A superresolution imaging technique that allows the structure of a respiratory virus to be seen in living cells could help unlock the virus's secrets, including how it enters cells and replicates, and why certain lung cells escape relatively unscathed. By the time children turn 2, most have had respiratory syncytial virus (RSV), and suffered coldlike symptoms. But for some, especially premature babies and the sick, the virus can be much more serious, leading to pneumonia and bronchitis. A superresolution optical image of a specific hRSV viral filament produced with dSTORM technology. The viral filament is approximately 4 µm in length, typical of hRSV. Images courtesy of Eric Alonas and Philip Santangelo. RSV can be difficult to study because the infectious particle can take different forms, ranging from 10-µm filaments to ordinary spheres. The virus can insert more than one genome into the host cells, and the RNA orientation and structure are disordered, which makes it difficult to characterize. If scientists could observe how the virus enters live cells and replicates, and view how many genomes it inserts into its hosts, they might get the information they need to develop new antiviral drugs, or perhaps even a vaccine. "We want to develop tools that would allow us to get at how the virus really works," said Philip Santangelo, an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Institute of Technology and Emory University School of Medicine. "We really need to be able to follow the infection in a single living cell without affecting how the virus infects its hosts, and this technology should allow us to do that." Although RSV will be the first target, the technique could be used to study other RNA viruses, including influenza and Ebola, the team said. “We’ve shown that we can tag the genome using our probes,” Santangelo said. “What we’ve learned from this is that the genome does get incorporated into the virion, and that the virus particles created are infectious. We were able to characterize some aspects of the virus particle itself at superresolution, down to 20 nanometers, using direct stochastic optical reconstruction microscopy [dSTORM] imaging.” The team, which included scientists from Vanderbilt and Emory universities, used a probe that quickly attaches to RNA within cells. The probe's multiple fluorophores indicate the presence of the viral RNA, allowing observation of where it goes in host cells — and how infectious particles leave to spread infection. This microscope image shows a cell infected with RSV. The RNA tagged by the probe is shown in red, while the nucleoprotein is green. “Being able to see the genome and the progeny RNA that comes from the genome with the probes we use really give us much more insight into the replication cycle,” Santangelo said. “This gives us much more information about what the virus is really doing. If we can visualize the entry, assembly and replication of the virus, that would allow us to decide what to go after to fight the virus.” The research depended on a new method for labeling RNA viruses using multiply labeled tetravalent RNA imaging probes (MTRIPS). The probes consist of a chimeric combination of DNA and RNA oligonucleotide labeled internally with fluorophores tetravalently complexed to neutravidin. The chimeric combination was used to help the probes evade cellular defenses. “There are lots of sensors in the cell that look for foreign RNA and foreign DNA, but to the cell, this probe doesn’t look like anything,” Santangelo said. “The cell doesn’t see the nucleic acid as foreign.” Introduced into cells, the probes quickly diffuse through a cell infected with RSV and bind to the virus’s RNA. Although binding tightly, the probe doesn’t affect the normal activities of the virus. Instead, it allows researchers to follow the activity for days using standard microscopy techniques. The MTRIPS can be used to complement other probe technology, such as GFP and gold nanoparticles. Among the mysteries the researchers would like to tackle is why certain lung cells are severely infected, while others appear to escape ill effects. “If you look at a field of cells, you see huge differences from cell to cell, and that is something that’s not understood at all,” Santangelo said. “If we can figure out why some cells are exploding with virus while others are not, perhaps we can figure out a way to help the bad ones look more like the good ones.” The work appeared online in December in ACS Nano. For more information, visit: www.gatech.edu