Cellular entry of viruses and non-viral gene delivery vectors
1. Visualizing individual influenza virus particles in living cells (past achievement)
2. Entry mechanisms of influenza viruses (past achievement and current project)
3. Endocytic trafficking of influenza viruses (past achievement and current project)
4. Nuclear import of influenza genome (past achievement)
5. Entry mechanism(s) of polioviruses (current project)
6. Entry mechanism(s) of polyomaviruses (current project)
7. Cellular entry and trafficking of polymeric gene-delivery vectors (current project)
1. Visualizing individual influenza virus particles in living cells (past achievement)
Each year, 10-20% of U.S. residents are infected by influenza; among these, about 36,000 people die of flu-related complications. This reminds us of one of the biggest human tragedies of all time - the notorious 1918 flu pandemic that claimed the lives of 20 million people worldwide. The exact reason why the 1918 influenza virus was so dangerous and virulent is still unknown. It may be just a matter of time before another strain of influenza virus emerges with a similar devastating power. The situation clearly demands an improved understanding of infection mechanisms of influenza viruses, to provide new insights of the disease and to suggest new strategies for treatment.
Our research currently focuses on the type A influenza virus, an enveloped virus with a lipid-bilayer membrane, a protein matrix, and a segmented genome, comprised of eight single-stranded RNAs packed into ribonucleoprotein (vRNP) complexes. A scheme of influenza A is shown is Fig. 1. Influenza viruses infect cells via receptor-mediated endocytosis, followed endocytic trafficking of influenza to acidic endosomes, and fusion of the viral membrane with endosomes to deliver the viral genome (vRNPs) into the cell. The vRNPs are then imported to the nucleus to initiate viral gene expression and replication . Despite intensive studies of influenza infection, many important aspects of cellular entry process of influenza still remain elusive.
Figure 1. A schematic of the influenza virus. The proteins coded by the genes are indicated next to the vRNPs.
We have developed imaging methods to visualize single influenza virus particles inside living cells. By tracking individual viruses in real time using advanced fluorescence microscopy, we have monitored the transport, acidification and fusion of influenza viruses in living cells (Fig. 2, movies 1 and 2 ). Our single-virus trajectories directly reveal a three-stage active-transport behavior of the viruses preceding viral fusion, each stage having a distinct molecular mechanism (Fig. 3, movie 1). Stage I is an actin-dependent movement in the cell periphery, primarily on the cell surface. Stage II is a rapid, dynein-directed translocation on the microtubule that delivers the virus from cell periphery to the perinuclear region. Stage III depicts an intermittent movement involving both plus- and minus-end-directed microtubule-based motilities in the perinuclear region. By monitoring the local chemical environment of individual viruses, including the pH level and the protein content of the endocytic compartment containing the virus, we have discovered new trafficking and sorting mechanisms for viruses inside the cell's endocytic network (Fig. 3, Movie 2 ).
Figure 2. Stacked, time-lapsed images of two viruses in living cells. The sudden color change from blue/pink to white indicates a dramatic fluorescence dequenching, signaling the fusion of the virus with an endosome. The viruses are labeled with membrane dyes.
Figure 3. Endocytic trafficking and fusion of individual viruses. (a) The trajectory of a DiD-labeled virus inside a cell. The color of the trajectory codes time with the colored bar indicating a uniform time axis from 0 s (black) to 500 s (yellow). The red star indicates the fusion site. (b) Time trajectories of the velocity and the DiD fluorescence intensity of the virus. The labels I, II, and III indicate stage I, II, and III movement, respectively. The fluorescence dequenching signal of the lipophilic dye, DiD, near 400 s indicates viral fusion. (c) The trajectory of a Cy3/CypHer5-labeled virus inside a cell. The green star indicates the initial acidification site (to pH 6). (d) Time trajectories of the velocity and the fluorescent emission ratio of CypHer5 (a pH-dependent dye) and Cy3 (a pH-independent dye) of the virus. Scale bars: 10 m m.
