Dr luis enjuanes - molecular basis of coronavirus virulence and vaccine design to protect against sars - Comment
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Information covered in this presentation slides: 1. Molecular basis of coronavirus virulence and vaccine design to protect against sars xrb. Universidad de barcelona 13 julio 2012 luis enjuanes cnb-csic. Madrid 2. Coronavirus tgev & sars-covreplication transcription packaging virulence virus-host interaction 3. Coronavirus induced diseases man central nervous system animals eye upper and lower as in humans & respiratory tract peritonitis immune system cells hepatitis nephritis gastrointestinal cardiomiopathy tract 4. Rna virus genome size in nucleotidesnido_coronavirus nido_torovirus nido_roniviridae closteroviridae benyvirusnido_arteriviridae sequiviridae pomovirus togaviridae comoviridae flaviviridae furovirus tobravirus pecluvirus cheravirus hordeivirus potyviridae iflavirus dicistroviridae marmaviridae bromoviridae idaeovirus picornaviridae caliciviridae flexiviridae tetraviridae hepeviridae astroviridae tobamovirus unclassified tymoviridae luteoviridae nodaviridae sobemovirus umbravirus tombusviridae barnaviridae leviviridae narnaviridae 5. Mutation of coronavirus exon motifs mhv rep 1a rep 1b nsp14 cpd he s 4a 4b 5a e m n an i c1 d89 e91 zn f d242 d272 q521yeast mut 1 .. Aidaefv-103- zn f .mut 2. Vfvghglnndfk-44-hdsiedahtalmhv-a59 gfdaega-111-vvcsvctk-11-ygcwrhs-3-dylynplivdiq-23-hvassdaimtrsars-cov gfdvegc-111-rtcclcdk-11-yacwnhs-3-dyvynpfmidvq-23-hvascdaimtrhcov-229e gmdvega-111-khcq-cgt-11-yccfkha-3-dyvynpyvidiq-23-hvasgdaimtrtgev gfdvega-111-qkce-cgk-11-yacfkha-3-dylynpycidiq-23-hvasgdaimtribv gfdveat-111-qvcs-cgs-11-yacwkhc-3-dfvynpllvdiq-23-hvasvdaimtreckerle et al., 2007, 2010 6. Distribution of mutations after passagemhv rep 1a rep 1b nsp14 cpd he s 4a 4b 5a e m n an iwt p5 c1 c2 p17 c1mut 1 p1 c1 p5 c2 p17 c1mut 2 p1 c1 p5 c2 p17 c2eckerle et al., 2007 7. Coronavirus structure and gene expression 3b orf 1a orf 1b s 3a e m n 7 utr + cap aaa mrna + aaa - uuu g + aaa - uuu s + aaa - uuu 3a + aaa - uuu 3b + aaa - uuu e + aaa - uuu m + aaa - uuu n + aaa - uuu 7 8. Transcription mechanism template switch a high frequency recombination engineered an infectious cdna clone 9. Regulation of sgmrna levels trs-l trs-b grna 5’ l cs-l cs-b an 3’5’ an 3’ + multifactor regulation 1. Trs rna structure3’ cl ccs-b un 5’ - 2. Trs primary sequence ctrs-b 3. Long distance interactions 4. Protein-rna binding 5’ ccs-b 3’ mrna ..................... 10. Transcription and leader-body cs identity gene 3a + 5’ l cuaaac cuaaac an 3’ + gauuug un 5’ - 11. Transcription and leader-body cs identity trs-l gene 3a + 5’ l cuaaac cuaaac an 3’ + gauuug 5’ - 12. Wild type trs-l structure prediction u c16 17 a18 apical heptaloop a15 a19 including the cs-l a14 a20 g13 c21 c12 g22 micro stem u11 a23 c10 a24 a9 a25 u26 a8 internal loop a27 c7 u28 c6 u29 a5 u30 c4 g31 a3 u32 g2 c33 g1 u34 wt 13. Wt and mutants trs-l structures u a u u a u a u a u a c c a c c c c a a a aa a u a a a a a a a c a a a a a aa a a a a a a a g c c g c g c g c g c g a a a g g a c g c g g c c g c g a a a u a a u u a u a c g c g c ac a a a c a a c a u a c g aa a c a a a u a u a a a u a u a u u aa a u a c a u a u a a a a uc a c u c u c u c u c g c g uc u c u c c u c g u a u c g a u a u a u a u c g a u a u c g c g c g u c g a u c g a u a u c g a u a u a