Understanding the impacts of IFITMs on lipid order and viral fusion Öffentlichkeit
Abbott, Elizabeth (Spring 2024)
Abstract
Interferon-induced transmembrane proteins (IFITMs) are host restriction factors which exhibit antiviral activity against a broad range of enveloped viruses. Specifically, these small trans-membrane proteins modulate the physical properties of the host cell membrane at the virus-cell fusion site in ways which stabilize the virus-host hemifusion diaphragm and inhibit the transition from this intermediate structure to a productive fusion pore. Despite significant research in this area, the exact mechanism of IFITM-mediated restriction remains unclear. To delineate the mechanism of virus restriction, we performed several biochemical and biophysical experiments with an emphasis on understanding the exact effects of IFITM incorporation on the lipid order of the host cell membrane. Here, we show that IFITM3 overexpression generally decreases lipid order in giant plasma membrane-derived vesicles (GPMVs), while IFITM1 slightly increases lipid order. Functional experiments involving virus-like particles (VLPs) and pseudoviruses show that the glycoproteins of several arenaviruses, which are known to be resistant to restriction by IFITMs, show robust fusion activity even in the presence of IFITMs. In contrast, the fusion of Vesicular Stomatitis Virus (VSV) is restricted. GPMVs derived from control cells and IFITM1-expressing cells show comparable levels of pseudoviral binding, reinforcing published literature that IFITMs do not block binding but rather specifically abrogate fusion. We also show proof of concept results demonstrating the use of GPMVs to form supported lipid bilayers (SLBs) compatible with imaging of single viral particle binding. Finally, we propose a microfluidics-based single-virus fusion assay which would allow us to probe the kinetics of fusion, with a focus on understanding how IFITM incorporation in the target membrane can affect the kinetics of viral membrane fusion.
Table of Contents
1. Introduction 1
1.1 Interferon-Induced Transmembrane Proteins 1
1.2 The viral life cycle & IFITM restriction mechanisms 1
1.3 Hemifusion stabilization 2
1.4 IFITMs and lipid order 3
1.5 IFITM resistance & Arenaviruses 4
1.5 Study Aims 4
2. Methods 5
2.1 Cell line maintenance and transfection 5
2.2 VLP and pseudovirus production and purification 6
2.3 GPMV production and SLB formation and characterization 7
2.4 BlaM virus-cell fusion assay 8
2.5 Viral infectivity assay 8
2.6 Lipid order measurements 9
2.7 Saponin lysis experiments in a microfluidic flow chamber 10
3. Results 10
3.1 IFITM3 overexpression significantly reduces lipid order of plasma membrane-derived vesicles measured with Nile Red 10
3.2 IFITM3 restricts fusion of VSV, but not LASV 14
3.3 GPMVs can be successfully labeled using biotin or DiD 17
3.4 Biotin-labeled GPMVs can produce a fluid SLB on a glass coverslip 19
3.5 VLPs bind to a supported lipid bilayer derived from GPMVs 19
3.6 Infectious IAV particles demonstrate hemifusion with GPMVs 22
3.7 Pseudoviruses incorporating mCherry-YFP-Vpr dual-labeled core as a reporter of full fusion events 24
4. Discussion 28
5. Acknowledgements 32
6. References 33
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