For almost any biological process, seeing is believing. Because of the role viruses play in infectious disease, many biologists would like to observe them doing their thing in real time. Fluorescence imaging provides one of the few noninvasive ways to observe how viruses transport their contents into a cell and how new viruses are assembled. Such studies require high-quality microscopes with sensitive cameras that can shoot multiple frames per second. As with cells, researchers have to make sure that the position of fluorescent labels and the experimental setup do not interfere with the behavior of the system that they are studying.
Viruses are small, and they can be structurally and functionally complex. While fluorescence imaging offers the unique opportunity to observe viruses both directly and noninvasively, there are plenty of pitfalls, says Börries Brandenburg, a senior scientist at Crucell, a biotechnology company in Leiden, Netherlands. “You need to know your virus,” he says. Though smaller than most cells, viruses are organized systems. Therefore, researchers must design experiments carefully to make sure that the fluorescence they observe is linked to the biological processes that they hope to understand.
The Scientist sought out the experts to bring you a guide on imaging viral behavior in living systems.
Choosing a label
The first step in tracking a virus is labeling it. To do so successfully, you will need to consider both the size of virus and what you’d like to observe. Viruses range in size from approximately 25 nm, such as picornaviruses, up to nearly a micrometer long, such as paramyxoviruses. Inserting a gene that encodes a fluorescent protein into the viral genome provides one tried-and-true way to make a virus glow. With larger viruses, such as the 400 nm vaccinia, researchers can generally insert the genes for classic fluorescent protein labels, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), without interfering with the virus’s structure and function. Smaller viruses such as influenza, which averages about 100 nm in diameter, are much harder to label with expressed proteins because the size of the protein can interfere with normal viral function, Brandenburg says.
One way to tackle these smaller viruses is to use fluorescent small molecules to chemically label lipids, proteins, or carbohydrates within them. Such labeling molecules attach to particular functional groups, but they react nonspecifically and therefore don’t allow you to know exactly which molecule you are tagging. Alternatively, newer, site-specific methods allow researchers to attach a chemical label to a molecule of interest. Hidde Ploegh and his colleagues at the Whitehead Institute in Cambridge, Massachusetts, developed a site-specific labeling method that uses bacterial enzymes that recognize specific amino-acid sequences to label proteins. They are currently using the approach to examine the biogenesis of influenza virus particles. In a recent paper (PLoS Pathogens,8:e1002604, 2012), Ploegh and his colleagues describe how they engineer the five-amino-acid recognition sequence for the bacterial enzyme into two influenza envelope proteins. Using a standard peptide synthesizer, they then create a fluorescent tag appended to a short peptide sequence. Finally they treat the virus with the bacterial enzyme in the presence of the tag. The method specifically labels only the proteins with the recognition sequence, Ploegh says. It also uses an enzyme that researchers can readily produce in the lab, off-the-shelf reagents, and solid-phase peptide synthesis—tools that are available to most biochemists and cell biologists.
Written By: Sarah Webbcontinue to source article at the-scientist.com