Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses

Journal of Virology

Volumn 88, Issue 24, 2014, Pages 14396-14411

  Wolferstätter, Michael ^a   Schweneker, Marc ^a   Späth, Michaela ^a   Lukassen, Susanne ^a   Klingenberg, Marieken ^a   Brinkmann, Kay ^a,c   Wielert, Ursula ^a   Lauterbach, Henning ^a   Hochrein, Hubertus ^a   Chaplin, Paul ^a   Suter, Mark ^b   Hausmann, Jürgen ^a  

^a Bavarian Nordic GmbH   (Germany)

^b UNIVERSITY OF ZURICH   (Switzerland)

^c Exosome Diagnostics GmbH   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA INTERFERON; BETA INTERFERON; DOUBLE STRANDED RNA; GAMMA INTERFERON; INTERFERON REGULATORY FACTOR 3; PROTEIN KINASE R; RECOMBINANT MODIFIED VACCINIA VIRUS ANKARA; RECOMBINANT VACCINE; UNCLASSIFIED DRUG; VIRUS PROTEIN; CYTOKINE;

ANIMAL CELL; ANIMAL EXPERIMENT; ANIMAL MODEL; APOPTOSIS; ARTICLE; CONTROLLED STUDY; CYTOKINE PRODUCTION; CYTOKINE RESPONSE; EMBRYO; ENZYME ACTIVATION; FEMALE; HOST RESISTANCE; HUMAN; HUMAN CELL; IMMUNOGENICITY; IMMUNOREACTIVITY; IMMUNOSTIMULATION; INNATE IMMUNITY; INTERFERON INDUCTION; MOUSE; NONHUMAN; PROTEIN ENGINEERING; PROTEIN EXPRESSION; PROTEIN PHOSPHORYLATION; PROTEIN RNA BINDING; PROTEIN SYNTHESIS INHIBITION; VACCINIA VIRUS; VIRUS REPLICATION; VIRUS TRANSCRIPTION; VIRUS VIRULENCE; ANIMAL; BAGG ALBINO MOUSE; C57BL MOUSE; CELL LINE; GENETICS; IMMUNOLOGY; METABOLISM; SECRETION (PROCESS);

VACCINIA VIRUS ANKARA;

ANIMALS; CELL LINE; CYTOKINES; EIF-2 KINASE; HUMANS; IMMUNITY, INNATE; MICE, INBRED BALB C; MICE, INBRED C57BL; RNA, DOUBLE-STRANDED; VACCINIA VIRUS;

EID: 84911500873     PISSN: 0022538X     EISSN: 10985514     Source Type: Journal    
DOI: 10.1128/JVI.02082-14     Document Type: Article

References (76)

1. Desmet CJ, Ishii KJ. 2012. Nucleic acid sensing at the interface between innate and adaptive immunity in vaccination. Nat. Rev. Immunol. 12: 479-491. http://dx.doi.org/10.1038/nri3247.
2. Iwasaki A. 2012. A virological view of innate immune recognition. Annu. Rev. Microbiol. 66:177-196. http://dx.doi.org/10.1146/annurev-micro-092611-150203.
3. Pichlmair A, Lassnig C, Eberle CA, Górna MW, Baumann CL, Burkard TR, Bürckstümmer T, Stefanovic A, Krieger S, Bennett KL, Rülicke T, Weber F, Colinge J, Müller M, Superti-Furga G. 2011. IFIT1 is an antiviral protein that recognizes 5'-triphosphate RNA. Nat. Immunol. 12:624-630. http://dx.doi.org/10.1038/ni.2048.
4. Haller O, Kochs G, Weber F. 2006. The interferon response circuit: induction and suppression by pathogenic viruses. Virology 344:119-130. http://dx.doi.org/10.1016/j.virol.2005.09.024.
5. Iwasaki A, Medzhitov R. 2010. Regulation of adaptive immunity by the innate immune system. Science 327:291-295. http://dx.doi.org/10.1126/science.1183021.
6. Frenz T, Waibler Z, Hofmann J, Hamdorf M, Lantermann M, Reizis B, Tovey MG, Aichele P, Sutter G, Kalinke U. 2010. Concomitant type I IFN receptor-triggering of T cells and of DC is required to promote maximal modified vaccinia virus Ankara-induced T-cell expansion. Eur. J. Immunol. 40:2769-2777. http://dx.doi.org/10.1002/eji.201040453.
7. Garcia-Arriaza J, Najera JL, Gomez CE, Tewabe N, Sorzano CO, Calandra T, Roger T, Esteban M. 2011. A candidate HIV/AIDS vaccine (MVA-B) lacking vaccinia virus gene C6L enhances memory HIV-1-specific T-cell responses. PLoS One 6:e24244. http://dx.doi.org/10.1371/journal.pone.0024244.
8. Chakrabarti A, Jha BK, Silverman RH. 2011. New insights into the role of RNase L in innate immunity. J. Interferon Cytokine Res. 31:49-57. http://dx.doi.org/10.1089/jir.2010.0120.
9. Garcia MA, Gil J, Ventoso I, Guerra S, Domingo E, Rivas C, Esteban M. 2006. Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol. Mol. Biol. Rev. 70:1032-1060. http://dx.doi.org/10.1128/MMBR.00027-06.
10. Kato H, Takahasi K, Fujita T. 2011. RIG-I-like receptors: cytoplasmic sensors for non-self RNA. Immunol. Rev. 243:91-98. http://dx.doi.org/10.1111/j.1600-065X.2011.01052.x.
11. Rehwinkel J, Reis e Sousa C. 2010. RIGorous detection: exposing virus through RNA sensing. Science 327:284-286. http://dx.doi.org/10.1126/science.1185068.
12. Langland JO, Jacobs BL. 2002. The role of the PKR-inhibitory genes, E3L and K3L, in determining vaccinia virus host range. Virology 299:133-141. http://dx.doi.org/10.1006/viro.2002.1479.
13. Malmgaard L, Melchjorsen J, Bowie AG, Mogensen SC, Paludan SR. 2004. Viral activation of macrophages through TLR-dependent and-independent pathways. J. Immunol. 173:6890-6898. http://dx.doi.org/10.4049/jimmunol.173.11.6890.
14. Williams BR. 1999. PKR; a sentinel kinase for cellular stress. Oncogene 18:6112-6120. http://dx.doi.org/10.1038/sj.onc.1203127.
15. Gil J, Esteban M. 2000. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5:107-114. http://dx.doi.org/10.1023/A:1009664109241.
16. Goh KC, deVeer MJ, Williams BR. 2000. The protein kinase PKR is required for p38 MAPK activation and the innate immune response to bacterial endotoxin. EMBO J. 19:4292-4297. http://dx.doi.org/10.1093/emboj/19.16.4292.
17. Barry G, Breakwell L, Fragkoudis R, Attarzadeh-Yazdi G, Rodriguez-Andres J, Kohl A, Fazakerley JK. 2009. PKR acts early in infection to suppress Semliki Forest virus production and strongly enhances the type I interferon response. J. Gen. Virol. 90:1382-1391. http://dx.doi.org/10.1099/vir.0.007336-0.
18. Gilfoy FD, Mason PW. 2007. West Nile virus-induced interferon production is mediated by the double-stranded RNA-dependent protein kinase PKR. J. Virol. 81:11148-11158. http://dx.doi.org/10.1128/JVI.00446-07.
19. Ludwig H, Mages J, Staib C, Lehmann MH, Lang R, Sutter G. 2005. Role of viral factor E3L in modified vaccinia virus Ankara infection of human HeLa cells: regulation of the virus life cycle and identification of differentially expressed host genes. J. Virol. 79:2584-2596. http://dx.doi.org/10.1128/JVI.79.4.2584-2596.2005.
20. Cooper JA, Wittek R, Moss B. 1981. Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs. J. Virol. 39:733-745.
21. Mahr A, Roberts BE. 1984. Arrangement of late RNAs transcribed from a 7.1-kilobase EcoRI vaccinia virus DNA fragment. J. Virol. 49:510-520.
22. Boone RF, Parr RP, Moss B. 1979. Intermolecular duplexes formed from polyadenylylated vaccinia virus RNA. J. Virol. 30:365-374.
23. Colby C, Duesberg PH. 1969. Double-stranded RNA in vaccinia virus infected cells. Nature 222:940-944. http://dx.doi.org/10.1038/222940a0.
24. Moss B. 2013. Poxviridae, p 2129-2159. In Knipe DM, Howley PM (ed), Fields virology. Lippincott Williams & Wilkins, Philadelphia, PA.
25. Lynch HE, Ray CA, Oie KL, Pollara JJ, Petty IT, Sadler AJ, Williams BR, Pickup DJ. 2009. Modified vaccinia virus Ankara can activate NF-kappaB transcription factors through a double-stranded RNA-activated protein kinase (PKR)-dependent pathway during the early phase of virus replication. Virology 391:177-186. http://dx.doi.org/10.1016/j.virol.2009.06.012.
26. Willis KL, Langland JO, Shisler JL. 2011. Viral double-stranded RNAs from vaccinia virus early or intermediate gene transcripts possess PKR activating function, resulting in NF-kappaB activation, when the K1 protein is absent or mutated. J. Biol. Chem. 286:7765-7778. http://dx.doi.org/10.1074/jbc.M110.194704.
27. Yuen L, Moss B. 1987. Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes. Proc. Natl. Acad. Sci. U. S. A. 84:6417-6421. http://dx.doi.org/10.1073/pnas.84.18.6417.
28. Smith GL, Symons JA, Alcami A. 1998. Poxviruses: interfering with interferon. Sem. Virol. 8:409-418. http://dx.doi.org/10.1006/smvy.1997.0145.
29. Chang HW, Watson JC, Jacobs BL. 1992. The E3L gene of vaccinia virus encodes an inhibitor of the interferon-induced, double-stranded RNAdependent protein kinase. Proc. Natl. Acad. Sci. U. S. A. 89:4825-4829. http://dx.doi.org/10.1073/pnas.89.11.4825.
30. Rivas C, Gil J, Melkova Z, Esteban M, Diaz-Guerra M. 1998. Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme. Virology 243:406-414. http://dx.doi.org/10.1006/viro.1998.9072.
31. Romano PR, Zhang F, Tan SL, Garcia-Barrio MT, Katze MG, Dever TE, Hinnebusch AG. 1998. Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain. Mol. Cell. Biol. 18:7304-7316.
32. Sharp TV, Moonan F, Romashko A, Joshi B, Barber GN, Jagus R. 1998. The vaccinia virus E3L gene product interacts with both the regulatory and the substrate binding regions of PKR: implications for PKR autoregulation. Virology 250:302-315. http://dx.doi.org/10.1006/viro.1998.9365.
33. Beattie E, Paoletti E, Tartaglia J. 1995. Distinct patterns of IFN sensitivity observed in cells infected with vaccinia K3L-and E3L-mutant viruses. Virology 210:254-263. http://dx.doi.org/10.1006/viro.1995.1342.
34. Beattie E, Kauffman EB, Martinez H, Perkus ME, Jacobs BL, Paoletti E, Tartaglia J. 1996. Host-range restriction of vaccinia virus E3Lspecific deletion mutants. Virus Genes 12:89-94. http://dx.doi.org/10.1007/BF00370005.
35. Lee SB, Esteban M. 1994. The interferon-induced double-stranded RNAactivated protein kinase induces apoptosis. Virology 199:491-496. http://dx.doi.org/10.1006/viro.1994.1151.
36. Beattie E, Tartaglia J, Paoletti E. 1991. Vaccinia virus-encoded eIF-2 alpha homolog abrogates the antiviral effect of interferon. Virology 183: 419-422. http://dx.doi.org/10.1016/0042-6822(91)90158-8.
37. Davies MV, Elroy-Stein O, Jagus R, Moss B, Kaufman RJ. 1992. The vaccinia virus K3L gene product potentiates translation by inhibiting double-stranded-RNA-activated protein kinase and phosphorylation of the alpha subunit of eukaryotic initiation factor 2. J. Virol. 66:1943-1950.
38. Mayr A, Stickl H, Müller HK, Danner K, Singer H. 1978. The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism (author's transl). Zentralbl. Bakteriol. B 167: 375-390. (In German.)
39. Gomez CE, Najera JL, Krupa M, Perdiguero B, Esteban M. 2011. MVA and NYVAC as vaccines against emergent infectious diseases and cancer. Curr. Gene Ther. 11:189-217. http://dx.doi.org/10.2174/156652311795684731.
40. Kennedy JS, Greenberg RN. 2009. IMVAMUNE: modified vaccinia Ankara strain as an attenuated smallpox vaccine. Expert Rev. Vaccines 8:13-24. http://dx.doi.org/10.1586/14760584.8.1.13.
41. Suter M, Meisinger-Henschel C, Tzatzaris M, Hülsemann V, Lukassen S, Wulff NH, Hausmann J, Howley P, Chaplin P. 2009. Modified vaccinia Ankara strains with identical coding sequences actually represent complex mixtures of viruses that determine the biological properties of each strain. Vaccine 27:7442-7450. http://dx.doi.org/10.1016/j.vaccine.2009.05.095.
42. Yang YL, Reis LF, Pavlovic J, Aguzzi A, Schäfer R, Kumar A, Williams BR, Aguet M, Weissmann C. 1995. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14:6095-6106.
43. Hochrein H, Schlatter B, O'Keeffe M, Wagner C, Schmitz F, Schiemann M, Bauer S, Suter M, Wagner H. 2004. Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and-independent pathways. Proc. Natl. Acad. Sci. U. S. A. 101:11416-11421. http://dx.doi.org/10.1073/pnas.0403555101.
44. Meisinger-Henschel C, Späth M, Lukassen S, Wolferstätter M, Kachelriess H, Baur K, Dirmeier U, Wagner M, Chaplin P, Suter M, Hausmann J. 2010. Introduction of the six major genomic deletions of modified vaccinia virus Ankara (MVA) into the parental vaccinia virus is not sufficient to reproduce an MVA-like phenotype in cell culture and in mice. J. Virol. 84:9907-9919. http://dx.doi.org/10.1128/JVI.00756-10.
45. Meisinger-Henschel C, Schmidt M, Lukassen S, Linke B, Krause L, Konietzny S, Goesmann A, Howley P, Chaplin P, Suter M, Hausmann J. 2007. Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J. Gen. Virol. 88:3249-3259. http://dx.doi.org/10.1099/vir.0.83156-0.
46. Meylan E, Curran J, Hofmann K, Moradpour D, Binder M, Bartenschlager R, Tschopp J. 2005. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437:1167-1172. http://dx.doi.org/10.1038/nature04193.
47. Schweneker M, Lukassen S, Späth M, Wolferstätter M, Babel E, Brinkmann K, Wielert U, Chaplin P, Suter M, Hausmann J. 2012. The vaccinia virus O1 protein is required for sustained activation of extracellular signal-regulated kinase 1/2 and promotes viral virulence. J. Virol. 86:2323-2336. http://dx.doi.org/10.1128/JVI.06166-11.
48. Posfai G, Plunkett G, III, Feher T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M, Burland V, Harcum SW, Blattner FR. 2006. Emergent properties of reduced-genome Escherichia coli. Science 312:1044-1046. http://dx.doi.org/10.1126/science.1126439.
49. Cottingham M. 2012. Genetic manipulation of poxviruses using bacterial artificial chromosome recombineering, p 37-57. In Isaacs S (ed), Vaccinia virus and poxvirology: methods and protocols, vol 890. Humana Press, Totowa, NJ.
50. Baur K, Brinkmann K, Schweneker M, Pätzold J, Meisinger-Henschel C, Hermann J, Steigerwald R, Chaplin P, Suter M, Hausmann J. 2010. Immediate-early expression of a recombinant antigen by modified vaccinia virus Ankara breaks the immunodominance of strong vectorspecific B8R antigen in acute and memory CD8 T-cell responses. J. Virol. 84:8743-8752. http://dx.doi.org/10.1128/JVI.00604-10.
51. Chakrabarti S, Sisler JR, Moss B. 1997. Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23:1094-1097.
52. Maniatis T, Falvo JV, Kim TH, Kim TK, Lin CH, Parekh BS, Wathelet MG. 1998. Structure and function of the interferon-beta enhanceosome. Cold Spring Harbor Symp. Quant. Biol. 63:609-620. http://dx.doi.org/10.1101/sqb.1998.63.609.
53. Schulz O, Pichlmair A, Rehwinkel J, Rogers NC, Scheuner D, Kato H, Takeuchi O, Akira S, Kaufman RJ, Reis e Sousa C. 2010. Protein kinase R contributes to immunity against specific viruses by regulating interferon mRNA integrity. Cell Host Microbe 7:354-361. http://dx.doi.org/10.1016/j.chom.2010.04.007.
54. Colby C, Jurale C, Kates JR. 1971. Mechanism of synthesis of vaccinia virus double-stranded ribonucleic acid in vivo and in vitro. J. Virol. 7:71-76.
55. Bayliss CD, Condit RC. 1993. Temperature-sensitive mutants in the vaccinia virus A18R gene increase double-stranded RNA synthesis as a result of aberrant viral transcription. Virology 194:254-262. http://dx.doi.org/10.1006/viro.1993.1256.
56. Xiang Y, Simpson DA, Spiegel J, Zhou A, Silverman RH, Condit RC. 1998. The vaccinia virus A18R DNA helicase is a postreplicative negative transcription elongation factor. J. Virol. 72:7012-7023.
57. Arsenio J, Deschambault Y, Cao J. 2008. Antagonizing activity of vaccinia virus E3L against human interferons in Huh7 cells. Virology 377: 124-132. http://dx.doi.org/10.1016/j.virol.2008.04.014.
58. Ludwig H, Suezer Y, Waibler Z, Kalinke U, Schnierle BS, Sutter G. 2006. Double-stranded RNA-binding protein E3 controls translation of viral intermediate RNA, marking an essential step in the life cycle of modified vaccinia virus Ankara. J. Gen. Virol. 87:1145-1155. http://dx.doi.org/10.1099/vir.0.81623-0.
59. Elde NC, Child SJ, Eickbush MT, Kitzman JO, Rogers KS, Shendure J, Geballe AP, Malik HS. 2012. Poxviruses deploy genomic accordions to adapt rapidly against host antiviral defenses. Cell 150:831-841. http://dx.doi.org/10.1016/j.cell.2012.05.049.
60. Backes S, Sperling KM, Zwilling J, Gasteiger G, Ludwig H, Kremmer E, Schwantes A, Staib C, Sutter G. 2010. Viral host-range factor C7 or K1 is essential for modified vaccinia virus Ankara late gene expression in human and murine cells, irrespective of their capacity to inhibit protein kinase R-mediated phosphorylation of eukaryotic translation initiation factor 2alpha. J. Gen. Virol. 91:470-482. http://dx.doi.org/10.1099/vir.0.015347-0.
61. Meng X, Chao J, Xiang Y. 2008. Identification from diverse mammalian poxviruses of host-range regulatory genes functioning equivalently to vaccinia virus C7L. Virology 372:372-383. http://dx.doi.org/10.1016/j.virol.2007.10.023.
62. McAllister CS, Samuel CE. 2009. The RNA-activated protein kinase enhances the induction of interferon-beta and apoptosis mediated by cytoplasmic RNA sensors. J. Biol. Chem. 284:1644-1651. http://dx.doi.org/10.1074/jbc.M807888200.
63. McAllister CS, Taghavi N, Samuel CE. 2012. Protein kinase PKR amplification of interferon beta induction occurs through initiation factor eIF-2alpha-mediated translational control. J. Biol. Chem. 287:36384-36392. http://dx.doi.org/10.1074/jbc.M112.390039.
64. Oie KL, Pickup DJ. 2001. Cowpox virus and other members of the orthopoxvirus genus interfere with the regulation of NF-kappaB activation. Virology 288:175-187. http://dx.doi.org/10.1006/viro.2001.1090.
65. Shisler JL, Jin XL. 2004. The vaccinia virus K1L gene product inhibits host NF-kappaB activation by preventing IkappaBalpha degradation. J. Virol. 78:3553-3560. http://dx.doi.org/10.1128/JVI.78.7.3553-3560.2004.
66. Zhang P, Samuel CE. 2008. Induction of protein kinase PKR-dependent activation of interferon regulatory factor 3 by vaccinia virus occurs through adapter IPS-1 signaling. J. Biol. Chem. 283:34580-34587. http://dx.doi.org/10.1074/jbc.M807029200.
67. Delaloye J, Roger T, Steiner-Tardivel QG, Le RD, Knaup RM, Akira S, Petrilli V, Gomez CE, Perdiguero B, Tschopp J, Pantaleo G, Esteban M, Calandra T. 2009. Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome. PLoS Pathog. 5:e1000480. http://dx.doi.org/10.1371/journal.ppat.1000480.
68. Oganesyan G, Saha SK, Guo B, He JQ, Shahangian A, Zarnegar B, Perry A, Cheng G. 2006. Critical role of TRAF3 in the Toll-like receptordependent and-independent antiviral response. Nature 439:208-211. http://dx.doi.org/10.1038/nature04374.
69. Assarsson E, Greenbaum JA, Sundstrom M, Schaffer L, Hammond JA, Pasquetto V, Oseroff C, Hendrickson RC, Lefkowitz EJ, Tscharke DC, Sidney J, Grey HM, Head SR, Peters B, Sette A. 2008. Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes. Proc. Natl. Acad. Sci. U. S. A. 105:2140-2145. http://dx.doi.org/10.1073/pnas.0711573105.
70. Nemeckova S, Hainz P, Otahal P, Gabriel P, Sroller V, Kutinova L. 2001. Early gene expression of vaccinia virus strains replicating (Praha) and non-replicating (modified vaccinia virus strain Ankara, MVA) in mammalian cells. Acta Virol. 45:243-247.
71. Stewart WE, Scott WD, Sulkin SE. 1969. Relative sensitivities of viruses to different species of interferon. J. Virol. 4:147-153.
72. Thacore HR. 1976. Rescue of vesicular stomatitis virus from homologous and heterologous interferon-induced resistance in human cell cultures by poxviruses. Infect. Immun. 14:311-314.
73. Brandt TA, Jacobs BL. 2001. Both carboxy-and amino-terminal domains of the vaccinia virus interferon resistance gene, E3L, are required for pathogenesis in a mouse model. J. Virol. 75:850-856. http://dx.doi.org/10.1128/JVI.75.2.850-856.2001.
74. Rice AD, Turner PC, Embury JE, Moldawer LL, Baker HV, Moyer RW. 2011. Roles of vaccinia virus genes E3L and K3L and host genes PKR and RNase L during intratracheal infection of C57BL/6 mice. J. Virol. 85:550-567. http://dx.doi.org/10.1128/JVI.00254-10.
75. Chen RA, Ryzhakov G, Cooray S, Randow F, Smith GL. 2008. Inhibition of IkappaB kinase by vaccinia virus virulence factor B14. PLoS Pathog. 4:e22. http://dx.doi.org/10.1371/journal.ppat.0040022.
76. Chen RA, Jacobs N, Smith GL. 2006. Vaccinia virus strain Western Reserve protein B14 is an intracellular virulence factor. J. Gen. Virol. 87: 1451-1458. http://dx.doi.org/10.1099/vir.0.81736-0.