The flu gets cold

Erin Tran, a staff scientist who works in the Laboratory of Cell Biology/Biophysics Section of the NIH National Cancer Institute, will present the research at the Biophysical Society's 61st Annual Meeting in New Orleans, Louisiana, which runs from Feb. 11-15, 2017. Tran has been using cryo-electron microscopy (cryo-EM) methods to determine whether the spike-shaped HA proteins in their native environment embed themselves within the viral membrane, mimicking what the body might see when it is exposed to influenza viruses. The work is part of an ongoing collaboration between the laboratories of Peter Palese and Florian Krammer at Mount Sinai and Sriram Subramaniam at NIH.

"In our case, this process starts with viruses suspended in liquid," Tran said. "A few drops of the virus suspension -- containing thousands of viruses -- are placed onto a tiny, circular copper grid. Extra liquid is blotted away with filter paper, and the grid is immediately plunged into a pool of liquid ethane, which is maintained at a temperature around -180° C. This freezes the viruses in a very thin layer of glass-like ice and allows us to image them in their native state."

Once frozen, the viruses are imaged using an electron microscope.

"We get a three-dimensional image using a procedure that involves collecting a series of images of the virus at different orientations relative to the incident electron beam," she said. "HA spike protein images from single viruses are 'noisy,' but by averaging images from multiple viruses we can obtain a more accurate representation of the shape of the HA surface protein."

Tran and colleagues were surprised by the flexibility and resilience shown by the influenza HA protein. When the proteins are made from two different DNA strains, and consequently called chimeric, the team found a dramatic twist.

"The head portion of the protein is offset from the stalk portion by nearly 60 degrees, yet viruses that express these chimeric HAs are still functional and the chimeric protein is still able to bind antibodies that target either the head or stem of the protein," said Tran. "[The work] shows that cryo-electron tomography can be used as an effective way to characterize influenza vaccine candidates in the native context."

Their next goal is to find out whether the structural changes they see in this particular chimeric HA protein are representative of a broader set of chimeric HAs.

"In general, we're interested in using cryo-electron microscopy and tomography to visualize the mechanisms viruses use to infect cells," said Tran. "We're exploring a variety of enveloped viruses, including HIV and Ebola, to better understand mechanisms by which their entry into cells can be blocked."