|Zhuang Research Lab|
While purified systems provide a powerful platform for manipulating and understanding biological processes, ultimately one wants to understand these systems in their physiological contexts, namely in living cells and organisms. To this end, we are developing live cell imaging and single particle tracking approaches to study the dynamics of biomolecular processes in living cells. Our current focus is on virus-cell interactions. Viruses are masterful manipulators of living organisms. By means of only small number of proteins and nucleic acids, viruses are able to reprogram the cell's machinery to do their bidding, ultimately replicating thousands of copies of themselves in order to spread an infection. As parasites that largely take advantages of the endogenous functions of cells, viruses are often excellent probes for studying cellular processes.
Many virus-cell interactions are transient and asynchronous, involving intermediate steps on the pathway, each featuring dynamic interactions between different viral and cellular components. What could be a better approach to study viral infection than to take a ride with the virus particle on its journey into the cell? To realize this goal, we are developing advanced real-time imaging methods to track the behavior of individual virus particles and their interactions with cellular machinery in live cells. This allows us to follow the fate of individual viruses, to capture fleeting interactions between viruses and cellular components, to dissect these interactions into microscopic steps, and ultimately to determine the molecular mechanisms underlying each step. Our current research is primarily focused on two viruses, influenza and polio, representing enveloped and non-enveloped viruses, respectively.
Influenza is an important human pathogen, killing hundreds of thousands people annually and causing some of the deadliest pandemics in human history. Flu infection begins by the binding of a virus particle to receptors on the cell surface (sialic acids in this case). Like the majority of animal viruses, influenza virus enters cells by endocytosis. After internalization, virus particles are sequestered into endocytic organelles until the acidity is sufficient to trigger viral fusion with the endosome, releasing the viral genome into the cytoplasm. The viral genome is then transported to the nucleus for replication and expression. The entire process from cell attachment to genome arrival at the replication sites is referred to as viral entry and is a subject of fundamental importance as well as a therapeutic target for treatment of many viral diseases.
By labeling influenza viruses with lipophilic fluorescent dyes we are able to track individual virus particles in live cells and observe their fusion with endosomes. We have observed three active-transport stages prior to viral fusion, each with a distinct molecular mechanism (see figure below).
Figure 1. Tracking individual influenza viruses in live cells. (A) Stacked, time-lapse image of two influenza viruses in a live cell. The motion of the viruses is evident from the track and the sudden color change from blue/pink to yellow/white indicates a dramatic increase in fluorescence, signaling the fusion of the viruses with endosomes. (B) Speed and fluorescence intensity time traces of a virus particle which exhibited actin-dependent (during the period t1) and microtubule-dependent (during periods t2 and t3) movement before fusion with an endosome, as indicated by the dequenching of DiD signal. Figures adapted from PNAS 100, 9280-9285 (2003).
Click here to see a movie showing the transport and fusion of a virus particle.
Multicolor imaging of viruses and cellular components allows us to directly visualize the interactions between virus particles and cellular structures important for infection. By simultaneously tracking influenza virus particles and endocytic structures in the cell, we have shown that the influenza virus enter cells via multiple pathways: About 60% of virus particles enter cells using clathrin-mediated endocytosis (see Figure 2) while the remaining 40% enter by a clathrin- and caveolin-independent pathway. Both pathways lead to viral fusion with similar efficiency.
Figure 2. Multicolor snapshots showing the internalization of an influenza virus (upper row and red in lower row) by the de novo formation of a clathrin-coated pit (middle row and green in lower row) in a living cell. Figures adapted from Nat. Struct. Mol. Biol. 11, 567-573 (2004).
Click here to see a movie showing the clathrin-mediated endocytosis of this virus particle.
An important factor for endocytosis is the endocytic adaptor proteins, which mediate the recognition of cargo receptors and the recruitment of components of the endocytic machinery. We found that instead of the commonly used AP-2 adaptor, influenza viruses use a cargo-specific adaptor epsin1 to mediate its clathrin-dependent uptake (see Figure 3).
Figure 3. Influenza virus uses epsin1 but not AP-2 as its adaptor for clathrin mediated endocytosis. (A) Multicolor snapshots showing the colocalization of an influenza virus (red, in white circle) with clathrin (cyan, upper panels) and epsin1 (green, lower panels) before entering the cell and fusing with an endosome. (B) Knockdown of epsin1 or over expression of a dominant negative mutant of epsin1 lacking the ubiqitin interaction motifs significantly reduces the fraction of viruses entering through the clathrin pathway, whereas knocking down AP-2 has little effect. Figures adapted from PNAS 105, 11790-11795 (2008) and Cell 124, 997-1009 (2006).
Click here to see a movie showing the epsin1- and clathrin-mediated entry of this virus.
We have also studied the post entry trafficking of the influenza virus. By tracking endosomal markers together with endocytic cargo particles, we have discovered distinct populations of early endosomes and differential targeting of cargos to these endosomal populations. We found that influenza viruses are preferentially targetted, via microtubule-dependent transport, to a dynamic, rapidly maturing population of early endosomes. Viral fusion takes place as these compartments mature toward late endosomes. After fusion, the genetic material of the virus, the viral RNA complexes with nucleoproteins, are transported to the nucleus by diffusion. Together, the above results provide a detail picture of the entry and trafficking mechanisms of influenza virus.
Poliovirus, while no longer a major human threat, is a model for non-enveloped viruses including rhino viruses (an agent that causes common cold), enteroviruses, and hepatoviruses (such as hepatitis A). Despite decades of research, the means by which poliovirus (PV) enters the cell remains poorly understood. By combining an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release, we have characterized the early events in PV infection. We observed that genome release by PV is highly efficient and rapid, and does not limit the overall infectivity or the infection rate. The virus particles enter the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release then takes place within 100-200 nm of the plasma membrane (see figure below). These results settle a long-lasting debate whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell.
Figure 4. Poliovirus entry. The left panels show the images of doubly labeled polioviruses at two different time points (upper panel: 10 min and lower panel: 60 min) post infection. The capsid label is shown in red and the viral RNA in green. At 60 min post infection, the viral RNA release is complete and the viruses exhibit only capsid label. The right panel shows a model for polio viral entry: After binding to cell-surface receptors, poliovirus undergoes conformational changes of the capsid, followed by clathrin- and caveolin-independent but actin-dependent endocytosis. The release of the viral genome takes place only after internalization. Figures adapted from PLoS Biol. 5, 1543-1555 (2007).
Other Viruses and Non-viral Gene Delivery Vectors
Using a similar approach, we have also investigated the entry and trafficking mechanism of a number of other viruses and non-viral gene-delivery vectors, including the dengue virus, cationic polymers, lipids and cell penetrating peptides.
Live cell imaging provides a most direct visualization of cellular dynamics and cell-pathogen interactions. We are advancing the technology such that more cellular process can be brought under the scope of live cell imaging. Our current and future efforts are focused on:
Through these studies, we aim to understand the entry, trafficking and assembly mechanisms of viruses at the molecular level and to elucidate the related cellular trafficking pathways through the use of virus probes.
Selected recent publications: