All enveloped viruses initiate infection by fusing to cell membranes, creating a fusion pore, and depositing their genomes into cells after the pore enlarges. Two classes of viral fusion proteins have been identified based on similarity in crystal structures. This study encompasses viral proteins of both classes. All class I viral proteins contain alpha-helical six-helix bundles in their final, post fusion structure;these bundles are critical for fusion and extremely thermostable. Viruses containing class I proteins include human immunodeficiency virus (HIV), influenza, Ebola, and leukemia viruses. Class II viral fusion proteins are rich in beta-strands and do not form bundles. Viruses that contain these proteins include Dengue, West Nile, almost all encephalitis viruses, and Semliki Forest Virus (SFV). The study of class I will focus on the mechanism of HIV fusion;HIV currently infects about 40 million people worldwide. The study of class II will investigate the triggering mechanism of SFV;its fusion protein is closely related to that of Dengue, which infects up to 100 million Deople per year worldwide. Determining how HIV and SFV fuse to target cells to initiate infection is important both for understanding the ubiquitous and fundamental cellular process of membrane fusion, and for controlling and preventing important diseases. In the case of HIV, factors leading to bundle formation will investigated in a cell-cell fusion system. Intermediates of fusion will be captured, and assays of the spread of fluorescent dyes and electrical measurements of fusion pores will be employed to follow the steps of fusion and how they can be disrupted. Bundle formation is triggered by the binding of Env to chemokine receptors;trigger strength will be altered through control of chemokine receptor affinity for Env, and the consequences to rate of bundle formation and pore growth will be determined. Mutation will reduce the stability of the HIV Env bundle;the reason infection requires this stability will be determined. The long cytoplasmic tail of HIV Env will be mutated or eliminated to determine whether configurational freedom of the membrane-spanning domain strongly controls the pore enlargement necessary for viral infection. The hypothesis that external calcium is required for HIV fusion because it bridges phosphatidylserine (PS) in the viral envelope and target membrane will be tested by fusing pseudovirus with varied PS levels to cells. For all class II viral fusion proteins, low pH is a necessary trigger for fusion. In the case of SFV, it has recently been found that low pH alone is not sufficient;a negative trans-membrane potential across the target membrane is also essential for fusion. The step that requires voltage as a second trigger will be isolated and identified for SFV. A histidine residue conserved among all class II fusion proteins may be or part of the voltage sensor;it will be mutated and its consequences for voltage dependence will be determined. Because structures are similar for all class II viral fusion proteins, the SFV studies can be extended to other class II proteins as new experimental technologies develop.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027367-29
Application #
7662355
Study Section
Biophysics of Synapses, Channels, and Transporters Study Section (BSCT)
Program Officer
Chin, Jean
Project Start
1980-04-01
Project End
2011-07-31
Budget Start
2009-08-01
Budget End
2011-07-31
Support Year
29
Fiscal Year
2009
Total Cost
$473,580
Indirect Cost
Name
Rush University Medical Center
Department
Physiology
Type
Schools of Medicine
DUNS #
068610245
City
Chicago
State
IL
Country
United States
Zip Code
60612
Leung, Michael Y K; Cohen, Fredric S (2011) Increasing hydrophobicity of residues in an anti-HIV-1 Env peptide synergistically improves potency. Biophys J 100:1960-8
Markosyan, Ruben M; Cohen, Fredric S (2010) Negative potentials across biological membranes promote fusion by class II and class III viral proteins. Mol Biol Cell 21:2001-12
Markosyan, Ruben M; Leung, Michael Y; Cohen, Fredric S (2009) The six-helix bundle of human immunodeficiency virus Env controls pore formation and enlargement and is initiated at residues proximal to the hairpin turn. J Virol 83:10048-57
Markosyan, Ruben M; Kielian, Margaret; Cohen, Fredric S (2007) Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell. J Virol 81:11218-25
Abrahamyan, Levon G; Mkrtchyan, Samvel R; Binley, James et al. (2005) The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle. J Virol 79:106-15
Mkrtchyan, Samvel R; Markosyan, Ruben M; Eadon, Michael T et al. (2005) Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion. J Virol 79:11161-9
Markosyan, Ruben M; Cohen, Fredric S; Melikyan, Grigory B (2005) Time-resolved imaging of HIV-1 Env-mediated lipid and content mixing between a single virion and cell membrane. Mol Biol Cell 16:5502-13
Melikyan, G B; Barnard, R J O; Markosyan, R M et al. (2004) Low pH is required for avian sarcoma and leukosis virus Env-induced hemifusion and fusion pore formation but not for pore growth. J Virol 78:3753-62
Markosyan, R M; Bates, P; Cohen, F S et al. (2004) A study of low pH-induced refolding of Env of avian sarcoma and leukosis virus into a six-helix bundle. Biophys J 87:3291-8
Cohen, F S; Melikyan, G B (2004) The energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement. J Membr Biol 199:1-14

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