Movie 1. Transport and fusion of a DiD-labeled influenza virus in a CHO cell. The fluorescence image of the virus is overlaid on a static DIC image of the cell. A standard Gamma-II color table is used to indicate relative fluorescence intensity. For clarity, the fluorescence signal has been dimmed outside of a small region surrounding one virus. Viral fusion is indicated by a significant increase in fluorescence signal near the end of the movie. The length of the movie is 500 s.
Movie 2. Transport and acidification of a Cy3/CypHer5-labeled virus in a CHO cell. The fluorescence is false-colored so that the virus appears green-yellow in a neutral environment and red in an acidic environment with pH = 6. The length of the movie is 450 s.
Reference:
- M. Lakadamyali, M. J. Rust, H P. Babcock, X. Zhuang, " Visualizing infection of individual influenza viruses ," Proc. Natl. Acad. Sci. USA 100 , 9280-9285 (2003).
- M. Lakadamyali, M. J. Rust, X. Zhuang, " Endocytosis of influenza viruses ," Micro. Infection 6 , 929-936 (2004).
2. Entry mechanisms of influenza viruses (past achievement and current project)
While many viruses are known to infect cells via receptor-mediated endocytosis, the exact endocytic mechanisms have remained unclear for most of them. Due to the presence of multiple endocytic pathways in cells, including phagocytosis, macropinocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, and clathrin- and caveolae-independent pathways, determining the exact endocytic mechanisms used by viruses has been challenging .
By tracking the interaction of single influenza viruses with cellular endocytic structures using real-time fluorescence microscopy, we observed individual viral entry events directly, isolated those entry events that led to viral fusion - an obligatory step for infection, and characterized the endocytic mechanism underlying each event. Using this approach, we have shown that influenza can exploit clathrin-mediated and clathrin/caveolin-independent pathways in parallel, both pathways leading to viral fusion (Figs. 4 & 5, Movies 3 , 4 , & 5 ). These experiments allowed us to quantitatively determine the partition of the viruses among different endocytic pathways. .
We have also addressed another important question: How are viruses targeted to cellular endocytic structures for entry? We observed a novel phenomenon: those influenza viruses using the clathrin pathway enter cells predominantly via the de novo formation of clathrin-coated pits (CCPs) at the viral binding sites (Movies 3 and 4). Remarkably, CCP formation at these sites is much faster than elsewhere on the cell surface. The kinetics and spatial distribution of the CCP formation suggest that these CCPs are most likely induced by viral binding.
Considering the lack of specific influenza receptors with internalization motifs, we suggest a potentially general mechanism to promote CCP formation, and thereby viral entry, that may be used by many virus types: The multivalent binding of the virus to cells induce local curvature on the plasma membrane, which can be detected by curvature-sensing components of CCPs, thereby promoting formation of a CCP. Currently, we are testing this hypothesis by monitoring CCP formation around objects with various radii of curvature, by perturbing the curvature-sensing capability of CCP components, and by monitoring different components of the CCPs forming at the sites of bound viruses in real time. These experiments will elucidate the mechanism(s) that influenza exploit to induce CCP formation.
Figure 4. Snapshots of a virus (red) internalized by a CCP (green). Overlay of green and red signals appears yellow. T = 0 s: the virus (red) binds to the cell. t = 115 s: a CCP labeled with EYFP (green) begins to form at the virus site. T = 175 s: the clathrin coat rapidly disassembles. T = 181 s, 202 s, and 235 s: transport of the virus on microtubules.
Figure 5. Time-trajectories of viruses fused with endosomes. ( a ) A virus internalized via a CCP. ( b ) A virus internalized without association with a CCP. 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.
Movie 3 and Movie 4 . The internalization of influenza viruses via CCPs. Both movies show the de novo formation of a CCP (green) around the virus (red, in white circles) and the uncoating of clathrin before the virus exhibit a rapid, unidirectional movement towards the perinuclear region (stage II movement).
