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 24th 2008

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

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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

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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

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

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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

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• 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!

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)

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• 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

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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.

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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

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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

Viral miRNAs may directly reduce host cell gene expression

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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

Expression of celluar miRNAs may be restructured to benefit the virus replication

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Figure: 2

• Cellular miRNAs directly or indirectly affect virus replication

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

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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!