RNA Virus Genome Replication and mrna Production

Transcription

1 RNA Virus Genome Replication and mrna Production Thomas Pietschmann - TWINCORE - Center for Experimental and Clinical Infection Research Hannover

2 Preview Introduction Mechanisms of Viral RNA Synthesis The Switch from mrna Production to Genome RNA synthesis Origins of Diversity in RNA Viruses Therapeutic intervention with RNA replication

3 Who is who?

4 Strategies for replication and mrna synthesis of RNA viruses How is this accomplished and regulated? Flint et al. Principles of Virology

5 A. Mechanisms of Viral RNA Synthesis 1. RNA-dependent RNA polymerases 2. The nature of the RNA template 3. Accessory Proteins in RNA-dependent RNA synthesis 4. Initiation and elongation 5. Cellular Sites of viral RNA synthesis

6 Identification of RNA dependent RNA Polymerases (RdRp) PNAS 1963 David Baltimore Nobel laureate 1975 Discovery of reverse transcriptase Specific to virus infected cells Not sensitive to actinomycin D Virus specific RdRp

7 RdRp RNA-dependent RNA polymerase synthesis of new genomes and mrnas unique enzyme process has no parallels in the cell is encoded by all RNA viruses except retroviruses protein with catalytic subunit for chain elongation vs. multi protein assembly including accessory proteins Q: Which requirements have (-)RNA viruses compared to (+)RNA viruses with respect to the RdRp? (-)RNA virus particles must contain RdRp!

8 Sequence Relationship Among RNA Polymerases Four common motifs (A to D) Motif C contains active site (Gly-Asp-Asp for +RNA viruses) Motif A and C are also found in DNA polymerases All four classes of nucleic acid polymerases might share a common topology! Flint et al. Principles of Virology

9 Structure of Polioviral 3D pol Flint et al. Principles of Virology

10 Different Polymerases Have a Similar Structure DNA -> DNA DNA polymerase I Klenow Fragment DNA -> RNA T7 RNA polymerase RNA -> DNA HIV-I Reverse Transciptase RNA -> RNA Poliovirus polymerase Flint et al. Principles of Virology

11 A. Mechanisms of Viral RNA Synthesis 1. RNA-dependent RNA polymerases 2. The nature of the RNA template 3. Accessory Proteins in RNA-dependent RNA synthesis 4. Initiation and elongation 5. Cellular Sites of viral RNA synthesis

12 Naked or Nucleocapis RNA (+)RNA viruses nakedrna helicase for unwinding template/product basepairing (ATP dependent mechanism) (-)RNA Viruses nucleocapsid RNA nucleoproteins: cooperative, single-stranded RNA-binding proteins, prevent basepairing Common goal: ssrna for additional round of RNA synthesis

13 Secondary Structures in Viral RNA Important Identification: for: RNA 2nd structure synthesisprediction translation Sequencecomparison assembly (conserved structure) Chemical/enzymatic probing Flint et al. Principles of Virology

14 Hepatitis C Virus IRES Cryo-electron microscopy reconstruction Tight binding to 40S subunit and induction of conformational changes Fraser and Doudna, Nat. Reviews Microbiology 2007

15 A. Mechanisms of Viral RNA Synthesis 1. RNA-dependent RNA polymerases 2. The nature of the RNA template 3. Accessory Proteins in RNA-dependent RNA synthesis 4. Initiation and elongation 5. Cellular Sites of viral RNA synthesis

16 Funtion of Accessory Proteins of RdRps 1. Direction of polymerase to the correct intracellular site cellular membranes in cytoplasm (+RNA viruses) nucleus (influenza) 2C anchors RNA to membranes 3AB recruits 3D pol to membranes Vesicle-associated Poliovirus replication complexes purified from infected cells Bienz et al J.Virol. 1990

17 Funtion of Accessory Proteins of RdRps 1. Direction of polymerase to the correct intracellular site cellular membranes in cytoplasm (+RNA viruses) nucleus (influenza) 2. Targeting of RNA polymerase to the correct initiation site on RNA templates 5' and 3' ends cis-acting replication elements (cre) protein-protein interactions 3. RNA helicases unwind dsrna and secondary structure in template RNA 4. Stimulating the activity of the polymerase

18 Poliovirus CREs and associated Proteins Chemical and enzymatic probing of poliovirus RNA 5 end Factors interacting with the poliovirus RNA 5 end Andino, R. et al Cell 1990 cloverleaf RNP of 3CD and cellular protein

19 A. Mechanisms of Viral RNA Synthesis 1. RNA-dependent RNA polymerases 2. The nature of the RNA template 3. Accessory Proteins in RNA-dependent RNA synthesis 4. Initiation and elongation 5. Cellular Sites of viral RNA synthesis

20 Initiation Requirements for Polymerases DNA-dependent DNA polymerases need primer DNA-dependent RNA polymerases de novo RNA-dependent RNA polymerases usually de novo Protein priming (Polio VPg pupu) Priming by capped RNA fragments (Influenza, Bunya)

21 Poliovirus Genome Flint et al. Principles of Virology

22 Polyovirus (-) Strand RNA Synthesis Cellular factors poly(rc)-binding protein 2 poly(a)-binding protein 1 VPg protein priming Flint et al. Principles of Virology

23 Polyovirus (+) Strand RNA Synthesis Flint et al. Principles of Virology

24 Initiation of Influenza Virus mrna Synthesis Cap snatching mrna mrna + virus mrna + virus +GTP Part. Alkali digest Plotch et al Cell 1981

25 A. Mechanisms of Viral RNA Synthesis 1. RNA-dependent RNA polymerases 2. The nature of the RNA template 3. Accessory Proteins in RNA-dependent RNA synthesis 4. Initiation and elongation 5. Cellular Sites of viral RNA synthesis

26 Ribosome-RNA Polymerase Collisions How is this regulated? Flint et al. Principles of Virology

27 Imbalanced (-) and (+) Strand RNA synthesis usually x more (+) RNA differences in initiation different polymerases required How is this regulated?

28 B. The Switch from mrna Production to Genome RNA Synthesis 1. Different RNA polymerases for mrna synthesis and genome replication 2. Suppression of intergenic stop-start reactions by nucleocapis protein 3. Suppression of termination induced by a stem-loop structure

29 Sindbis Virus Genome Structure And Expression Flint et al. Principles of Virology

30 The Three Polymerases of Sindbis Virus Flint et al. Principles of Virology

31 B. The Switch from mrna Production to Genome RNA Synthesis 1. Different RNA polymerases for mrna synthesis and genome replication 2. Suppression of intergenic stop-start reactions by nucleocapis protein 3. Suppression of termination induced by a stem-loop structure

32 Vesicular Stomatitis Virus leader Flint et al. Principles of Virology

33 Vesicular Stomatitis Virus mrna and UV map Virus particle ± UV UV-dosis No VSV UV-dosis No VSV Transcription + Translation G UV target size of each mrna Andrews PNAS 1976 short long exposure N P M

34 Vesicular Stomatitis Virus mrna and UV map Gradient of mrna quantity Termination, polyadenylation and reinitiation at intergenic regions Flint et al. Principles of Virology

35 Switch from mrna synthesis to genome replication Two different RNA polymerases N protein suppresses chain termination Flint et al. Principles of Virology

36 B. The Switch from mrna Production to Genome RNA Synthesis 1. Different RNA polymerases for mrna synthesis and genome replication 2. Suppression of intergenic stop-start reactions by nucleocapis protein 3. Suppression of termination induced by a stem-loop structure

37 Arenavirus RNA Synthesis Arenavirus is (-)RNA virus, but genome is in fact ambisense! 2 genomic RNA segments Suppresion of termination at stem-loop structures N protein permitts synthesis of full length RNA Flint et al. Principles of Virology

