding cellular targets (Boss et al., 2011) (Boss & Renne, 2011) 21. Preventing Apoptosis EBV miR-BART5 rLCV miR-BART5 PUMA Block Apoptosis (Choy et al., 2008) Repress RNAhybrid miRANDA Predicted target of EBV miR- BART5 It represents the conservation of miRNAs from different viruses (Choy et al., 2008) 22. Alteration of cell cycle HCMV miR-US25-1: ~ 15 targets identified Most targets in cell cycle Cyclin E2 is one of the targets (Grey et al., 2010) This is the first report of a virus miRNA targeting the 5UTR of an mRNA (Grey et al., 2010) (Grey et al., 2010) 23. Transformation 12 KSHV miRNAs Thrombospondin Angiogenesis anti-angiogenic factor frequently mutated in various cancers KSHV (Samols et al., 2007) 24. Viral miRNA mimicking a host miRNA Known as analogs Three Viruses known to encode: KSHV MDV1 BLV (Kincaid & Sullivan, 2012) Mimicking host miRNAs provides obvious benefits to a virus by allowing it to access a pre-existing target network of numerous host transcripts that may have been selected for a particular functional outcome (e.g., prevention of apoptosis or evasion of immune signaling) (Kincaid & Sullivan, 2012) 25. miR-155 mimicsviral analogues of a human oncomir? Kincaid & Sullivan, 2012 miR-155 originally identified as product of bic gene (Clurman and Hayward, 1989) Expressed in many lymphomas acute myeloid leukemia (AML) solid tumors of the breast, colon and lung Represents an oncomiR miR-155 KSHV miR-K11 MDV1 miR-M4 Strikingly, although the lymphotropic EBV does not encode a miR-155 mimic of its own, the viral LMP-1 protein strongly induces expression of the cellular miR-155 in latently infected B-cells (Gatto et al., 2008) 26. Potential effects of cellular miRNAs on Viral replication (Gottwein & Collen., 2008) 27. Antiviral miRNAs HIV-1 PFV-1 Influenza VSV - 5-human encoded miRNAs - target nef, vpr, vif, vpu (Hariharan et al. , 2005) - miR-24 & miR-93 - Restrict replication (Otsuka et al. , 2007) - miR-32 - Restrict replication (Lecellier et al. , 2005) - hsa-miR-507&136 - target PB2 & HA. - (Scaria et al., 2007) Cellular miRNAs as Antivirals 28. miR-122: Cellular miRNA enhancing Viral Replication (Gupta et al., 2012) 29. Attenuated vaccines Oncolytic Virotherapy Determination of Cell Tropism Antiviral Drug Development Applications 30. MIRA (MicroRNA Attenuation) Technology miRNA-controlled viruses showed no evidence of pathogenesis, but their limited replication made them ideal vaccine candidates (Barnes et al., 2008) Poliovirus 31. Live attenuated IAV vaccine Incorporated MRE miR-93 (a ubiquitous miRNA) ORF of influenza A nucleoprotein coding regions H1N1 (Perez et al., 2009) (Perez et al., 2009) 32. Engineering microRNA responsiveness for Oncolytic Virotherapy Colorectal Carcinoma Measles Virus (Leber et al., 2011) VSV (Edge et al., 2008) 33. Engineering microRNA to determine cell specificity for viruses Determine role of haematopoietic cell in viral replication? DENV strain engineered to encode four HPC- specific miR-142- targeting sites in the 3 UTR of the virus miR-142 completely blocked spread of the infection in vivo, suggesting that replication in haematopoietic cells is required for viral dissemination 34. Janssen et al., 2013 Miravirsen is a LNA modified phosphorothiolate antisense oligonucleotide targeting and blocking miR-122 35. Miravirsen- the 1st miRNA targeted drug First drug to exploit miRNA for therapeutic use As a host targeting agent miravirsen poses a high barrier to resistance Can work in all HCV genotypes because miR-122 binding sites are conserved Has successfully completed Phase II clinical trial (Janssen et al., 2013) 36. Summary MicroRNAs are short 22 nt ncRNAs MicroRNAs are involved in post transcriptional gene regulation Encoded both by viruses and hosts Degree of complementarity between a miRNA & its target determine the regulatory mechanism With notable exceptions, there is a striking lack of evolutionary conservation of most viral miRNAs Have been used for therapeutic purpose As new discoveries in viral miRNA function are made new questions emerge, making the complexities governing viral-host interactions, at the least, a little more transparent (Boss et al., 2011) 37. Future Perspectives Understand why some members of the same virus subfamilies do and do not encode miRNAs (e.g., HTLV in the Delta Retroviridae or VZV in the alpha herpesviruses How do viral miRNAs work synergistically with viral proteins to regulate the viral replication cycle? Strategies to develop new therapeutic interventions Viral miRNA target identification Relevant in vivo model systems for viral miRNAs that recapitulate all modes of infection