title: Reduction of virion-associated σ1 fibers on oncolytic reovirus variants promotes adaptation toward tumorigenic cells.
abstract: Wild-type mammalian orthoreovirus serotype 3 Dearing (T3wt) is nonpathogenic in humans but preferentially infects and kills cancer cells in culture and demonstrates promising antitumor activity in vivo. Using forward genetics, we previously isolated two variants of reovirus, T3v1 and T3v2, with increased infectivity toward a panel of cancer cell lines and improved in vivo oncolysis in a murine melanoma model relative to that of T3wt. Our current study explored how mutations in T3v1 and T3v2 promote infectivity. Reovirions contain trimers of σ1, the reovirus cell attachment protein, at icosahedral capsid vertices. Quantitative Western blot analysis showed that purified T3v1 and T3v2 virions had ∼ 2- and 4-fold-lower levels of σ1 fiber than did T3wt virions. Importantly, using RNA interference to reduce σ1 levels during T3wt production, we were able to generate wild-type reovirus with reduced levels of σ1 per virion. As σ1 levels were reduced, virion infectivity increased by 2- to 5-fold per cell-bound particle, demonstrating a causal relationship between virion σ1 levels and the infectivity of incoming virions. During infection of tumorigenic L929 cells, T3wt, T3v1, and T3v2 uncoated the outer capsid proteins σ3 and μ1C at similar rates. However, having started with fewer σ1 molecules, a complete loss of σ1 was achieved sooner for T3v1 and T3v2. Distinct from intracellular uncoating, chymotrypsin digestion, as a mimic of natural enteric infection, resulted in more rapid σ3 and μ1C removal, unique disassembly intermediates, and a rapid loss of infectivity for T3v1 and T3v2 compared to T3wt. Optimal infectivity toward natural versus therapeutic niches may therefore require distinct reovirus structures and σ1 levels.
  
  
  OBJECTIVE
  Wild-type reovirus is currently in clinical trials as a potential cancer therapy. Our molecular studies on variants of reovirus with enhanced oncolytic activity in vitro and in vivo now show that distinct reovirus structures promote adaptation toward cancer cells and away from conditions that mimic natural routes of infection. Specifically, we found that reovirus particles with fewer molecules of the cell attachment protein σ1 became more infectious toward transformed cells. Reduced σ1 levels conferred a benefit to incoming particles only, resulting in an earlier depletion of σ1 and a higher probability of establishing productive infection. Conversely, reovirus variants with fewer σ1 molecules showed reduced stability and infectivity and distinct disassembly when exposed to conditions that mimic natural intestinal proteolysis. These findings support a model where the mode of infection dictates the precise optimum of reovirus structure and provide a molecular rationale for considering alternative reovirus structures during oncolytic therapy.
authors: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors: Adil
authors: A
authors: Mohamed
authors: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors: Carmit
authors: C
authors: Teicher
authors: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
authors: Sarah
authors: S
authors: Haefliger
authors: Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada Li Ka-Shing Institute of Virology, University of Alberta, Edmonton Alberta, Canada shmulevi@ualberta.ca.
authors: Maya
authors: M
authors: Shmulevitz
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002918:Chymotrypsin; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D012089:Mammalian orthoreovirus 3 / Q000235:genetics* / Q000472:pathogenicity; D051379:Mice; D009154:Mutation / Q000235:genetics; D050504:Oncolytic Viruses / Q000235:genetics* / Q000472:pathogenicity; D034622:RNA Interference; D014771:Virion / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D036022:Capsid Proteins; C031728:sigma 1 protein, reovirus; D002918:Chymotrypsin
references: J Biol Chem. 2004 Mar 5;279(10):8547-57
references: 14670972
references: Virology. 1969 Dec;39(4):791-810
references: 4311639
references: J Virol. 1970 Jul;6(1):1-11
references: 5529847
references: J Virol. 1985 Feb;53(2):391-8
references: 2982027
references: J Virol. 2005 Dec;79(23):14981-5
references: 16282499
references: Mol Ther. 2007 Aug;15(8):1522-30
references: 17457318
references: Mol Ther. 2007 Aug;15(8):1512-21
