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.

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

UNLABELLED: 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 previously using inhibitory compounds. There has been some debate on the mechanism by which CHIKV enters the cell: several studies suggest that CHIKV enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. Here we applied live-cell microscopy and monitored the cell entry behavior of single CHIKV particles in living cells transfected with fluorescent marker proteins. This approach allowed us to obtain detailed insight into the dynamic events that occur during CHIKV entry. We observed that almost all particles fused within 20 min after addition to the cells. Of the particles that fused, the vast majority first colocalized with clathrin. The average time from initial colocalization with clathrin to the moment of membrane fusion was 1.7 min, highlighting the rapidity of the cell entry process of CHIKV. Furthermore, these results show that the virus spends a relatively long time searching for a receptor. Membrane fusion was observed predominantly from within Rab5-positive endosomes and often occurred within 40 s after delivery to endosomes. Furthermore, we confirmed that a valine at position 226 of the E1 protein enhances the cholesterol-dependent membrane fusion properties of CHIKV. To conclude, our work confirms that CHIKV enters cells via clathrin-mediated endocytosis and shows that fusion occurs from within acidic early endosomes. IMPORTANCE: Since its reemergence in 2004, chikungunya virus (CHIKV) has spread rapidly around the world, leading to millions of infections. CHIKV often causes chikungunya fever, a self-limiting febrile illness with severe arthralgia. Currently, no vaccine or specific antiviral treatment against CHIKV is available. A potential antiviral strategy is to interfere with the cell entry process of the virus. However, conflicting results with regard to the cell entry pathway used by CHIKV have been published. Here we applied a novel technology to visualize the entry behavior 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 from within acidic early endosomes is observed. Furthermore, the membrane fusion capacity of CHIKV is strongly promoted by cholesterol in the target membrane. Taking these findings together, this study provides detailed insight into the cell entry process of CHIKV.

Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

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.

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

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.