Assembly of endocytic machinery around individual influenza viruses during viral entry

Abstract

Most viruses enter cells via receptor-mediated endocytosis. However, the entry mechanisms used by many of them remain unclear. How viruses are targeted to cellular endocytic machinery is also largely unknown. We have studied the entry mechanisms of influenza viruses by tracking the interaction of single viruses with cellular endocytic structures in real time using fluorescence microscopy. Our results show that influenza can exploit clathrin-mediated and clathrin-and caveolin-independent endocytic pathways in parallel, both pathways leading to viral fusion with similar efficiency. Remarkably, viruses taking the clathrin-mediated pathway enter cells via the de novo formation of clathrin-coated pits (CCPs) at viral binding sites. CCP formation at these sites is much faster than elsewhere on the cell surface, suggesting a virus-induced CCP formation mechanism that may be commonly exploited by many other types of viruses.

Internalization of Large Double-Membrane Intercellular Vesicles by a Clathrin-dependent Endocytic Process

“…This protein forms a typical curved lattice around endocytic vesicles that are internalized at the plasma membrane (PM). In addition, the involvement of clathrin in several uncharacteristic endocytic processes has been reported, including the internalization of viruses, pathogenic bacteria, and large latex beads (Aggeler and Werb, 1982;Ehrlich et al, 2004;Rust et al, 2004;Veiga and Cossart, 2005). Here, we describe another function of the clathrin-dependent endocytic machinery that results in the internalization of large, doublemembrane vesicles at lateral PMs of cells that are coupled by gap junctions (GJs).…”

“…In addition, this internalization process occurs on lateral membranes that contact neighboring cells. Recent consistent reports have shown clathrin-related internalization of large particles, such as viruses and pathogenic bacteria (Ehrlich et al, 2004;Rust et al, 2004;Veiga and Cossart, 2005) and of large latex beads (Aggeler and Werb, 1982).…”

“…How GJ protrusion/invagination is initiated is currently not known; however, GJs are internalized primarily into one of two coupled cells, indicating a highly regulated process. Actin polymerization has been linked to clathrin-dependent endocytosis, membrane protrusion, and invagination events as well as to the clathrin-mediated uptake of viruses and bacteria into host cells (Bonifacino and Glick, 2004;Ehrlich et al, 2004;Merrifield, 2004;Rust et al, 2004;Gouin et al, 2005;Veiga and Cossart, 2005;Yarar et al, 2005), suggesting that actin polymerization might also be involved in the internalization of GJs. The mechanism for double-membrane fission also remains unclear.…”

“…Internalization of double-membrane GJ vesicles is an intriguing process with similarities to phagocytosis or intracellular pathogen (protozoa, bacteria, or viruses) invasion and cell-to-cell spreading (Johnson and Huber, 2002;Cossart et al, 2003;Gruenheid and Finlay, 2003;Rust et al, 2004;Gouin et al, 2005). AGJ vesicle formation requires active double-membrane protrusion and/or invagination, neck restriction, and double-membrane fission/resealing.…”

HIV-1–cellular interactions analyzed by single virus tracing

“…With this technique, the individual steps of the entry process and the dynamics of these processes can be elucidated. SVT has been utilized to investigate the entry and trafficking of several viruses including adeno-associated virus (Bräuchle et al 2002;Seisenberger et al 2001), adenovirus (Suomalainen et al 1999), simian virus 40 (Damm et al 2005), murine polyoma virus (Ewers et al 2005), HIV (McDonald et al 2002), influenza virus (Lakadamyali et al 2003;Rust et al 2004), rabies virus (Finke et al 2004) and murine leukemia virus (Lehmann et al 2005). From the trajectories of individual viruses, the interactions between these particles and different cellular components can be directly observed.…”