Visualizing Heat-Treated Viral Capsids Using Transmission Electron Microscopy

Abstract

Source: Moore, M. D., et al. Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity. J. Vis. Exp.(2017).

This video demonstrates how purified viral capsids are heat-treated, prepared, and imaged using transmission electron microscopy (TEM) to reveal structural changes and heat-induced damage.

Protocol

1. TEM Sample Preparation
   1. Obtain carbon support film (nickel) grids designed for TEM.
      NOTE: Consult with the core facility on recommended reagents and their handling for use with the facility microscope. Be sure to store grids in a box containing desiccant or a vacuum chamber to minimize exposure to moisture.
   2. Tear approximately 5 cm x 5 cm pieces of filter paper. Obtain glass Petri dishes and proper self-closing reverse tweezers designed for use in microscopy.
   3. Cut approximately a 2.5 cm x 5 cm piece of paraffin film. Place on the benchtop.
   4. Heat treatment of norovirus GII.4 Sydney capsids
      1. Obtain purified human norovirus major capsid protein (VP1) of GII.4 Sydney (Accession: JX459908) assembled in capsids. 
         NOTE: Capsids in the study were obtained courtesy of R. Atmar (Baylor College of Medicine, Houston, TX).
      2. Dilute capsids to 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4) to a maximum volume of 15 µL in individual capped polymerase chain reaction (PCR) tubes. Ensure that the tube contains at least 600 ng of capsid. Ensure that there is 600 ng of capsid per treatment-ligand combination.
      3. Preheat the thermal cycler to the desired heat treatment. 
         NOTE: For this protocol, treatment at 68 °C for different times (0 - 25 min) was chosen.
      4. Preheat an additional thermal cycler to 4 °C for immediate cooling. Add tubes with capsid solution to the thermal cycler for the desired time. Immediately transfer to a pre-cooled 4 °C cycler for 5 min after heating.
      5. Briefly spin down in a centrifuge to get all droplets to the bottom of the tube.
      6. Pipette the entire ~15 µL drop of treated capsid solution onto paraffin film.
   5. Grab the grid edge with tweezers, allowing them to close on the edge to hold the grid, and place the grid carbon-side down on top of the drop of treated solution. Let it incubate for 10 min to allow the capsid to attach to the grid.
      1. Depending on the purity of the capsid preparation, add washes of the 10 mM HEPES solution if needed in the form of 10-15 µL droplets placed in line after the treated capsid droplet.
      2. After 10 min, pick up the grid with the droplet. Place the grid nearly perpendicular to a piece of filter paper to wick away the droplet. Pick the droplet of 10 - 15 µL HEPES buffer up with the grid, hold for 30 s, then wick away with another piece of filter paper to wash.
   6. Place a 15 µL droplet of 2% uranyl acetate solution on the paraffin film in an empty area.
      1. Pick up the droplet of uranyl acetate with the grid and hold for 45 s. Wick away the uranyl acetate, being sure to remove most of the solution. Place the grid carbon-side up on a piece of filter paper, being careful that the grid does not "stick" to the moisture on the tweezers. Place the filter paper with the grid on it in an open glass Petri dish.
         NOTE: Do not use plastic Petri dishes, as the grids will be electrostatically attracted to them and become stuck to the dish.
   7. Repeat for all treatments. Place the Petri dish halves with the grids in a desiccator overnight to dry prior to observing with TEM.
   8. Consult the microscopy facility at the respective institution regarding observing the prepared grids. Some facilities permit the user to be trained on the operation of their facility's instrument. Specific details regarding the setup and observation of a specific microscope are beyond the scope of this article.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Transmission electron microscopy sample preparation
Nickel electron microscopy grids with carbon support film	Ladd Research	10880-100
Self-closing reverse electron microscopy tweezers	Ted Pella	5372-NM
Qualitative filter paper	Sigma-Aldrich (Whatman)	WHA1002055
Glass petri dishes	Sigma-Aldrich	BR455743
100 mM stock HEPES solution (pH 7.2)	N/A	N/A
2% uranyl acetate	N/A	N/A