1. Viral and Cellular MicroRNAs as Determinants of Viral Pathogenesis and Immunity Role of miRNAs in interaction between viruses and their hosts Ines Hahn, Department of Pulmonary Medicine, Laboratory for Experimental Lung Research Hannover Medical School Eva Gottwein and Bryan R. Cullen Cell Host & Microbe, June 2008 November 24 th 2008

2. What are microRNAs ? microRNAs (miRNAs) are small, single-stranded, non-coding RNA molecules about 19-23 nucleotides in length encoded in the genomes of plants and animals regulate the expression of genes by binding to the 3´ untranslated regions (3'-UTR) of specific mRNAs mammalian genomes are predicted to encode 200-500 miRNAs Hannover Medical School

3. 1.miRNAs are initially transcribed as part of a long RNA molecule: Primary-miRNA (500-3000nt) 2.In the nucleus, the dsRNA-specific ribonuclease Drosha processes primary-miRNAs into 70-100nt hairpin RNAs: pre-miRNAs 3.Drosha acts in conjunction with RNA-binding protein DGCR8 miRNA formation and processing www.ambion.com Pir-miRNA: Primary microRNA

4. Hannover Medical School 4.pre-miRNAs are transported to the cytoplasm via an exportin 5-dependent mechanism 5.In the cytoplasm, the pre-miRNAs are digested by a second double strand- specific ribonuclease: Dicer 6.Dicer processing results in a sequence- specific, single-stranded, mature miRNA molecule 7.The single-stranded, 19-23nt mature miRNA is bound by a complex that is similar to the RNA-induced silencing complex (RISC) that participates in RNA interference

5. 8.RISCs consist minimally of the miRNA and one of four Argonaute (Ago) proteins 9.In animals, the RISC-bound miRNA binds mRNAs through a sequence that at least partially matches the 3`-UTR of target mRNA 10.Seed region: 2-7 nt from the miRNA 5`end 11.The bound mRNA remains untranslated, resulting in reduced expression of the corresponding gene Hannover Medical School

6. Function of miRNA as regulators of viral and/or host cell gene expression miRNA are attractive candidates as virally encoded regulators of viral and/or host cell gene expression due to: –their small size –lack of immunogenicity –remarkable functional flexibility Hannover Medical School miRNAs may regulate viral replication and pathogenesis: –viruses may use cellular miRNA for their replication –viral miRNAs may directly regulate viral and/or host cell gene expression to benefit the virus –expression of celluar miRNAs may be induced/inhibited to benefit the virus On the other hand: cellular miRNA can be disadvantageous to the virus!

7. Viral miRNA expression in human pathogenic herpesviruses Viral miRNAs exclusively derive from dsDNA viruses mainly of the herpesvirus family e.g.: –Herpes simplexvirus type 1 (HSV) –Human and murine cytomegalovirus (hCMV, mCMV) –Karposi Sarcoma Herpesvirus (KSHV) –Human Epstein-Barr Virus (EBV) Hannover Medical School Some herpesviruses express multiple viral miRNAs e.g. hCMV, mCMV, EBV, KSHV Herpesvirus miRNAs, like cellular miRNAs, are processed from POL II transcripts Herpesviral miRNAs derive from non-coding regions, open reading frames of protein-coding mRNAs, intronic regions

8. Viral miRNA expression during latency and productive infection Expression of miRNA in γ -herpesvirus primarily associated with viral latency (e.g. KSHV, EBV) –EBV: expresses 23 miRNAs; two miRNA clusters are differentially expressed in different forms of EBV latency –KSHV: expresses 12 distinct miRNAs during latency; expression of two miRNAs are enhanced upon induction of lytic replication miRNAs from most α- and β-herpesviruses are expressed during productive virus replication (e.g. HSV-1, hCMV, mCMV) → miRNAs of α- and β-herpesviruses (HSV) were also associated with latency in a publication by Bryan R. Cullen in a Nature paper, published August 2008 The sequences of cellular miRNAs and of their target sites are usually highly conserved whereas herpesviral miRNA sequences are generally poorly conserved Hannover Medical School

9. Viral miRNA expression during latency and productive infection γ-herpesvirus e.g. KSHV, EBV α- and β-herpesviruses e.g. HSV-1, hCMV, mCMV expression of miRNA primarily associated with viral latency ≠ miRNAs expressed during productive virus replication clustered miRNA ≠ miRNAs are dispersed throughout the viral genome in closely related viruses miRNAs are arranged in the same genomic location ≠ encoded in mostly different locations miRNA sequences are poorly conserved, even closely related viruses exhibit no evident sequence homology = miRNAs sequences are poorly conserved, even closely related viruses exhibit no evident sequence homology ☺The exception prooves the rule.

10. Hannover Medical School RISC Viral miRNAs may directly regulate viral gene expression - perfect complementarity to the 3´UTR of the mRNA - Virus-derived miRNAs may target viral transcripts for degradation with RISC Figure: 1A BART: BamA rightward transcripts

11. Hannover Medical School Viral miRNAs may directly regulate viral gene expression - perfect complementarity - Example 1: Polyoma virus SV40 expresses two miRNAs: miR-S1-5p and miR-S1-3p Both miRNAs are perfectly complementary to mRNAs coding for T cell antigens mRNAs transcribed antisense to pre- miRNA-precursor → Cleavage of these transcripts by RISC BART: BamA rightward transcripts Example 2: EBV miR-BART2: perfect complementarity to the 3´UTR of the mRNA for the EBV DNA polymerase BALF5, which is transcripted antisense to miR-BART2 → Cleavage of BALF5 transcript by miR- BART2 RISC Figure 1A:

12. Hannover Medical School RISC Viral miRNAs may directly regulate viral gene expression - imperfect complementarity to the 3´UTR of the mRNA - Viral miRNAs may inhibit translation of viral transcripts carrying imperfect matches to the miRNA IE: Immediate Early Figure: 1B

13. Hannover Medical School Viral miRNAs may directly regulate viral gene expression - imperfect complementarity - Example 2: EBV miR-BART1-5p, miR-BART16, miR-BART17-5p mRNA with imperfect match: Latent membrane protein 1 (LMP1) → Downmodulation LMP1 expression, balancing LMP1 growth stimulation and the potential toxic effect of the protein IE: Immediate Early Example 1: hCMV miR-UL112 mRNA with imperfect match: IE72 → cleavage of mRNA transcript by miR- UL112 RISC, Inhibition of IE72 expression Figure 1B:

14. Viral miRNAs may directly reduce host cell gene expression Hannover Medical School Figure: 1C virus-derived miRNAs may inhibit host mRNAs or function as orthologs of cellular miRNA, thereby inhibiting host cell mRNA targets for host cell miRNA

15. hCMV miR-UL112 downregulates the gene expression of host cell MHC class I-related chain B (MICB) → reduced susceptibility of virus-infected cells to killing by NK cells Interaction of miR-UL112 and MICB mRNA appears to be independent of an intact seed region therefore target novel sites on cellular mRNAs –hCMV miR-UL112 appears to target both cellular and viral targets at the translational level! Department of Pulmonary Medicine, Laboratory for Experimental Lung Research Hannover Medical School Viral miRNAs may directly reduce host cell gene expression - novel mRNA targets - Viral miRNA with perfect complementarity to a cellular transcript triggering degradation have not been found so far! Example 1: Fig 1C

16. Most viral miRNAs do not share seed homology with cellular miRNAs therefore target novel sites on cellular mRNAs Department of Pulmonary Medicine, Laboratory for Experimental Lung Research Hannover Medical School Viral miRNAs may directly reduce host cell gene expression - viral orthologs of cellular miRNA - Example 2: Fig 1C KSHV miR-K12-11 is an ortholog of cellular miR-155: identical along their 5`- terminal 8 nt i.e. including the entire seed region: miR-155: product of the Bic gene Loss of miR-155 expression results in defects in adaptive immune responses Constitutive expression of miR-155 resultes in B cell lymphomas (oncomir)

17. Expression of celluar miRNAs may be restructured to benefit the virus replication Hannover Medical School Figure: 2 Cellular miRNAs directly or indirectly affect virus replication

18. Expression of celluar miRNAs may be restructured to benefit the virus replication Hannover Medical School cellular miRNA facilitating Hepatitis C Virus (HCV) replication: miR-155 HCV: positive strand RNA virus miR-155 expressed in the liver miR-155 binds to 5`-UTR of the HCV genomic RNA via an unknown mechanism Of note: individual knockdown of Drosha, DGCR8, Dicer or Ago protein results in reduced HCV replication Example: Fig 2

19. Celluar miRNAs may function as antiviral miRNA to inhibit virus replication Several viruses are accessible to inhibition by experimentally introduced siRNAs, making it likely that they are also accessible to inhibition by miRNA-RISC There is no evidence for antiviral miRNAs so far Reported interactions between viruses and cellular miRNAs resulting in reduced viral replication might be random Viruses should be able to escape such inhibitory interactions by mutation and viral escape from experimentally introduced siRNA has been reported for a range of viruses Celluar miRNAs may inhibit virus replication by repressing cellular factors Hannover Medical School

20. Conclusion miRNA are produced by both viruses and their hosts can either benefit the virus or the host Viral miRNAs can –regulate viral gene repression and replication –affect cellular gene expression Cellular miRNAs can alter viral life cycle (e.g. miR-155 / HCV) Department of Pulmonary Medicine, Laboratory for Experimental Lung Research Hannover Medical School Thanks for the attention!