u g c a u g c g c g c g c g u g c g u g u g c g u g u g u g u wt ms2 il1 ms1 ms3 il3 il2 14. Conclusion the extent of sg mrna synthesis: Correlates with trs-l stability with the availability of trs-l requires a specific secondary structure 15. Transcription mechanism template switch a high frequency recombination engineered an infectious cdna clone 16. Transcription and leader-body cs identity gene 3a + 5’ l cuaaac cuacac an 3’ + g a ug u g u un 5’ - 17. Transcription and leader-body cs identity gene 3a + 5’ l cuaaac cuacac an 3’ + g aug u g u 18. Requirement of complementarity between cs-l and ccs-b for sgmrna synthesis cs-b mutants cs-l mutants titer* titer* 5’ c u a a a c 3’ 5’ c u a a a c 3’ b-c1g g 8x108 l-c1g g - b-u2g g 1x107 l-u2g g - b-a3c c 5x108 l-a3c c - b-a4c c 6x108 l-a4c c 2x104 b-a5c c 3x108 l-a5c c 4x104 b-c6g g 4x108 l-c6g g 2x103 non-watson-crick mutants complementary mutants titer* titer* 5’ c u a a a c 3’ 5’ g u a a a c 3’ l-c1u u 2x103 d-c1g - 3’ c a u u u g 5’ wt 5x108 - l-a3u u 3x103 - 5x104 2x105 l-a4u u l-a5u u 2x104 5’ c u a a a g 3’ 4x105 d-c6g l-c6u u 1x108 3’ g a u u u c 5’ 2x10 3 * pfu/ml 19. Main conclusion δbase-pairing (δg) is a driving forcein cov transcription 20. Complementary and synthesis of sgmrna mrna cs2 5’ cgaacuaaacgaaa 3’ 75 potential base-pairing score cs1 acuaaac cs2 cuaaac 65 m1 acuaaac m2 aacuaaac 55 m3 gaacuaaac mrna 3a.2 m4 cgaacuaaac 45 m7 cuaaacg m8 cuaaacga 35 m9 cuaaacgaa m10 cuaaacgaaa 25 m5 cgaacuaaacga m6 cgaacuaaacgaaa 15 24550 24684 24685 24710 24734 24746 genome position, nt mutants mock cs1 cs2wt 1 2 3 4 7 8 9 10 5 6 506 mrna-3a.2 220 mrna cs2 21. Complementarity between nascent rna and trs-l sequence and mrna levels 14 m4 10 m3 -δg, kcal/mol m2 m1 6 cs-s2 5’ trs mutants δ 2 0 50 100 150 200 250 300 mrna, u.r. 22. Mrna levels and base-pairing score 110 90 base-pairing relative units 70 50 30 mrna 10 s 3a e m n 7 mrna 23. Regulation of sg mrna-n levels trs-l trs-n n-genegrna 5’ l cs-l de pe cs-n an 3’ 460nt 7nt 5’ 3’ cl taatgtata attacatat an 3’ + ccs-n un 5’ - ctrs-n 24. Complementarity and enhancer activitysgmrna-n, relative units 200 150 100 50 0 5 10 15 20 25 ∆g, kcal/mol 25. Requirement of pe and de flanking sequences sgmrna-n, %mutant ad de pe 0 50 100 150 200e2-trs-ntrs-nde-113-158de-45-158de-173-20pe-20 26. Optimized trm de pe csorf 1a 77orf 1a orf 1b orf 1b n aaa 173 9 20 20 9 7 6 6 original trm 642 nt 250 nt 27. Enhancement mechanism pe cs-n 3’ de 5’ ad 5’ 28. Enhancement mechanism cs-n 3’ 5’ ad 5’ 29. Requirement of long distance interaction trm mutant ad de pe cs-n n rep-1 trs-n ad ad-trs-n trmopt trmopt-n 0 5 10 15 rep-1 trs-n ad-trs-n trmopt-n sgmrna-n/grna 30. Enhancement mechanism pe cs-n 3’ de 5’ ad 5’ 80% enhancement 31. Active domain regions requirement a 25 sgmrna-n, relative units 20 b 15 10 5 c 0 + - δa δb δc 32. Active domain region c requirement δa δa-c’ 25 sgmrna-n, relative unitsb 20 15 10c 5 0 δa δa-c’ δ[a-b] 33. Active domain region b requirement ∆a ∆a-b*2r ∆a-b*1r ∆a-b*1r4 ∆a-b*1r3b 25 sgmrna-n, relative units 20 15 10 5 0 ∆a ∆a-b*2r ∆a-b*1r ∆a-b*2r4 ∆a-b*1r3 (-) 34. Intragenomic rna-rna interactions involving b rna motif cb-m b-m cb-m b-mnt 218-229 nt 26408 - 26421 nt 477-489 nt 26408 - 26421 ∆g = -12.9 ∆g = -12.7 35. Tgev replicons with mutated b and cb rna motifs 218-229 477-486 26,212-26,221 5’ 3’ b de pe n wt/b 5’ 3’ de pe n ∆ wt/∆b 5’ 3’ cb-218 de pe n * b cb-218*/b 5’ 3’ de pe n ∆ cb-218*/∆b 5’ * 3’ * b* de pe n cb-218*/b* 5’ 3’ cb-477 b de pe n cb-477*/b 5’ * 3’ de pe n ∆ cb-477*/∆b 5’ * 3’ b* de pe n cb-477*/b* 5’ * 3’ b* de pe n cb-477*/b*-14 5’ * 3’ 36. Sgmrna n, relative units 0 5 10 15 20 25 30 35 40 45 5’ w t/b w t/∆ b 218 477 cb -2 18 cb * /b -2 18 */∆ cb b -2 18 * /b * cb 47 7* cb /b b -4 26,212 77 */∆ cb b de -4cb 77 -4 */ b pe 77 * */ b n *- 14 3’ transcription and complementary levels 37. Evolution of viruses with mutated b and cb rna motifs 38. Relevance of the interaction between b and cb rna motifs on sgmrna and viral titers 39. Mechanism of discontinuous transcription ad cb-m b-m trs-l de pe cs-n5’(+) 3’(+) pe cs-n 3’(+) 5’(-) de ccs-n 5’(+) cs-l b-m cb-m ad cb-m cs-l 5’(+) 40. Cov transcription is regulated at three levelstrs-l stability and secondary structure complementarity between nascentminus rnas and trs-llong distance rna-rna interactions 41. Trm activity within infectious viruses orf 1a orf 1b s 3a 3b e m n 7wt-tgev n n a de pe 0 2 4 6 cstrs-3a ad de pe cstrmopt-3atrmopt*-3atrmopt-19-3a sgmrna-3a/grna 42. Geographical area origin of sars 43. Severe acute respiratory syndrome neumonia high fever lymphopenia death 44. Spreading of sars …July 5th, 03 end epidemy by whonov 02 jan 03 feb 03 dec03 to jan04foshan guangzhou hong kong four laboratory cases dr. A vietnam canada four non-laboratorymost first nephro- isolated cases singapore recent major logist usacommon outbreak two additional ancestor hospital hotel m sse in hk beijin apr f 26y, bsl-4 (>100 contacts) beijin apr m 44y, bsl-4 45. Racooncivet cat ferrets 46. Coronavirus evolution genus αgenus β genus ϒ 47. Engineering of a sars-cov infectious cdna clone as a bac t by c t by a 10338 11163 8a l 3b m 7a 8b 9b cmv rep 1a rep 1b s 3a e 6 7b n an pbac-sars-cov-urb* 48. Basis of sars-cov virulence 8a 3b m 7a 8b 9bl rep 1a rep 1b s 3a e 6 7b n an deleted gene structural: E accessory: 6, 7a, 7b, 8a, 8b, 9b combinations: E, 6, 7a, 7b, 8a, 8b, 9b 49. Bsl-3 cnb. Csic. Madrid 50. Growth kinetics of sars-cov deletion mutants vero e6 caco-2 107 wtvirus titer, pfu/ml δ6-9b 105 δe 103 δe,6-9b 101 0 24 48 72 0 24 48 72 time post-infection, hour genes: E, 6, 7a, 7b, 8a, 8b, 9b are non essential 51. Morphogenesis of sars-cov in ergic sars-cov sars-cov-∆e ∆ sars-cov-∆ [6-9b] ∆ sars-cov-∆ [e,6-9b] ∆ 52. Activity wheel used to quantify clinical illness 53. ∆sars-cov-∆e-infected hamster wheel activity infection 1200 ∆e 1000revolutions/ hr 800 mock 600 wt 400 200 0 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 day 54. Attenuation of sars-cov deletion mutants in transgenic mice expressing hace-2 12000 pfu intranasally clinical disease lethality 110 δe 100 startting weight, % δe,6-9b survival, % δe 100 75 90 δe,6-9b 50 80 wt δ6-9b 25 0 δ6-9b 70 2 4 6 8 2 4 6 8 time postinoculation, days time postinoculation, days 55. Summarye gene conditions tissue specific tropism ∆sars-cov-∆e is attenuated gene e is a virulence factor 56. Mechanism of e protein virulence e protein and host gene expression 8a 3b m 7a 8b 9b l rep 1a rep 1b s 3a e 6 7b n an ±e 57. Differential gene expression in ∆edifferential gene expression in δe and wt and wt infected cells infected cells wt vs mock δe vs mock δe vs wt 14 12 10 vero e6 8 6 15 hpilog experiment 4 2 14 12 10 ma104 8 6 65 hpi 4 2 2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14 log control 58. Δgenes differentially