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Presenter: Nguyen Trong Binh

Genetic analyses of the function of PB1 subunit of the influenza virus RNA-dependent RNA polymerase. Presenter: Nguyen Trong Binh. NA. HA. NP. PA. PB2. PB1. M2. vRNP complex. M1. Structure of influenza A virus. Envelope. PB1. The schematic diagram of influenza virus RNA polymerase.

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Presenter: Nguyen Trong Binh

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  1. Genetic analyses of the function of PB1 subunit of the influenza virus RNA-dependent RNA polymerase Presenter: Nguyen Trong Binh

  2. NA HA NP PA PB2 PB1 M2 vRNP complex M1 Structure of influenza A virus Envelope

  3. PB1 The schematic diagram of influenza virus RNA polymerase Endonuclease 1 716 a.a. PA PA PB2 NLS NH2 COOH PB1 RNA polymerase consensus motif 1 757 a.a. PB1 NLS NH2 COOH Cap binding 318 483 1 496 759 a.a. 448 PB2 NLS NH2 COOH PB1 binding

  4. PB1 The schematic diagram of influenza virus RNA polymerase PA PB2 PB1 RNA polymerase consensus motif 1 757 a.a. PB1 NLS NH2 COOH Putative cRNA promoter binding Putative vRNA promoter binding Putative nucleotide binding

  5. Aim Functional analysis of PB1 subunit using mutant viruses • By determining the functional site of PB1 • involved in polymerization, nucleotide recognition,and so on. • Reverse-genetics: Using mutant viruses to determine the replicational and transcriptional activities • (2) Forward-genetics: Using ribavirin which inhibits RNA synthesis to determinethe catalytic active site of PB1 by isolation of ribavirin-resistant PB1 mutants

  6. Viral polymerase activity is regulated by the viral promoter PA RNA synthesis activity PB2 PB1 Capping activity Flu A promoter The enzymatic activities are regulated by the interaction with promoter sequence Regulatroy mechanism? To find important residues by comparing Flu A with Flu B in promoter binding site Flu A recognized only Flu A promoter FluB promoter

  7. Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses Homology 85% 50~60% 1 757 a.a. PB1 NLS NH2 COOH Putative cRNA promoter binding Putative vRNA promoter binding 1-15 PA binding site 16 27 44 The a.a. positions 1-50 was highly conserved between influenza A and B viruses, but the amino acid positions 16, 27, and 44 differ between two viruses.

  8. The viral RNA synthesis activity of mutant viruses N16A, D27V, and 44I 3 2.5 3 2.5 2 2 Relative amount of mRNA 1.5 ** ** ** Relative amount of cRNA 1.5 ** ** * 1 1 0.5 0.5 0 0 mock wt N16A D27V N44I mock wt N16A D27V N44I 4 3.5 3 2.5 Relative amount of vRNA 2 1.5 1 0.5 0 mock wt N16A D27V N44I Student’s t test (*, P < 0.05; **, P < 0.01) C. vRNA A. mRNA B. cRNA Binh et al, 2013. Frontiers in Virology These mutations affect the RNA synthesis activity of viral mRNA, cRNA, and vRNA equally, there could be two possibilities:

  9. Hypothesis • Amino acids at the positions 27 and 44 affect cRNA/mRNA synthesis from vRNA through recognition of the promoter on vRNA, but do not affect vRNA synthesis from cRNA through recognition of the promoter on cRNA • (2) These mutations affect independently the synthesis of mRNA, cRNA, and vRNA, but the sum of effects leads similar outputs in the synthesis of mRNA, cRNA, and vRNA.

  10. Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses 1-15 PA binding site 16 27 44 D Q Q: structure of Q is to close to that of N D: position 27

  11. 3 2.5 2.5 2 2 1.5 Relative amount of cRNA Relative amount of mRNA 1.5 1 1 0.5 0.5 0 0 mock N D Q A ** ** * mock N D Q A (wt) (wt) 3 2.5 2 Relative amount of vRNA 1.5 1 0.5 0 mock N D Q A (wt) The viral RNA synthesis of mutant viruses at the position 16 Student’s t test (*, P < 0.05; **, P < 0.01) A. mRNA B. cRNA C. vRNA Binh et al, 2013. Frontiers in Virology Negatively charged amino acid may decrease the viral RNA polymerase activity.

  12. Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses 1-15 PA binding site 16 27 44 E (2 types of negative charge D and E) The amino acid at the position 27 in the wild type virus is aspartate, and aspartate is conserved among influenza A viruses, except for an H4N8 strain.

  13. 3 3 2.5 ** ** * 2.5 2 ** ** * Relative amount of cRNA 1.5 2 Relative amount of mRNA 1 1.5 0.5 1 0 0.5 4 mock D E N V 3.5 (wt) 0 3 mock D E N V 2.5 (wt) Relative amount of vRNA 2 1.5 1 0.5 0 mock D E N V (wt) The viral RNA synthesis of mutant viruses at the position 27 Student’s t test (*, P < 0.05; **, P < 0.01) A. mRNA B. cRNA C. vRNA Binh et al, 2013. Frontiers in Virology Uncharged amino acids at the position 27 enhanced the viral RNA synthesis.

  14. 1-15 PA binding site Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses 16 27 44 D Q Q: structure of Q is to close to that of N D: position 27

  15. 3 3.5 2.5 3 3 2 2.5 2.5 Relative amount of mRNA 1.5 2 2 Relative amount of vRNA 1 Relative amount of cRNA 1.5 1.5 0.5 1 1 0 0.5 0.5 mock N I D Q 0 (wt) 0 mock N I D Q ** ** ** mock N I D Q (wt) (wt) The viral RNA synthesis of mutant viruses at the position 44 Student’s t test (*, P < 0.05; **, P < 0.01) A. mRNA B. cRNA C. vRNA ** Binh et al, 2013. Frontiers in Virology 44Q stimulated only vRNA synthesis.

  16. B. Position 16 C. Position 27 A. influenza B amino acids 2 2 2 1.5 1.5 1.5 mRNA/vRNA mRNA/vRNA 1 mRNA/vRNA 1 1 0.5 0.5 0.5 0 0 0 mock N A D Q mock D N E V mock wt N16A D27V N44I (wt) (wt) 2 D. Position 44 1.5 mRNA/vRNA 1 0.5 0 mock N I D Q (wt) The effect on the primary transcription activity by incoming vRNP Binh et al, 2013. Frontiers in Virology These mutations did not affect primary transcription activity.

  17. Input (2%) mock wt D27N D27E D27V N16A N16D N16Q N44I N44D N44Q 1 2 3 4 5 6 7 8 9 10 11 WB: anti-PB2 WB: anti-tubulin IP: anti-PB2 antibody mock wt D27N D27E D27V N16A N16D N16Q N44I N44D N44Q + - + + + + + + + + + + 1 2 3 4 5 6 7 8 9 10 11 12 anti-PB2 PB1 PB2 WB: anti-PB1 anti-PB2 PA WB: anti-PA 1.0 1.0 1.1 0.9 0.9 1.0 1.0 0.9 1.1 1.2 PB1/PA ratio Effect of mutations on assembly of viral RNA polymerase complexes The assembly of PB1 with PA and PB2 was not affected by these mutations.

  18. Summary 1 • Amino acids at the position 27 and 44 are involved in RNA polymerase activity and may contribute to subtype specificity. • As the primary transcription activity of mutant viruses were not affected, this result indicates that replication activity and/or vRNP stability might be affected. • The assembly of PB1 with PA and PB2 was not affected by these mutations. • Amino acid residues at the position 27 and 44 are involved in the viral genome replication, possibly via the cRNA promoter recognition with little effects on the transcription activity and the assembly of the RNA polymerase complex

  19. Aim Functional analysis of PB1 subunit using mutant viruses (1) Reverse-genetics: Using mutant viruses to determine the replicational and transcriptional activities (2) Forward-genetics: Using ribavirin which inhibits RNA synthesis to determine the catalytic active site of PB1 by isolation of ribavirin-resistant PB1 mutants Ribavirin

  20. Screening of ribavirin-resistant PB1 mutant B. EGFP fluorescence in the first screening A. Mini-replicon assay system for screening Binh et al, 2014. BBRC

  21. Characterization of D27N mutant A. Sequencing of ribavirin-resistant PB1 mutant B. EGFP fluorescence C. Luciferase activity D. Effect of D27N mutation on assembly of PB1 subunit Binh et al, 2014. BBRC

  22. Summary 2 • D27N mutant was isolated by forward genetics in the presence of ribavirin. • The luciferase assay of influenza virus RNA polymerase activity of D27N mutant was higher than that of wild type in the presence of ribavirin. • The expression level of D27N in the presence of ribavirin was similar to that of wild type.

  23. Models and Discussion Stimulation Promoter RNA polymerase consensus motif 1 757 a.a. PB1 NLS NH2 COOH Putative cRNA promoter binding Putative vRNA promoter binding Putative nucleotide binding Ribavirin

  24. Acknowledgments

  25. Thank you for your attention!

  26. CAG pro PB2 PB1 PA NP seg7 seg6 seg3 seg8 seg4 seg5 seg2 seg1 Generation of recombinant viruses by reverse-genetics Plasmids expressing influenza viral proteins Plasmids expressing influenza vRNA Point mutation Pol.I Transfection 293 T cell PB1 PB2 PA NP M1 NS1 NS2 HA NA M2 Nucleus ER HA Translation Encapsidation vRNA (-) Golgi NA vRNPs M1: NS2 M2 M1 cRNA (+) vRNP Budding Replication Packaging Nuclear export vRNP Transcription Recombinant virus mRNA (+)

  27. Transcription Replication PB2: recognize and bind the cap PA: cleave the cap PB1: nucleotide addition Cap vRNA Host cellular pre-mRNA cRNA 5’ Cap vRNA 3’ UUUUU UUUUU 5’ 3’ Cap Progeny vRNA 5’ UUUUU 3’ AAAAA Cap AAAAA… Cap Viral mRNA Transcription and replication of influenza virus genome

  28. Method 2. Analysis of primary transcription activity by qRT-PCR MDCK cells Infection with WSN, MOI=2.5 Adding cycloheximide (CHX) into medium 9 h post infection RNA extraction by AGPC method mRNA, and vRNA were analyzed by qRT- PCR MDCK cell Cytoplasm Nucleus X CHX Progeny vRNP X X Incoming vRNP mRNA Polymerase + NP

  29. Mechanism of action methotrexate Alternate pathway Preferred pathway MTX MTX Y Y Y MTX Folic acid receptor Y MTX Reduced folic acid receptor MTX(Glu)n Purine biosynthesis FH4 RNA FH2 DNA CH2FH4 TTP dUMP Cytoplasm Nucleus TMP Folylpolyglutamate synthase MTX polyglutamate [MTX (Glu)n] MTX Dihydrofolate reductase FH2 FH4

  30. Effect of methotrexate on D27N mutant A. Mini-replicon assay system random mutation CAG pro PB2 PB1 PA vNS-Luc pol.I pro NP transfection 293T cell Methotrexate (MTX) PA NP PB2 PB1 vRNP Luc transcription Luciferase m7Gppp translation Luciferase B. Luciferase activity

  31. Significant impact on our life by influenza virus infection B. Virus classification A. Illustration of the host range of influenza A virus Familly: Orthomyxoviridae Genera: Influenza A Influenza B Influenza C • Nucleoprotein (NP) and matrix (M1) proteins • Other important characteristics • -FluA : a wide variety of hosts • -FluB : only humans • -FluC : humans but also from swine • -HA and NA of FluA viruses amino acid sequence variability than those of FluB viruses. FluC viruses: only a single hemagglutinin-esterase-fusion protein (HEF). Isolated by Shope and Lewis in 1930 Wahlgren, 2011. Infection Ecology and Epidemiology.

  32. Nucleotides (A G U C R) WT WT 27N 27N WT 27N R R G G Models and Discussion vRNA promoter cRNA promoter Improved nucleotide recognition Strong activity Progeny vRNA cRNA

  33. Why was the N-terminal region of PB1 chosen? B. Structure of PB1 subunit 1 139 493 267 Putative cRNA promoter binding 494 757 1 83 249 256 Putative vRNA promoter binding 179 297 458 519 Putative nucleotide binding 286 483 678 757 1 15 RdRp catalytic domain PB2 binding PA binding A. Panhandle model of vRNA and cRNA promoters of PB1 subunit between influenza A and B viruses 2. FluB vRNA 3. FluA cRNA 4. FluB cRNA 1. FluA vRNA

  34. 757 1 139 493 267 Putative cRNA promoter binding 494 757 1 83 249 256 Putative vRNA promoter binding 179 297 458 519 Putative nucleotide binding 35 1 286 483 678 757 1 15 678 RdRp catalytic domain Sugiyama et al, 2009. EMBO PB2 binding PA binding Structure of PB1 subunit PAC-PB1N PB1C-PB2N 2 15 716 239 Obayashi et al, 2008. Nature

  35. The viral RNA synthesis of mutant viruses at the position 27 (various moi) when the amounts of viral RNAs of D27V were analyzed at various moi (0.5, 2.5, 10, and 25), those of D27V were increased

  36. Comparison of RNA synthesis of mutant influenza A viruses containing amino acids specific for influenza B viruses (3 and 6 hpi)

  37. The viral RNA synthesis of mutant viruses at the position 16 (3 and 6 hpi)

  38. The viral RNA synthesis of mutant viruses at the position 27 (3 and 6 hpi)

  39. The viral RNA synthesis of mutant viruses at the position 44 (3 and 6 hpi)

  40. The viral RNA synthesis of mutant viruses at the position 27 in the presence of ribavirin (IC50= 20 μM)

  41. Virion of influenza A virus

  42. Structure of influenza A virus genome and viral proteins

  43. Function of the influenza A virus components

  44. Virion of influenza B virus Hemmaglutinin (HA) Matrix (M1) Neuramidase (NA) Polymerase complex (PB1, PB2, and PA) Nucleoprotein (NP) BM2 ion channel Nuclear export protein (NEP) NB ion channel

  45. Structure of influenza B virus genome and viral proteins 8 segments coding for 11 proteins. Segments size range from 890 to 2,341nt. Genome total size is 13.5Kb Viral RNA polymerase (PB1, PB2 and PA) transcribes one mRNA from each genome segment. Transcription is primed by cap cleaved from cellular mRNAS by Cap snatching. mRNA are polyadenylated by the viral polymerase stuttering on a poly U track. MP and NS mRNA can be spliced, giving rise to mRNA coding for BM2 and NEP proteins. PB1-F2 is translated from the +1 ORF of PB1 mRNA, NB is translated from the -1 ORF of NA mRNA.

  46. Virion of influenza C virus

  47. Structure of influenza C virus genome and viral proteins No NA-encoding gene

  48. Catalytic sites of influenza A virus PB1 subunit http://www.uniprot.org/uniprot/P03431

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