Movie 5 . The internalization of an influenza virus without association with a CCP.
Reference:
- M. J. Rust, M. Lakadamyali, F. Zhang, X. Zhuang, " Assembly of endocytic machinery around individual influenza viruses during viral entry ," Nature Struct. Mol. Biol. 11 , 567-573 (2004)
- M. Lakadamyali, M. J. Rust, X. Zhuang, " Endocytosis of influenza viruses ," Micro. Infection 6 , 929-936 (2004).
3. Endocytic trafficking of influenza viruses (past achievement and current project)
Influenza viruses are believed to fuse their envelope with acidic endosomes to release their genome into the cell. It is therefore of great interest to understand how influenza viruses are trafficked toward those endosomes. The dynamic nature of the endocytic network makes it highly desirable to address this question by real-time imaging. By tracking single viruses simultaneously with different types of endosomes specifically labeled with fluorescent endocytic markers or Rab GTPases, we observed that influenza viruses are sorted to a small sub-population of endosomes that are highly dynamic and rapidly maturing. By largely avoiding the other relatively static "early" endosomes, these viruses put themselves in a position to quickly achieve fusion. How such a "smart" choice is made is currently under investigation.
4. Nuclear import of influenza genome (past achievement)
The influenza genome is comprised of eight single-stranded RNAs packed into ribonucleoprotein (vRNP) complexes. After the vRNPs are released into the cell by viral fusion, they need to be imported to the nucleus for viral gene expression and replication . We have extended the single-particle tracking technique to investigate the nuclear trafficking of the viral genome. By directly tracking individual vRNP particles in live cells (Movie 6), we have determined the transport mechanisms of vRNPs in cells - vRNP particles diffuse rapidly both inside the cytoplasm and inside the nucleus without exhibiting directed movement (Fig. 6). We have observed heterogeneous interactions between the vRNPs and nuclear pore complexes (NPCs) with dissociation rate constants spanning two orders of magnitude.
Figure 6. Tracking the movement of single vRNPs. The measured mean square displacement (<Dr2>) vs. time (Dt) for 4 example vRNPs in the cytoplasm of a cell. Lines are the best fit to <Dr2> = 4DDt, with D being the diffusion coefficient.
The nuclear import of the influenza vRNPs are regulated, a property that is critical for the replication of the influenza viruses. While it is known that the influenza matrix protein, M1, is responsible for down-regulating the nuclear-import of vRNP, how M1 achieves this goal has remained elusive. Our single-particle tracking experiments have provided new insights into the regulation mechanism - M1, down-regulates the nuclear-import of vRNP by inhibiting the interaction of vRNPs with the nuclear pore complexes, but not by inhibiting the transport of vRNPs (Fig. 7).
Figure 7. Regulated nuclear import of influenza vRNPs. ( a ) A DIC image of an injected cell. ( b ) A fluorescence image of the same cell, taken 30 minutes after the microinjection of dye-labeled vRNPs, showing nuclear import of the labeled vRNPs. (c) A fluorescence image of a cell taken 2 minutes after injection of vRNP. The ring at the nuclear envelope indicates association of vRNPs to the nuclear envelope. (d) A fluorescence image of a cell that was co-injected with labeled vRNP and anti-NPC antibodies. (e) A fluorescence image of a pre-infected cell taken 10 min after injection with vRNPs. The cell was infected with influenza viruses 6 hours prior to injection. No fluorescent ring is observed at the nuclear envelope.
Movie 6 . A movie showing the motion of individual labeled vRNP in a BS-C-1 cell. The movie was taken at 10 fps. The gray ring shows the location of the nuclear envelope as determined by DIC microscopy. The diffusive movement of the vRNPs and the preferential binding of the vRNPs to the nuclear envelope are clearly evident.
Reference:
5. Entry mechanisms of polioviruses (current project)
Poliovirus is the causal agent of Poliomyelitis. The polio infection in the spinal cord can cause paralysis. Poliovirus is a non-enveloped virus consist of an icosahedral protein shell that encapsidates a single-stranded RNA genome. Despite the identification of cellular receptors for polioviruses, their entry mechanism is still poorly understood. Indeed, whether the polio genome is released at the plasma membrane or whether endocytosis is required for infection, is still under debate.
We have recently developed a fluorescent labeling method that does not perturb the infectivity of poliovirus, but allow individual polioviruses to be visualized in living cells and the release of a single polio genome to be observed in real time. We are currently investigating the polio entry mechanism by tracking the behavior of individual poliovirus particles in living cells and by tracking the interaction of polioviruses with relevant cellular machinery potentially responsible for the viral entry. We shall complement these real-time imaging studies with conventional infectivity tests and structural analysis of polioviruses.
Figure 8: A poliovirus
6. Exploring the entry mechanisms of polyomaviruses (current project)
An understanding of the human polyomaviruses, BKV and JCV, is of great importance due to their active infection of immunocompromised persons. Active infection has been linked to hemorrhagic cystitis, ureteric stenosis, polyomavirus-associated nephropathy, and progressive multifocal leukoencephalopathy. Polyomaviruses are non-enveloped viruses with icosahedral protein capsids. The polyoma viral genome is a double-stranded and circular DNA. The infection mechanisms of these viruses are not well-understood, but different viruses within the polyomavirus family were seen to utilize different endocytic mechanisms.
Using the single particle-tracking method that we have developed for investigating influenza and polio viral entry, we are currently investigating the cellular entry mechanism of human polyomaviruses, BKV and JCV, and comparing their entry behavior with that of the relatively well studies SV40 viruses.
Figure 9: A polyomavirus
7. Cellular entry and trafficking of polymeric gene-delivery vectors (current project)
Gene delivery refers to the introduction of exogenous DNA into a host cell for the production of an absent or defective protein. It has emerged as a promising therapeutic method for a variety of diseases including cystic fibrosis, Parkinson's disease, and certain cancers. Using similar vector technology, small interfering RNA (siRNA) provides a possibility of RNA-based therapeutics. Exciting results have shown that siRNA has therapeutic potential for diseases ranging from influenza to Huntington 's. For both DNA and siRNA, the most important component of a delivery system is the vector, which assists the entry of DNA or siRNA into the cell and prevents their degradation. While viruses are the most efficient gene-delivery vectors, it is becoming increasingly clear that non-viral vectors may prove to be a safer, more easily controlled method of gene delivery.
Polymer-mediated gene delivery replaces the conventional viral vector with a positively charged polymer. This cationic polymer binds to the anionic DNA or siRNA, causing it to condense into a small complex with a net positive charge. At present, the best polymeric gene-delivery vectors is still a few orders of magnitude less efficient than viral vectors. A detailed understanding of the entry and trafficking mechanisms of polymer-DNA complexes is critical for the rational design of polymer vectors with much improved efficiency.
Using a combination of fluorescence microscopy, single-particle tracking, and drug inhibition we have characterized the endocytic pathway of polymer-mediated gene delivery using the cationic polymer vector, polyethylenimine (PEI). These techniques provide a real-time, dynamic picture of polymer-mediated gene delivery in live cells, allowing us to observe the individual steps on the gene delivery pathway and to directly assess the efficiency of each step. Our results indicate that PEI-DNA complexes are internalized and trafficked in cells via a novel, previously uncharacterized endocytic pathway, providing new insights into the barriers for polymer-mediated gene delivery.
Currently, we are extending our investigation into several newly developed gene-delivery polymers synthesized by our collaborators' laboratories, assessing their performance on each steps of the gene-delivery pathway to provide further insights for rational designs of polymers with improved gene delivery efficiency.
Movie 7 . Visualizing PEI-DNA complexes in live cells.
Our collaborators on the poliovirus and gene-delivery projects:
Links to our other research areas: