Cooperative Chikungunya Virus Membrane Fusion and Its Substoichiometric Inhibition by CHK-152 Antibody

Chikungunya virus (CHIKV) presents a major burden on healthcare systems worldwide, but specific treatment remains unavailable. Attachment and fusion of CHIKV to the host cell membrane is mediated by the E1/E2 protein spikes. We used an in vitro single-particle fusion assay to study the effect of the potent, neutralizing antibody CHK-152 on CHIKV binding and fusion. We find that CHK-152 shields the virions, inhibiting interaction with the target membrane and inhibiting fusion. The analysis of the ratio of bound antibodies to epitopes implied that CHIKV fusion is a highly cooperative process. Further, dissociation of the antibody at lower pH results in a finely balanced kinetic competition between inhibition and fusion, suggesting a window of opportunity for the spike proteins to act and mediate fusion, even in the presence of the antibody

Early Events in Chikungunya Virus Infection-From Virus Cell Binding to Membrane Fusion

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research

Northeastern environmental science

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 El fusion trimers are involved in initiating the fusion process

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

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in Africa, Asia and the Americas. The cell entry pathway hijacked by CHIKV to infect a cell has been studied before using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell, as several studies suggest that it enters via clathrin-mediated endocytosis, while some other studies show that CHIKV enters cells independently of clathrin. Here, we applied live-cell microscopy and monitored the cell entry behaviour of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain a detailed insight in the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 minutes post-addition to the cells. The vast majority of particles that fused first colocalized with clathrin. The time from initial colocalization with clathrin till the moment of membrane fusion was on average 1.7 minutes, highlighting the fast nature of the cell entry process of CHIKV. Furthermore, these results also show that the virus spends a profound time searching for a receptor. Membrane fusion was predominantly observed from within Rab5-positive endosomes and often occurred within 40 seconds post-delivery to endosomes. Furthermore, we confirmed that a valine at position E1-226 enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV cell entry occurs via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes. IMPORTANCE: Since its re-emergence in 2004 chikungunya (CHIKV) has rapidly spread around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, there is no vaccine or specific antiviral treatment available against CHIKV. A potential antiviral strategy is to interfere with the cell entry process of the virus. However, contradicting results were published with regard to the cell entry pathway used by CHIKV. Here, we applied a novel technology to visualize the entry behaviour of single CHIKV particles in living cells. Our results show that CHIKV cell entry is extremely rapid and occurs via clathrin-mediated endocytosis. Membrane fusion is seen from within acidic early endosomes. Furthermore, the membrane fusion capacity of CHIKV is strongly promoted by cholesterol in the target membrane. Taken together, this study provides an exquisite insight in the cell entry process of CHIKV