expressed in sars-cov-δe vs full-length virus infected cells fold change fold change gene vero ma-104 gene vero ma-104 stress response immunoregulation hsp10 hspe1 + 3.3 -- fkbp4 + 4.7 + 5.5 hsp20 b5 cryab -- + 3.2 ripk2 + 2.3 -- hsp27 b1 +19.0 -- zc3h12a -- + 2.9 hsp40 dnajb1 +14.8 +25.9 hsp40 dnaja1 + 8.7 + 7.2 inflammation hsp40 dnajb6 + 3.9 + 2.1 hsp40 dnaja4 -- + 2.6 nfkbiz + 2.9 -- hsp40 dnajb4 -- + 4.1 adamts1 + 2.1 -- hsp47 serpinh1 + 6.7 + 2.5 ccl2 -- - 3.2 hsp60 d1 + 4.1 + 4.7 cxcl2 -- - 2.2 hsp70 a1a +22.3 +29.3 apoptosis hsp70 a1b +18.8 -- dedd2 + 2.6 + 5.0 hsp70 a8 + 5.8 + 4.9 clu + 2.1 + 2.0 hsp70 a4l -- + 2.3 phlda1 + 3.2 -- hsp90 aa1 +15.1 + 6.5 stk17b -- + 2.2 hsp90 ab1 + 6.1 + 2.7 hsp105/110 +35.3 +13.4 signal transduction ubb + 3.9 + 4.4 dscr1 + 5.9 -- ubc + 2.3 + 2.7 dusp1 + 4.0 -- bag3 + 7.2 + 3.9 dusp10 -- + 2.7 cct4 + 3.3 -- ywhag + 2.5 -- cct3 + 3.3 -- wasl + 2.5 -- ahsa1 + 4.7 -- map2k3 + 2.1 -- cryab -- + 3.2 folr1 -- +15.6 tcp1 -- + 2.3 pde4b -- + 2.5 stip1 -- + 2.1 k-ras -- - 2.6 59. Unfolded protein response re cytoplasm nucleus activation in e vs wt- δ grp78 grp78 perk eif2 translational infected cells block perk eif2 p atf4 translation attenuation - chop grp78 gene grp78 cleavedgrp78 correct atf6 atf6 expression protein folding - chop grp78 grp78 grp78 xbp1 ire1 mrna ire1 sxbp1 sxbp1 protein degradation + p58ipk mrna 60. Effect of e protein on the stress produced δ by sars-cov-δe infection e protein provided in trans 12 hsp a1a hsp90 aa1 hsp h1 serpin h1 hsp e1 18s e+ fold change, relative units 10 e- 8 6 4 2 61. Sars-cov e protein and stress produced by rsv infection e protein provided in trans 350 7 hsp90 aa1 ubb hsp h1 serpin h1 hsp e1 18s 300 6 e+fold change, relative units 250 5 e- 200 4 150 3 100 2 50 1 2 24 48 2 24 48 2 24 48 2 24 48 2 24 48 2 24 48 time post-infection, hours 62. Conclusione protein reduced cellular stressinduced by: Sars-cov infection rsv infection thapsigargin - ca++ tunicamycin - glycosylation 63. Δsars-cov-δe infection and inflammation stress inflammatory response response +e -e 64. Mouse adapted sars-cov sars-cov-ma15high titers in lungsviremia, extrapulmonary spreadneutrophiliapathological changes in lungsdeath k. Subbarao and r. Baric labs 65. Construction of a mouse adapted sars-cov 8a 3b m 7a 8b 9b l rep 1a rep 1b s 3a e 6 7b n an nsp5 nsp9 10384 12814 nsp13 s (rbd) m 16117 22797 26428 nsp5 10793 engineered mutants wt δ[6-9b] δe δ[e,6-9b] 66. Conclusion δsars-cov-ma15-δe is attenuated andfullyprotects both young and old micetherefore it is a promising vaccine δcandidate, better than sars-cov-δe 67. Cnb. Csic. Madrid collaboratorsreplication proteomicsfernando almazan silvia juarez. Cnb-csicaitor nogales alexander akoulichev. Oxfordsilvia marquez genomicstranscription and assembly irene lopez-vidrieroisabel sola marta godoypedro a. Mateos juan carlos oliverossonia zuñigamartina becares animal models kanta subbarao. Nihvirus-host interaction: Sars stanley perlman. Univ. Iowamarta l. Dediegojose luis nietojose manuel jimenez electron microscopyjose angel regla maria teresa rejas. Cbm, csicraul fernandez cristina patiño. Cnb. Csic carlos m. Sanchez sarhay rosluis enjuanes margarita gonzalez |
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| Posted by : peter88 | Post date : 2020-01-22 18:43 | ||
| Category : Health & Medicine | Views : 325 | ||
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