Experiment Summary

Structural stability of the capsid core is a critical parameter for the productive infection of a cell by a retrovirus. Compromised stability can lead to premature core disassembly, exposure of replication intermediates to cytosolic nucleic acid sensors that can trigger innate antiviral responses, and failure to integrate the proviral genome into the host DNA. Thus, core stability is a critical feature of viral replicative fitness. While there are several well-described techniques to assess viral capsid core stability, most are generally time and labor intensive. Recently, we compared the relative stability of murine leukemia virus capsid cores using an in vitro detergent-based approach combined with ultracentrifugation against the popular fate of capsid assay. We found that both methods reached similar conclusions, albeit the first method was a significantly simpler and faster way to assess relative capsid core stability when comparing viral mutants exhibiting differences in core stability.

Materials and Reagents

1. 10 cm culture dishes
2. Pasteur pipettes
3. Microcentrifuge tubes
4. PVDF membrane
5. Sterile 0.45 μm Luer-Lok syringe filters
6. Sterile 20 ml syringes with Luer-Lok
7. Sterile 50 ml conical tubes
8. Sterile pipette tips
9. Polycarbonate tubes and lids
10. Serological pipettes, 10 ml
11. 293T cells
12. NIH 3T3
13. R187 Hybridoma
14. Anti-eGFP
15. Anti-Mouse IgG, HRP conjugated
16. Anti-Rat IgG, HRP conjugated
17. Dulbecco's modified Eagle's medium (DMEM) high glucose, with L-glutamine, sodium pyruvate and phenol red
18. ECL
19. Fetal bovine serum (FBS)
20. HCl, 36.5-38%
21. Hybridoma-SFM
22. KCl
23. KH₂PO₄
24. Methanol
25. Milli-Q Water
26. Na₂HPO₄
27. NaCl
28. NaOH, 10N certified
29. Penicillin/Streptomycin
30. Polyethylenimine (PEI)
31. Sodium dodecyl sulphate (SDS)
32. Sucrose
33. Tris-Base
34. Triton X-100
35. 10x PBS
36. 1x PBS
37. PBS-T
38. 20% (m/v) sucrose in PBS
39. 2% (v/v) Triton X-100 or 0.2% (m/v) SDS in 5% (m/v) sucrose in PBS
40. Complete DMEM
41. Transfer Buffer (25x)

Equipment

1. Pipettes
2. 0.22 μm Steritop® filters
3. Balance
4. Biosafety cabinet
5. Digital Imager
6. Fridge (4 °C)
7. Hemocytometer
8. Microscope
9. Refrigerated table-top centrifuge
10. Tissue culture incubator, humidity, temperature and CO2 regulated
11. Type 70Ti Rotor
12. Ultracentrifuge

Procedure

1. Refer to Figure 1 for an overview of the two methodologies used in this protocol.

Fig. 1 Overview of the retroviral capsid core stability assay.

2. Seed 10 cm dishes with viral producer cells (i.e., viable infected cells that produce representative viral particles) in 8 ml of complete DMEM. For this assay, we typically seed 2 dishes each with 2 x 10⁶ NIH 3T3 cells, chronically infected with MLV. Alternatively, the cells (such as 293T) can be transfected with a virus expressing plasmid.

3. Allow the cells to propagate and produce virus at 37 °C in 5% CO₂ until they reach confluence; this should take approximately 72-96 h. Confluence is determined by lack of surface area remaining on the well's growth surface.

4. Before collecting the viral supernatant, pre-cool the ultracentrifuge to 4 °C.

5. Collect the viral supernatant (roughly 15 ml) using a serological pipette, and transfer it into a 50 ml conical tube. In these conditions, this is an excess of virus that will allow for multiple attempts at SDS-PAGE analysis if necessary.

