A MODEL SYSTEM FOR PHYSIOLOGICAL EXOCYTOSIS

Summary

Principal Investigator: FREDRIC COHEN
Affiliation: Rush University Medical Center
Country: USA
Abstract: 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.
Funding Period: 1980-04-01 - 2010-07-31
more information: NIH RePORT

Top Publications

  1. ncbi Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion
    Samvel R Mkrtchyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA
    J Virol 79:11161-9. 2005
  2. ncbi The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle
    Levon G Abrahamyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612, USA
    J Virol 79:106-15. 2005
  3. ncbi Time-resolved imaging of HIV-1 Env-mediated lipid and content mixing between a single virion and cell membrane
    Ruben M Markosyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612
    Mol Biol Cell 16:5502-13. 2005
  4. ncbi Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell
    Ruben M Markosyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1653 W Congress Pkwy, Chicago, IL 60612, USA
    J Virol 81:11218-25. 2007
  5. ncbi The six-helix bundle of human immunodeficiency virus Env controls pore formation and enlargement and is initiated at residues proximal to the hairpin turn
    Ruben M Markosyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA
    J Virol 83:10048-57. 2009

Detail Information

Publications5

  1. ncbi Ternary complex formation of human immunodeficiency virus type 1 Env, CD4, and chemokine receptor captured as an intermediate of membrane fusion
    Samvel R Mkrtchyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA
    J Virol 79:11161-9. 2005
    ..Therefore the pronounced kinetic delay in HIV Env-induced fusion is caused predominantly by the time needed for ternary complexes to form...
  2. ncbi The cytoplasmic tail slows the folding of human immunodeficiency virus type 1 Env from a late prebundle configuration into the six-helix bundle
    Levon G Abrahamyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612, USA
    J Virol 79:106-15. 2005
    ..Because of this facilitation of maximal exposure and hindrance of bundle formation, the coiled coil is optimally exposed for a longer time for WT than for DeltaCT. This accounts for the greater peptide inhibition of WT-induced fusion...
  3. ncbi Time-resolved imaging of HIV-1 Env-mediated lipid and content mixing between a single virion and cell membrane
    Ruben M Markosyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612
    Mol Biol Cell 16:5502-13. 2005
    ..NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge...
  4. ncbi Fusion induced by a class II viral fusion protein, semliki forest virus E1, is dependent on the voltage of the target cell
    Ruben M Markosyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1653 W Congress Pkwy, Chicago, IL 60612, USA
    J Virol 81:11218-25. 2007
    ..It is proposed that the trans-negative voltage across the endosomal membrane facilitates fusion after low-pH-induced conformational changes of SFV E1 have occurred...
  5. ncbi The six-helix bundle of human immunodeficiency virus Env controls pore formation and enlargement and is initiated at residues proximal to the hairpin turn
    Ruben M Markosyan
    Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL 60612, USA
    J Virol 83:10048-57. 2009
    ..This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth...