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.