Chikungunya virus fusion properties elucidated by single-particle and bulk approaches

Abstract

Chikungunya virus (CHIKV) is a rapidly spreading, enveloped alphavirus causing fever, rash and debilitating polyarthritis. No specific treatment or vaccines are available to treat or prevent infection. For the rational design of vaccines and antiviral drugs, it is imperative to understand the molecular mechanisms involved in CHIKV infection. A critical step in the life cycle of CHIKV is fusion of the viral membrane with a host cell membrane. Here, we elucidate this process using ensemble-averaging liposome-virus fusion studies, in which the fusion behaviour of a large virus population is measured, and a newly developed microscopy-based single-particle assay, in which the fusion kinetics of an individual particle can be visualised. The combination of these approaches allowed us to obtain detailed insight into the kinetics, lipid dependency and pH dependency of hemifusion. We found that CHIKV fusion is strictly dependent on low pH, with a threshold of pH 6.2 and optimal fusion efficiency below pH 5.6. At this pH, CHIKV fuses rapidly with target membranes, with typically half of the fusion occurring within 2 s after acidification. Cholesterol and sphingomyelin in the target membrane were found to strongly enhance the fusion process. By analysing our single-particle data using kinetic models, we were able to deduce that the number of rate-limiting steps occurring before hemifusion equals about three. To explain these data, we propose a mechanistic model in which multiple E1 fusion trimers are involved in initiating the fusion process.

Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells

“…In the mosquito cell line C6/36, however, CHIKV infection was dependent on both Rab5-and Rab7-positive endosomes (32), indicating that fusion might also occur from Rab5/Rab7-positive maturing endosomes and Rab7-positive late endosomes. Furthermore, and in line with the findings for other alphaviruses, low-pH-dependent CHIKV fusion is strongly promoted by target membrane cholesterol and sphingomyelin (33,34).…”

“…Liposomes (large unilamellar vesicles) of 200 nm were prepared by freeze-thaw extrusion as described previously (24,34). Unless otherwise specified, liposomes consisted of phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine (PE) prepared from transphosphatidylation of egg PC, sphingomyelin (SPM) from porcine brain, and cholesterol from ovine wool (all from Avanti Polar Lipids, Alabaster, AL) in a molar ratio of 1:1:1:1.5.…”

“…Unless otherwise specified, liposomes consisted of phosphatidylcholine (PC) from egg yolk, phosphatidylethanolamine (PE) prepared from transphosphatidylation of egg PC, sphingomyelin (SPM) from porcine brain, and cholesterol from ovine wool (all from Avanti Polar Lipids, Alabaster, AL) in a molar ratio of 1:1:1:1.5. Fusion of pyrene-labeled CHIKV with liposomes was monitored in a Fluorolog 3-22 fluorometer (BFi OPTiLAS, Alphen aan den Rijn, The Netherlands) as described previously (24,34,38). Briefly, 4 ϫ 10 10 pyrene-labeled CHIKV particles were mixed with 6 ϫ 10 10 liposomes in a total volume of 665 l HNE buffer at 37°C with continuous stirring.…”

“…Virus for the bulk fusion assay was labeled biosynthetically with pyrene, as described previously (34). Briefly, BHK-21 cells were cultured in the presence of 15 g/ml of 1-pyrenehexadecanoic acid (Invitrogen) 48 h prior to infection.…”

“…Plaques were counted 2 days after infection. Furthermore, reverse transcriptase quantitative PCR (RT-qPCR) was used to determine the number of GCPs, as described previously (34).…”