6. Centrifuge this supernatant for 5 min at 500 x g at room temperature (20-25 °C) to clear cellular debris.

7. During this centrifugation step, prepare the appropriate number of syringes and 0.45 μm filters.

8. Transfer the cleared supernatant into a syringe and filter it directly into another 50 ml conical tube.

a) Approach 1: Add Triton X-100 to a final concentration of 10% (i.e., 1.7 ml Triton X-100 + 15 ml viral supernatant), mix by inversion or pipetting. Incubate at room temperature (20-25 °C) for 20 min.

b) Approach 2: Move to Step 9.

9. Transfer the solution into a Type 70Ti tube.

10. If necessary, top up each tube with media or PBS such that they contain approximately 7.5 ml below the maximum threshold.

11. Place a sterile Pasteur pipette into each 70Ti tube, with the thin side immersed in viral media. Placement and use of the Pasteur pipette in this fashion is shown in Figure 2 of a recent publication by our laboratory.

12. Slowly add the sterile 5% sucrose-detergent buffer of choice, 2 ml of this is sufficient. This allows for a short exposure to detergent before passing through the denser, 20% sucrose solution, which is detergent-free. In our experiments, we typically use 5% sucrose + 2-10% Triton X-100 or 0.2% SDS.

13. Using the same Pasteur pipette, slowly add the sterile 20% sucrose solution through the Pasteur pipette so it may form a cushioning layer below the detergent layer. Five milliliters of this solution is sufficient.

14. Insert the lids and caps onto the tubes and balance each tube appropriately for ultracentrifugation (within 0.05 g). Sterile media or PBS can be used to adjust the mass of viral samples.

15. Cap each tube, ensuring the O-rings and aluminum caps are sealed properly. Insert these tubes appropriately into the Type 70Ti rotor and insert the rotor into the ultracentrifuge.

16. Ultracentrifuge these samples with an acceleration and deceleration set to maximum, for 2 h at 4 °C at approximately 100,000 x g.

17. Remove the tubes from the rotor, visualize the pellets and circle with a marker. These pellets are rather translucent with a white tinge, as the envelope should have been stripped from them at this stage. As time passes after the centrifugation, these pellets become more difficult to see and may dislodge, so samples need to be processed as fast as possible.

18. Gently remove supernatants using a serological pipette and resuspend the pellet in buffer of choice. We typically use 250 µl Laemmli sample buffer for analysis by SDS-PAGE. Resuspend slowly and carefully to avoid the introduction of bubbles.

19. Store at -20 °C or -80 °C until ready for analysis.

Data Analysis

SDS-PAGE:

Viral capsid core stability can be determined by quantifying essential viral components; loss of viral envelope glycoprotein is expected to occur as the phospholipid bilayer will be dissociated from the capsid. Density of the bottom sucrose layer will govern the stringency and overall yield of this assay. For M-MLV, we targeted the p30 capsid protein (R187, rat monoclonal, 1 µg/ml) and viral envelope glycoprotein Env-eGFP (anti-eGFP, JL-8, mouse monoclonal, 0.2 µg/ml). For the p30 capsid antibody, R187 cells were propagated in the conditions outlined by the ATCC using Hybridoma-SFM. The supernatant may be directly used or purified using Protein A or G resins. The remaining antibodies, including secondaries, were purchased as outlined in the Materials and Reagents section. This approach and necessary background information are illustrated and interpreted in our recent publication in the Journal of Virology. The development of this protocol was to assess the impact on core stability caused by mutations in various viral structural proteins and in an accessory protein called glycosylated Gag (also known as glycogag or gPr80). Figure 2 below depicts the localization of each mutation in the viral proteins. A virus mutant named M-MLV [CTA] specifically depicts the loss of the CTG alternative start codon for gPr80, making this virus deficient in this protein. The other mutants express gPr80 lacking glycosylation sites. These mutations consequently impact the matrix (N113) and CA (N480 and N505) structural proteins.

Fig.2 Schematic representation of the glycosylated Gag (gPr80) protein.

Fig. 3 Assessment of viral capsid core stability.