Cholesterol-Dependent Membrane Fluidity in Sindbis Virus Governs Viral Particle Stability and Infectivity
- 1Department of Biology
- 2Indiana University
- 3Department of Chemistry
Abstract
Cholesterol is a key biomolecule that regulates lipid bilayer fluidity, and cholesterol-rich domains in host cells and virions are critical for virion entry and egress. Here, using Alphavirus Sindbis virus (SINV) as a model, we combine new experimental methods and single-particle biological characterization to revisit how envelope cholesterol affects particle stability and the biological consequences for infectivity. To this end, we generated two SINV populations with a 3-fold difference in envelope cholesterol levels (SINV LC and SINV HC ) to assess how cholesterol affects SINV stability and infectivity. Transmission electron microscopy and dynamic light scattering showed minimal differences in morphology and size, indicating that cholesterol minimally alters virion structure. Electrochemical measurements between SINV LC and SINV HC particles revealed similar Zeta Potentials (ζ-potential ~ -31 mV), which implies that the surface distribution of the E1-E2 glycoprotein heterotrimers within the envelope is cholesterol-independent. Titer measurements showed that SINV HC retains nearly an order of magnitude more infectious particles than SINV LC when pre-exposed to 30 minutes of thermal stress, corresponding to a 1.25-fold faster decay rate for the low-cholesterol virions, with SINV LC and SINV HC showing values of 0.34/min and 0.27/min, respectively. Single-particle fluorescence lifetime imaging microscopy analysis using the fluidity-sensitive dye DI-8-ANEPPS revealed a statistically significant increase (≥ 0.2 ns; p < 0.0001) in photon arrival times in the SINV HC population, indicating that elevated cholesterol enhances membrane rigidity. Because virion size and ζ-potential remained unchanged while fluorescence lifetimes increased with cholesterol level, these findings indicate that membrane fluidity is the primary parameter governing viral stability and subsequent infectivity.
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BibTeX
@misc{richardson2026cholesteroldependent,
title = {{Cholesterol-Dependent Membrane Fluidity in Sindbis Virus Governs Viral Particle Stability and Infectivity}},
author = {Ciara N. Richardson and Sarah J. Cox-Vázquez and Celeste Carlos and Barry Stein and Suchetana Mukhopadhyay and Ricardo Javier Vázquez},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15006138/v2},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15006138/v2},
note = {CC BY},
}
RIS
TY - UNPB
TI - Cholesterol-Dependent Membrane Fluidity in Sindbis Virus Governs Viral Particle Stability and Infectivity
AU - Ciara N. Richardson
AU - Sarah J. Cox-Vázquez
AU - Celeste Carlos
AU - Barry Stein
AU - Suchetana Mukhopadhyay
AU - Ricardo Javier Vázquez
PY - 2026
DA - 2026/10/21
PB - ChemRxiv
DO - 10.26434/chemrxiv.15006138/v2
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15006138/v2
AB - Cholesterol is a key biomolecule that regulates lipid bilayer fluidity, and cholesterol-rich domains in host cells and virions are critical for virion entry and egress. Here, using Alphavirus Sindbis virus (SINV) as a model, we combine new experimental methods and single-particle biological characterization to revisit how envelope cholesterol affects particle stability and the biological consequences for infectivity. To this end, we generated two SINV populations with a 3-fold difference in envelope cholesterol levels (SINV LC and SINV HC ) to assess how cholesterol affects SINV stability and infectivity. Transmission electron microscopy and dynamic light scattering showed minimal differences in morphology and size, indicating that cholesterol minimally alters virion structure. Electrochemical measurements between SINV LC and SINV HC particles revealed similar Zeta Potentials (ζ-potential ~ -31 mV), which implies that the surface distribution of the E1-E2 glycoprotein heterotrimers within the envelope is cholesterol-independent. Titer measurements showed that SINV HC retains nearly an order of magnitude more infectious particles than SINV LC when pre-exposed to 30 minutes of thermal stress, corresponding to a 1.25-fold faster decay rate for the low-cholesterol virions, with SINV LC and SINV HC showing values of 0.34/min and 0.27/min, respectively. Single-particle fluorescence lifetime imaging microscopy analysis using the fluidity-sensitive dye DI-8-ANEPPS revealed a statistically significant increase (≥ 0.2 ns; p < 0.0001) in photon arrival times in the SINV HC population, indicating that elevated cholesterol enhances membrane rigidity. Because virion size and ζ-potential remained unchanged while fluorescence lifetimes increased with cholesterol level, these findings indicate that membrane fluidity is the primary parameter governing viral stability and subsequent infectivity.
ER -