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. Also largely unknown is the way in which viruses are targeted to cellular endocytic machinery. 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.

Figure 1

Fluorescence images of clathrin-coated structures in BS-C-1 cells expressing EYFP-clathrin. For a clearer visualization of discrete fluorescent spots, a low-spatial frequency background due to cytoplasmic EYFP-clathrin has been subtracted from the images. Scale bars: 3 μm (a) The immunofluorescence image of clathrin-coated structures in a cell transiently transfected with EYFP-clathrin. (b) The EYFP fluorescence image of clathrin-coated structures in the same cell. (c) Overlay of the immunofluorescence signal (red) with the EYFP signal (green). Pixels with both red and green signal appear yellow. Counting spots in multiple cells shows that over 97% of the clathrin-coated structures that appeared in the immunofluorescence image colocalized with those in the EYFP image. The red-only spots in the upper-right corner of the image belong to a neighboring cell not transfected with EYFP-clathrin. (d) Overlay of the transferrin image (red) and EYFP image (green) of a cell stably expressing EYFP-clathrin. Alexa Fluor 647-labeled transferrin (Molecular Probes) was bound to cells at 4°C for 15 minutes. After removing unbound transferrin, cells were imaged at room temperature. An image of the first 5 s is shown. Over 96% of the transferrin spots colocalize with clathrin-coated structures and appear yellow.

Figure 2

Internalization of influenza viruses via multiple pathways. (a) Snapshots of a virus internalized via a CCP. A live movie of this virus is available as Supplementary Video 3. Scale bar: 10 μm. t = 0 s: the virus (red) binds to the cell. t = 50 s: the virus is undergoing stage I movement. t = 115 s: a CCP labeled with EYFP (green) begins to form at the virus site. t = 150 s, the clathrin coat reaches its peak fluorescence intensity. t = 175 s: the clathrin coat rapidly disassembles. t = 181 seconds: the virus is transported towards the nucleus on a microtubule (stage II movement). t = 202 s: the virus enters stage III transport involving both plus- and minus-ended-directed motilities on microtubules. t = 235 s: the virus continues stage III movement. (b) The time-trajectories of a virus internalized via de novo formation of a CCP. Black symbols are the velocity time-trajectories of the virus. Stage II movement is identified as the rapid unidirectional translocation from the cell periphery to the perinuclear region (red arrows). Green symbols are the integrated fluorescence intensity of EYFP-clathrin associated with the virus. (c) The time-trajectories of a virus internalized without association with a clathrin-coated structure. Symbols are as defined in b. Live movies of the two viruses in b and c are also available (Supplementary Videos 4 and 5).

Figure 3

Dynamics of the CCPs and CCVs formed de novo at the virus-binding sites. (a) A histogram of the time between viral binding and onset of CCP formation. (b) A histogram of the time between the onset of CCP formation and CCV uncoating. Inset: an averaged curve of 60 time-trajectories of the EYFP-clathrin intensity associated with viruses shows the life cycle of CCPs. Each EYFP-clathrin trajectory was normalized to the peak height and scaled in time so that the onset of the CCP formation occurs at t = 0 sec and the peak clathrin intensity occurs at t = 69 sec, which is the average duration of the clathrin-accumulation phase. (c) A histogram of the time between the first CCV uncoating event and the onset of stage II viral movement.

Figure 4

The effect of neuraminidase (NA) inhibitors on the endocytosis of influenza viruses. Grey columns show a histogram of the time between viral binding and the onset of stage II movement in the absence of neuraminidase inhibitors. Red columns show the histogram in the presence of 1 μM RWJ270201. The histogram in the presence of 1 μM oseltamivir is similar (data not shown).

Figure 5

Time-trajectories of viruses that successfully fused after endocytosis. (a) An example of a virus that was internalized via a CCP. (b) An example that was internalized without association with a CCP. Live movies of these two viruses are available (Supplementary Videos 6 and 7). Black symbols are the velocity time-trajectories of the viruses. Red symbols are the integrated DiD fluorescence intensities of the viruses. Viral fusion can be identified as a dramatic increase of the DiD signal. Green symbols are the integrated fluorescence intensities of EYFP-clathrin associated with the viruses.