טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentFridman Karen
SubjectUltrastructure and Function of AFF-1 and EFF-1 in
Membrane Remodeling
DepartmentDepartment of Biology
Supervisor Professor Benjamin Podbilewicz
Full Thesis textFull thesis text - English Version


Abstract

Cell fusion is a widespread process in eukaryotic development and necessary for the normal development of the organism. Enveloped viruses take advantage of fusion to enter the host cell. Fusion of biological membranes is mediated by proteins called fusogens. Fusogens are varied in their conformation and mechanism but they exert the same effect - bringing two lipid bi-layers close enough and fusing them.

In C. elegans a family of fusogens has been identified, the Fusion Family (FF). AFF-1 and EFF-1 are essential and sufficient to fuse cells in vivo and in heterologous cells. In order to study the structure and function of the FFs we constructed Vesicular Stomatitis Virus (VSV) pseudotypes displaying FFs (AFF-1 and EFF-1) on the viral envelope. We used SDS-PAGE with either Coomassie or silver staining to analyze the proteomic content of the pseudotypes. The protein gels and mass spectrometry analysis confirmed that AFF-1 and EFF-1 are present in the pseudotype-viruses in place of, and in comparable amounts to the endogenous VSV glycoprotein G (VSVG) fusogen.

Using negative stain electron microscopy (EM) we analyzed the structure of the proteins. We determined that AFF-1 and EFF-1 form distinct complexes that differ from the native VSVG. To verify that the FF proteins are in the viral membrane we performed immuno-gold staining. We found specific immunoreactivity that confirmed that the particles we see are AFF-1 and EFF-1. We found that AFF-1 and EFF-1 affect membranes differently. AFF-1 forms round or bended vesicles and EFF-1 forms long and thin nano-tubes.

Finally we showed that FF proteins in VSV pseudotypes can mediate viral infection of cells expressing FF proteins and that the infection of VSV particles complemented with EFF-1 (VSVΔG-EFF-1) can be partially inhibited with monoclonal antibodies. Furthermore, EFF-1 structure is affected by low pH. EFF-1 is smaller and thinner at pH 5.33. This result suggests that a change in pH could be necessary to induce FF mediated fusion.

Thus, we showed that AFF-1 and EFF-1 are highly expressed on VSV pseudotypes and that they can mediate viral fusion. We also showed that the FF proteins are different from VSVG and that AFF-1 and EFF-1 affect membranes differently. We propose a model for FF activity during the fusion of two membranes.