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Harvard Team Combines Imaging, Virology Assays to Study Poliovirus Genome Delivery

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An important avenue in virology research is the study of the mechanisms of viral entry into cells, but while this process is fairly well understood for enveloped viruses, it is still uncertain how nonenveloped viruses disrupt cellular membranes for viral genome release.
 
In a recent study, James Hogle, the Edward S. Harkness Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School, and his colleagues describe a new method for studying how nonenveloped viruses enter their target cells that combines imaging and virological assays in order to track the mechanisms behind viral entry.
 
The investigators published their work in the July issue of PLoS Biology.
 
Hogle’s team first produced a poliovirus and labeled both the RNA genome and the capsid with fluorescent dyes. The researchers then used real-time fluorescent microscopy to follow single virus particles during infection, to define how they enter cells, and to determine when and where in the target cell the viral genome gets released.
 
They complemented the microscopic studies with virologic assays, which demonstrated that the mechanisms they observed by microscopy were productive. The investigators found that poliovirus enters live cells in a process that requires ATP, an intact actin cytoskeleton, and tyrosine kinase cell-signaling pathways, but does not require clathrin, caveolin, flotillin, or functional microtubules.     
 
The researchers also found that RNA genome release is “surprisingly efficient” and occurs from vesicles that are very close to the cell surface, according to the PLoS article.
 
This week, Cell-Based Assay News spoke to Hogle about the study and his other projects.
 
Could you give me some background on the work done by you and your team?
 
My lab has been interested in a number of questions related to poliovirus for 25 years or more. My major focus is structural biology. Over the years, my lab has looked at the structure of poliovirus using x-ray crystallography and assembled an intermediate poliovirus particle using x-ray crystallography.
 
More recently, we have turned to electron microscopy to look at structures of cell entry intermediates. We have also done some biochemical and genetic characterizations of pathways leading to the entry of the virus into cells.
 
An obvious thing to do would be to use optical microscopy and/or electron microscopy in cells to actually look at what is happening during the entry process. We had not done that, predominantly because of the major issue that applies to most animal viruses — infection is very inefficient.
 
Although we were aware that improvements in optical microscopy had reached the point where one could look at a single virus particle entering the cell, we despaired of ever really learning anything useful by microscopy techniques alone.
 
For me, a real epiphany came when I heard a seminar given by my collaborator, Xiaowei Zhuang. Working with influenza virus, she and the students in her lab had determined that they needed to couple an assay for some function that was related to viral entry. They could then identify what particles were likely integral in establishing infection.
 
They loaded up enough dye in the outer membrane of the virus particles to quench the fluorescence. They were then able to follow the dimly lit virus as it attached to the cell.
 
For influenza virus, as is true for all enveloped viruses, the process of getting the viral genome into the cytoplasm is mediated by fusing the virus membrane with the cell membrane. When the two membranes merge during fusion, the dye in the viral envelope is diluted and lights up, because the quenching is released, and one sees a bright flash.
 
You are able to watch the particles, and specifically score those that lead to this bright flash. If you postulate that the viral particle itself engineers fusion in the appropriate compartment in the cell, it’s done its job. Anything that is inefficient about infection from then on is not the fault of the virus particle itself, but is related to some process of replication downstream.
 
For us, the equivalent would be to separately label the viral protein and the RNA, and then look for the point where the two labels part company. Labeling the viral capsid turns out to be very easy. You take purified virus and add any number of dyes that react with amino groups.
 
We had been told that we had to be careful about the amounts of dye that we used, and we found out that we could add enough dye that it would quench itself, without affecting the specific infectivity of the virus.    
 
Labeling the RNA was harder. We tried to sneak the label into purified virus, and were never successful. We had to go in and metabolically label the RNA. In terms of dyes, we had some pretty strict criteria that we were looking at.
 
We had to have a dye that would be able to enter the cell, so it had to be able to cross membranes. It could not be cytotoxic. It could not interfere with viral replication in the cell in the first round. It had to be incorporated in the virus at sufficient levels to be able to detect single particles. And it had to be able to do so without affecting the specific infectivity of the virus, even after labeling with the secondary capsid label.
 
Lily Lee, one of the authors on the paper, in my lab screened several dozen dyes from what was then Molecular Probes [now a part of Invitrogen], to find one that met all of our criteria. That dye [Syto82] works pretty well.
 
Syto82 bleaches very quickly, however. So we had to be very careful about the amount of exposure to the laser that specifically excited the RNA dye.     
 
Then, Boerries Brandenburg in Xiaowei’s lab was able to use microscopy to demonstrate that these techniques were successful. We wanted to couple that with another biological assay. This is based on an old technology, in which there is a dye that you can put inside the virus. If you shine light on the virus, the dye will cross-link the RNA and kill it. But as soon as the RNA is released from the virus during infection, it is no longer susceptible to the light.
 
Now you have two assays, one that you are visualizing and one that you are reading out, that you can look at and compare with one another. Are the kinetics of the two assays the same? They are.
 
You can also use a whole bunch of inhibitors of cell trafficking pathways, and see if they affect the two assays in the same way. They do. Now you can be confident that the process you visualize under the microscope is biologically relevant.
 
This is an example of a situation in which two labs with complementary areas of expertise worked together to do a single study. In order for a situation like that to work, it helps if each lab understands what the other one is doing.
 
 
Can you discuss your findings? 
 
We did get some very interesting results. First of all, it was never clear from earlier studies as to what pathway poliovirus used to deliver its genome into the cell. It was also unknown as to whether the virus crossed the membrane at the cell surface or crossed the membrane after internalization by some endocytic pathway.  
 
Some good evidence was found in Tom Kirchhausen’s lab at Harvard Medical School that viral entry was independent of a protein called dynamin, which pinches off invaginations to make clathrin-coated vesicles. It also pinches off some, but not all, caveolae to make the caveolin-dependent pathway work.
 
Some people interpreted these results to mean that the virus entered the cell at the cell surface.
 
One of the findings of our current study is that productive RNA release is very efficient in poliovirus. We anticipated that RNA release would be one of the major inefficient steps in the infection process.
 
We also found that RNA release occurs very close to the cell surface, but not at the cell surface. It has to be in a vesicle or an almost completely closed-off compartment, because it’s not accessible to changes in extracellular pH at that point.
 
We also found that the pathway it uses is dependent on actin, ATP, and a tyrosine kinase that we have yet to identify, and is more complex than was previously anticipated. It does not, however, require functional microtubules, clathrin, caveolin, or flotillin. It appears to be a rather unusual pathway. 
 
Another important finding, as far as I am concerned, is the fact that RNA release is so efficient that we are now free to use other visualization methods, such as light microscopy and electron microscopy, to look at the viral entry process. That is one of the things that my lab does.
 
A lot of what we know about how the cell takes things in has been learned in part from studying viral cargoes. Hopefully, if we continue to probe the compartment in which the viral genome enters the cell, we will either identify a new pathway or learn that a pathway previously thought to be of little significance has a greater biological relevance than previously believed. 
 
Did these results surprise you?
 
Yes. I had always thought that RNA release would be inefficient, and I have been working with poliovirus for a long time. So the fact that it is an efficient process surprised me.
 
In addition, the fact that RNA release occurs near, but not at, the cell surface was surprising. Many other well-characterized viral pathways require movement of the virus in a vesicle from the cell surface to the cell center in order for RNA release or fusion, depending on the mechanism, to be triggered. The idea that this is occurring almost certainly in a vesicle, but that the vesicle remains resident near the cell surface, was surprising.
 
Another surprising finding was that after RNA release takes place, the empty particle that is left is then transported by microtubules to the cell center, presumably to be degraded.
 
The real puzzle is — what triggers that? What keeps the virus from jumping on a microtubule and going to the cell center prior to RNA release? These things were to me, very surprising.
 
What do you see as areas for future research?
 
We have yet to identify the compartment or the tyrosine kinase. Xiaowei’s lab is doing some work on what happens before the virus particle gets internalized. There are also questions about the potential differences among viral entry pathways for different types of cells. Another study by a collaborator suggests that some viral cell entry pathways are different depending on the cell type that they are entering.
 
We have been studying the virus in HeLa cells. But what happens in a polarized epithelial cell? What happens in a neuron? Some hints suggest that differences exist between productive viral entry and transport in the central nervous system.
 
We may be interested in pursuing these questions using optical microscopy. My next major thrust will be to complement the optical microscopy with electron microscopy to look at viral entry in cells to try and answer these questions.

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