references: 17519890
references: Mol Ther. 2008 Mar;16(3):627-32
references: 18253152
references: J Struct Biol. 2008 May;162(2):277-89
references: 18321727
references: Gene Ther. 2008 Jun;15(12):911-20
references: 18323793
references: Gene Ther. 2008 Sep;15(18):1257-70
references: 18401435
references: J Virol. 1988 Jan;62(1):246-56
references: 3275434
references: Virology. 1988 Dec;167(2):601-12
references: 3201754
references: J Virol. 1989 Mar;63(3):1188-93
references: 2915380
references: Virology. 1989 May;170(1):62-70
references: 2470196
references: J Virol. 1989 Nov;63(11):4676-81
references: 2677401
references: J Virol. 1990 Apr;64(4):1830-3
references: 2157065
references: J Virol. 1990 Jun;64(6):2976-89
references: 2335823
references: Virology. 1990 Nov;179(1):95-103
references: 2219743
references: Virology. 1991 May;182(1):336-45
references: 2024469
references: Virology. 1991 Jun;182(2):810-9
references: 2024499
references: Microb Pathog. 1992 May;12(5):351-65
references: 1501574
references: J Cell Biol. 1993 Sep;122(5):1023-41
references: 8394844
references: Virology. 1994 Jun;201(2):303-11
references: 8184540
references: Virology. 1994 Aug 15;203(1):149-52
references: 8030270
references: J Virol. 1995 Aug;69(8):5057-67
references: 7609075
references: J Virol. 1995 Nov;69(11):6972-9
references: 7474116
references: J Virol. 1997 Mar;71(3):1834-41
references: 9032313
references: Virology. 1998 Mar 30;243(1):218-28
references: 9527931
references: EMBO J. 1998 Jun 15;17(12):3351-62
references: 9628872
references: J Virol. 1998 Oct;72(10):8205-13
references: 9733863
references: Science. 1998 Nov 13;282(5392):1332-4
references: 9812900
references: J Virol. 1999 May;73(5):3941-50
references: 10196289
references: J Am Med Assoc. 1958 May 10;167(2):147-56
references: 13538681
references: Science. 1959 Nov 20;130(3386):1387-9
references: 14440555
references: J Bacteriol. 1965 Mar;89:597-602
references: 14273633
references: Auris Nasus Larynx. 2004 Dec;31(4):407-12
references: 15571915
references: J Mol Biol. 2005 Jan 21;345(3):461-74
references: 15581891
references: J Infect Dis. 2005 Apr 15;191(8):1221-4
references: 15776366
references: J Gen Virol. 2005 May;86(Pt 5):1489-97
references: 15831962
references: Clin Cancer Res. 2008 Nov 1;14(21):7127-37
references: 18981012
references: Cancer Gene Ther. 2010 Jan;17(1):69-70
references: 19590528
references: Cell. 2010 Apr 30;141(3):472-82
references: 20398923
references: Nature. 2000 Apr 27;404(6781):960-7
references: 10801118
references: J Virol. 2000 Sep;74(18):8472-9
references: 10954547
references: J Virol Methods. 2000 Oct;90(1):59-67
references: 11011081
references: J Biol Chem. 2001 Jan 19;276(3):2200-11
references: 11054410
references: Cell. 2001 Feb 9;104(3):441-51
references: 11239401
references: J Virol. 2001 Jun;75(11):5335-42
references: 11333914
references: Cancer Res. 2002 Mar 15;62(6):1696-701
references: 11912142
references: Hum Gene Ther. 2002 Mar 20;13(5):641-52
references: 11916487
references: J Biol Chem. 2002 Jul 5;277(27):24609-17
references: 11986312
references: Cell. 2002 Nov 27;111(5):733-45
references: 12464184
references: Clin Cancer Res. 2003 Mar;9(3):1218-23
references: 12631628
references: Clin Cancer Res. 2010 Jun 1;16(11):3067-77
references: 20484020
references: Cancer Res. 2010 Jun 15;70(12):4912-21
references: 20501842
references: Invest New Drugs. 2010 Oct;28(5):641-9
references: 19572105
references: Curr Top Microbiol Immunol. 2010;343:91-119
references: 20397070
references: Clin Cancer Res. 2010 Nov 15;16(22):5564-72
references: 20926400
references: J Virol. 2010 Dec;84(24):12723-32
references: 20943982
references: Br J Cancer. 2011 Jan 18;104(2):290-9
references: 21179029
references: Clin Cancer Res. 2011 Feb 1;17(3):581-8
references: 21106728
references: J Biol Chem. 2012 Mar 9;287(11):8029-38
references: 22253447
references: Clin Cancer Res. 2012 Apr 1;18(7):2080-9
references: 22316603
references: J Virol. 2012 Jul;86(13):7403-13
references: 22532697
references: Adv Exp Med Biol. 2013;790:42-71
references: 23884585
references: J Virol. 2013 Aug;87(16):9279-89
references: 23760238
references: J Virol. 2013 Dec;87(24):13532-42
references: 24089575
references: J Virol. 2003 Jul;77(14):7964-77
references: 12829836
references: Nat Struct Biol. 2003 Dec;10(12):1011-8
references: 14608373
references: J Virol. 2003 Dec;77(24):13361-75
references: 14645591