title: Genetically engineered, biarsenically labeled influenza virus allows visualization of viral NS1 protein in living cells.
abstract: Real-time fluorescence imaging of viral proteins in living cells provides a valuable means to study virus-host interactions. The challenge of generating replication-competent fluorescent influenza A virus is that the segmented genome does not allow fusion of a fluorescent protein gene to any viral gene. Here, we introduced the tetracysteine (TC) biarsenical labeling system into influenza virus in order to fluorescently label viral protein in the virus life cycle. We generated infectious influenza A viruses bearing a small TC tag (CCPGCC) in the loop/linker regions of the NS1 proteins. In the background of A/Puerto Rico/8/34 (H1N1) (PR8) virus, the TC tag can be inserted into NS1 after amino acid 52 (AA52) (PR8-410), AA79 (PR8-412), or AA102 (PR8-413) or the TC tag can be inserted and replace amino acids 79 to 84 (AA79-84) (PR8-411). Although PR8-410, PR8-411, and PR8-412 viruses are attenuated than the wild-type (WT) virus to some extent in multiple-cycle infection, their growth potential is similar to that of the WT virus during a single cycle of infection, and their NS1 subcellular localization and viral protein synthesis rate are quite similar to those of the WT virus. Furthermore, labeling with membrane-permeable biarsenical dye resulted in fluorescent NS1 protein in the context of virus infection. We could exploit this strategy on NS1 protein of A/Texas/36/91 (H1N1) (Tx91) by successfully rescuing a TC-tagged virus, Tx91-445, which carries the TC tag replacement of AA79-84. The infectivity of Tx91-445 virus was similar to that of WT Tx91 during multiple cycles of replication and a single cycle of replication. The NS1 protein derived from Tx91-445 can be fluorescently labeled in living cells. Finally, with biarsenical labeling, the engineered replication-competent virus allowed us to visualize NS1 protein nuclear import in virus-infected cells in real time.
authors.affiliation: Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, Canada S7N 5E3.
authors.forename: Yang
authors.initials: Y
authors.lastname: Li
authors.forename: Xinya
authors.initials: X
authors.lastname: Lu
authors.forename: Junwei
authors.initials: J
authors.lastname: Li
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bérubé
authors.forename: Kerri-Lane
authors.initials: KL
authors.lastname: Giest
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Deborah H
authors.initials: DH
authors.lastname: Anderson
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhou
mesh_terms: D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D005818:Genetic Engineering; D016679:Genome, Viral; D054884:Host-Pathogen Interactions; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D008954:Models, Biological; D013194:Staining and Labeling / Q000379:methods*; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins; D003545:Cysteine
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 19;105(33):11790-5
references.pmid: 18689690
references.citation: Proc Natl Acad Sci U S A. 2000 May 23;97(11):6108-13
references.pmid: 10801978
references.citation: J Virol. 2007 Apr;81(7):3058-67
references.pmid: 17229704
references.citation: J Gen Virol. 2008 Oct;89(Pt 10):2359-2376
references.pmid: 18796704
references.citation: J Am Chem Soc. 2002 May 29;124(21):6063-76
references.pmid: 12022841
references.citation: Cell Host Microbe. 2009 May 8;5(5):439-49
references.pmid: 19454348
references.citation: J Clin Virol. 2008 Dec;43(4):381-90
references.pmid: 18926763
references.citation: Nat Biotechnol. 2005 Oct;23(10):1308-14
references.pmid: 16155565
references.citation: Nat Methods. 2006 Oct;3(10):817-24
references.pmid: 16990814
references.citation: J Virol. 2007 Jan;81(2):514-24
references.pmid: 17079289
references.citation: J Virol. 2009 Mar;83(6):2611-22
references.pmid: 19153240
references.citation: J Virol. 2007 Jul;81(13):7011-21
references.pmid: 17442719
references.citation: Nature. 2008 Aug 14;454(7206):890-3
references.pmid: 18615016
references.citation: RNA. 1995 May;1(3):304-16
references.pmid: 7489502
references.citation: Genes Dev. 1994 Aug 1;8(15):1817-28
references.pmid: 7958859
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):13093-8
references.pmid: 18725644
references.citation: J Gen Virol. 2009 Sep;90(Pt 9):2124-33
references.pmid: 19420157
references.citation: Nature. 2008 Dec 18;456(7224):985-8
references.pmid: 18987632
references.citation: Methods Mol Biol. 2007;356:209-20
references.pmid: 16988405
references.citation: Vaccine. 2002 Aug 19;20(25-26):3165-70
references.pmid: 12163268
references.citation: J Virol. 2008 Jul;82(13):6419-26
references.pmid: 18448539
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):14194-9
references.pmid: 16963558
references.citation: Proc Natl Acad Sci U S A. 2006 May 2;103(18):7100-5
references.pmid: 16627618
references.citation: J Virol. 2010 Feb;84(4):2157-63
references.pmid: 19939917
references.citation: RNA. 2001 Jun;7(6):920-31
references.pmid: 11421366
references.citation: J Gen Virol. 2007 Jan;88(Pt 1):13-18
references.pmid: 17170431
references.citation: Nat Methods. 2008 Jan;5(1):75-85
references.pmid: 18059278
references.citation: Nature. 2004 Jul 8;430(6996):209-13
references.pmid: 15241415
references.citation: Annu Rev Biochem. 1998;67:509-44
references.pmid: 9759496
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9280-5
references.pmid: 12883000
references.citation: J Virol. 1977 Oct;24(1):401-5
references.pmid: 904030
references.citation: J Virol. 2007 Dec;81(23):12730-9
references.pmid: 17881440
references.citation: Science. 1998 Jul 10;281(5374):269-72
references.pmid: 9657724
references.citation: J Virol. 1994 Apr;68(4):2433-41
references.pmid: 8139028
references.citation: Nat Rev Microbiol. 2007 Mar;5(3):197-208
references.pmid: 17304249
references.citation: J Gen Virol. 2007 Mar;88(Pt 3):942-950
references.pmid: 17325368
references.citation: J Virol. 2007 Jun;81(11):5995-6006
references.pmid: 17376915
references.citation: Nat Biotechnol. 2009 Jun;27(6):572-6
references.pmid: 19483680
references.citation: PLoS Pathog. 2008 Mar 07;4(3):e1000015
references.pmid: 18369466
references.citation: Science. 2005 Oct 7;310(5745):77-80
references.pmid: 16210530