title: The pre-transmembrane domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein is critical for membrane fusion and virus infectivity.
abstract: The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity.
authors.affiliation: Boyce Thompson Institute at Cornell University, Tower Road, Ithaca, NY 14853-1801, USA.
authors.forename: Zhaofei
authors.initials: Z
authors.lastname: Li
authors.forename: Gary W
authors.initials: GW
authors.lastname: Blissard
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000378:metabolism; D002460:Cell Line; D004252:DNA Mutational Analysis; D007930:Leucine / Q000235:genetics; D008561:Membrane Fusion / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000378:metabolism; D053586:Virus Internalization
chemical_list: D014760:Viral Fusion Proteins; D007930:Leucine
references.citation: Curr Opin Struct Biol. 2009 Apr;19(2):189-96
references.pmid: 19356922
references.citation: Nature. 1970 May 2;226(5244):466-7
references.pmid: 16057320
references.citation: FEBS Lett. 1992 Jun 15;304(2-3):221-4
references.pmid: 1618326
references.citation: Virology. 1999 Feb 15;254(2):297-314
references.pmid: 9986796
references.citation: Biochemistry. 2001 Aug 14;40(32):9570-8
references.pmid: 11583156
references.citation: Biochim Biophys Acta. 2008 Jul-Aug;1778(7-8):1624-39
references.pmid: 18222166
references.citation: J Virol. 2000 Sep;74(17):8038-47
references.pmid: 10933713
references.citation: Virology. 2004 Apr 10;321(2):274-86
references.pmid: 15051387
references.citation: J Virol. 1991 Nov;65(11):5820-7
references.pmid: 1920618
references.citation: J Cell Biol. 1996 Dec;135(6 Pt 2):1831-9
references.pmid: 8991094
references.citation: J Virol. 2008 Apr;82(7):3329-41
references.pmid: 18216100
references.citation: J Virol. 1992 Nov;66(11):6829-35
references.pmid: 1404622
references.citation: FEBS Lett. 2003 Jan 2;533(1-3):47-53
references.pmid: 12505157
references.citation: J Virol. 2008 Feb;82(3):1368-77
references.pmid: 17989172
references.citation: Biochemistry. 2005 Jan 25;44(3):947-58
references.pmid: 15654751
references.citation: J Virol. 2002 Dec;76(23):12300-11
references.pmid: 12414970
references.citation: Arch Virol. 2006 Jul;151(7):1257-66
references.pmid: 16648963
references.citation: Nat Struct Mol Biol. 2008 Oct;15(10):1024-30
references.pmid: 18776902
references.citation: J Virol. 2008 Mar;82(6):2883-94
references.pmid: 18199653
references.citation: J Virol. 2009 May;83(9):4447-61
references.pmid: 19244324
references.citation: J Virol. 1996 Jul;70(7):4607-16
references.pmid: 8676487
references.citation: J Virol. 1993 Aug;67(8):4566-79
references.pmid: 8392598
references.citation: Virology. 1999 Jan 5;253(1):65-76
references.pmid: 9887319
references.citation: Curr Top Microbiol Immunol. 1986;131:103-18
references.pmid: 3545692
references.citation: Virus Res. 2006 Sep;120(1-2):146-55
references.pmid: 16616792
references.citation: J Virol. 2000 Mar;74(5):2239-46
references.pmid: 10666254
references.citation: J Virol. 2006 Sep;80(17):8830-3
references.pmid: 16912330
references.citation: J Virol. 1999 Mar;73(3):2469-80
references.pmid: 9971832
references.citation: Nat Struct Mol Biol. 2008 Jul;15(7):690-8
references.pmid: 18596815
references.citation: J Virol. 2003 Dec;77(23):12807-18
references.pmid: 14610202
references.citation: Virology. 2001 Feb 1;280(1):52-61
references.pmid: 11162818
references.citation: J Virol. 2002 Jun;76(11):5729-36
references.pmid: 11992001
references.citation: Proc Natl Acad Sci U S A. 1991 May 1;88(9):3623-7
references.pmid: 2023911
references.citation: Nature. 1989 Nov 30;342(6249):555-8
references.pmid: 2586627
references.citation: J Virol. 2008 May;82(9):4449-60
references.pmid: 18287233
references.citation: J Virol. 2008 Jun;82(11):5118-26
references.pmid: 18353966
references.citation: Virology. 1999 Jun 5;258(2):455-68
references.pmid: 10366584
references.citation: Virology. 2006 Sep 1;352(2):427-37
references.pmid: 16777166
references.citation: Virology. 1995 Jun 1;209(2):592-603
references.pmid: 7778291