38 B. The Switch from mrna Production to Genome RNA Synthesis 1. Different RNA polymerases for mrna synthesis and genome replication (Sindbis virus, Alphaviridae) 2. Suppression of intergenic stop-start reactions by nucleocapis protein (VSV, Rhabdoviridae) 3. Suppression of termination induced by a stem-loop structure (Lassavirus, Arenaviridae)

39 C. Origins of Diversity in RNA Viruses 1. Misincorporation of nucleotides no proofreading capabilites of RdRps error rate (DdRps 1000x lower) "quasispecies (Influenza Virus: antigenic drift) 2. Segment reassortment only for segmented genomes (Influenza virus: antigenic shift) high frequency 3. RNA recombination from donor to acceptor strand 4. RNA editing bp change or insertion after RNA synthesis (e.g. Ebola GP protein)

40 D. Therapeutic intervention with RNA replication Hepatitis C Virus Family: Genus: Species: Size: Genome: Prevalence: Therapy: Flaviviridae Hepacivirus Hepatitis C virus (6 genotpyes) nm (+) RNA, ~9.6 kb 170 million patients PEG-IFN-α + Ribavirin

41 Viral Proteins 5 NTR (+) strand RNA genome 3 NTR IRES translation, processing structural non-structural proteins C E1 E2 p A 4B 5A 5B C E1 E2 p B 5A 5B 4A Virus particle Ion channel replicase complex

42 Viral Enzymes Primary Targets for Drug Development 5 NTR IRES (+) strand RNA genome 3 NTR C E1 E2 p A 4B 5A 5B Protease Protease Helicase Polymerase

43 Targets for Development of HCV-specific anti-viral treatments 5 NTR Polyprotein 3 NTR ss (+) RNA (9.6 kb) IRES 1) Protease inhibitors (peptidomimetic) 2) Polymerase inhibitors (nucleoside / non-nucleoside) 3) Alternative Replication Inhibitors Cyclophilin B inhibitors Target sites CsA NS5A NS5A inhibitors For inhibition of HCV NS5B Pawlotsky, Gastroenterology 2007

44 Interaction of NS5B with host cell proteins cytosol CsA lumen NS5B RNA binding Replicase complex Cyclophilin B Molecular mechanism Clinical development CsA inhibiert HCV Replikation (Replikon) (Watashi et al. Hepatology Nov;38(5): ) Cyclophilin B regulates HCV RNA replication via binding to NS5B CsA blocks this interaction thus inhibiting HCV RNA replication (Watashi et al. Mol Cell Jul 1;19(1): ) DEBIO-025 (DebioPharm) Phase 1b Trial, 1200 mg 2x dayly, 15 days All patients > 2 log decline of HCV RNA Maximal -3.6 log Genotype independent But: Hyperbilirubinemia, Thrombozytopenia NIM 811 (Novartis) Phase 1 study

45 NS5A a novel target for development of anti-viral compounds cytosol NS5A Domain I lumen Vap-A Fbl2 Replicase complex Molecular mechanism Pre-clinical development Phosphoprotein, RNA binding Phosphorylation of NS5A modulates HCV RNA Replication and VAP-A Interaction (Evans et al PNAS 2004 Aug 31;101(35) A-831 (Arrow Therapeutics) IC50 with GT 1b and GT1a replicons ca.0,27µm Resistance mutations within NS5A Phase I Study NS5A-Fbl2 interaction is essential for HCV replication (Wang et al. Mol Cell May 13; (18): )

46 Frequency of NS3 protease variants in patients treated with telaprevir (14 days therapy) Sarrazin, Kieffer et al., Gastroenterology 2007

47 Future treatment of HCV How to avoid resistance? Improve efficacy of antivirals Maintain effective plasma levels Optimal dosing ( adherence to therapy) Use combination therapies Avoid antagonistic effects Avoid overlapping resistance profiles

48 Summary Mechanisms of Viral RNA Synthesis The Switch from mrna Production to Genome RNA synthesis Origins of Diversity in RNA Viruses Therapeutic intervention with RNA replication