Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.
Dengue virus (DENV) is the most common arthropod-borne infection worldwide with 50–100 million cases annually. Despite its high clinical impact, little is known about the infectious cell entry pathway of the virus. Previous studies have shown conflicting evidence about whether the virus fuses directly with the cell plasma membrane or enters cells by receptor-mediated endocytosis. In this manuscript, we dissect the cell entry pathway of DENV by tracking single fluorescently labeled DENV particles in living cells expressing various fluorescent cellular markers, using real-time multi-color fluorescence microscopy. We show that DENV particles are delivered to pre-existing clathrin-coated pits by diffusion along the cell surface. Following clathrin-mediated uptake, the majority of DENV particles are transported to early endosomes, which mature into late endosomes, where membrane fusion occurs. This is the first study that describes the cell entry process of DENV at the single particle level and therefore provides unique mechanistic and kinetic insights into the route of entry, endocytic trafficking behavior, and membrane fusion properties of individual DENV particles in living cells. This paper opens new avenues in flavivirus biology and will lead toward a better understanding of the critical determinants in DENV infection.