Labeling of Replicating HSV-1 Genomes with a Nucleoside Analog

In This Article

Abstract

Source: Dembowski, J. A., Deluca, N. A. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (2017)

This video demonstrates the infection of stationary-phase mammalian cells with HSV-1 (Herpes Simplex Virus-1), followed by replication of the viral genome using host and viral replication machinery. A synthetic nucleoside analog is incorporated into newly synthesized viral DNA to label replicating genomes, which are subsequently isolated for proteomic analysis of associated host and viral proteins.

Protocol

1. Cell Culture, Viral Infection, and EdC (5-Ethynyl-2′-deoxycytidine) Labeling (Figure 1)

The following protocol involves working with viruses. Please refer to your institution's bio-safety protocols regarding safe handling of viruses and other biological agents. This protocol was approved by the Institutional Review Board of the University of Pittsburgh.

Trypsinize a confluent 150 cm² tissue culture flask of MRC-5 (Medical Research Council cell strain 5) cells and transfer the cells to a 600 cm2 tissue culture plate containing 100 mL DMEM (Dulbecco's Modified Eagle Medium) plus 10% FBS. Incubate at 37 °C in the presence of 5% CO₂ for 3 - 4 days to reach confluency and stationary phase. A confluent 600 cm2 dish contains ~7 x 10⁷ cells. Prepare 1 plate for each condition and 1 plate for each corresponding negative control.
NOTE: Cells must reach the stationary phase to inhibit cellular DNA replication to ensure that only vDNA (viral DNA) is labeled and purified in subsequent steps.
NOTE: Each sample requires a complimentary unlabeled negative control prepared using the same infection conditions and time point without the addition of EdC.
NOTE: A scaled down version of this experiment should be carried out in tandem to test for the labeling of cellular DNA by imaging using comparable cell growth, infection, EdC labeling conditions, and time points (see Step 2).
Dilute 7 x 10⁸ PFU (plaque-forming units) HSV-1 in 7 mL cold Tris-Buffered Saline (TBS). Remove growth medium and store at 37 °C. To infect, add the 7-mL diluted virus to MRC-5 cells and rock for 1 h at room temperature. Following adsorption, remove inoculum, rinse with 50 mL room temperature TBS, and replace the original growth medium. Incubate at 37 °C in the presence of 5% CO₂. This step should be carried out for each experimental condition and negative control.
NOTE: Increased EdC labeling can be achieved using HSV-1 mutants defective for the expression of the viral dUTPase (UL50 gene) and/or uracil glycosylase (UL2 gene). HSV-1 dUTPase has low substrate specificity and can decrease the activation of several nucleoside analogs.
Label viral genomes with EdC. Follow the directions to label incoming genomes (1.3.1.), replicating genomes (1.3.2.), or viral replication forks (1.3.3.).
NOTE: Only Step 1.3.1, 1.3.2, or 1.3.3 should be carried out depending on whether incoming genomes, replicated genomes, or replication forks are to be analyzed in subsequent steps, respectively.
NOTE: EdU or f-ara EdU can be substituted for EdC. The toxicity of nucleoside analogs should be monitored and minimized.
1. Use prelabeled stocks to carry out infection in Step 1.2 and proceed to Step 2 or 3 within 4 h post infection (hpi) to investigate unreplicated vDNA (Figure 1A).
  NOTE: Prepare prelabeled virus stocks using the previously described protocol25. Virus stocks must be passed through a G-25 column to remove any residual EdC.
2. Label replicating vDNA by adding 5 - 25 µM EdC to the cell culture medium of infected cells for 2 - 4 h (Figure 1B). Before adding, dilute EdC in 1 mL of growth medium.
3. To label viral replication forks, after the onset of vDNA replication (≥4 hpi), pulse label replicating vDNA with 5 - 25 µM EdC for 5-20 min. To chase, rinse 3x with chase medium containing 25 - 100 µM 2´deoxycytidine (deoxyC), and then incubate in the presence of chase medium for an additional 20-40 min (Figure 1C).
  NOTE: Pulse and chase medium should be equilibrated to 37 °C and 5% CO₂ before use.
  NOTE: For pulse chase experiments, it is important to consider the amount of time it takes for nucleosides to enter the cell and be phosphorylated before they can be incorporated into replicating DNA. This must be determined empirically.
  NOTE: To determine the resolution of pulse chase experiments, calculate the viral replication rate under the experimental conditions used. This can be determined by quantitative PCR of viral genomes during a single step growth time course.

Results

Figure 1: Approaches to label HSV-1 DNA. (A) To assay the state of incoming virus, resting MRC-5 cells in G0 are infected with prelabeled HSV-1 and genomes are assayed at less than 4 hpi to study unreplicated viral DNA. (B) To assay the state of replicated virus, MRC-5 cells are infected with unlabeled HSV-1 and EdC (orange stars) is added to the growth medium of infected cells and incubated for 2 - 4 h after the onset of viral DNA replication (≥4 hpi). (C) To assay viral replication forks, infected cells are pulse labeled with EdC for 5-20 min. Replication forks can then be chased with deoxyC. EdC labeled DNA is orange. This figure has been modified from Dembowski and DeLuca

Materials

List of materials used in this article
Name	Company	Catalog Number	Comments
MRC-5 cells	ATCC	CCL-171
Fetal Bovine Serum (FBS)	Gibco	26140-179
Dulbecco's Modified Eagle Medium (DMEM)	Gibco	12800-082	Substituted with 10% FBS, 2 mM L-glutamine, 12 mM (for growth in flasks) or 30 mM (for growth in dishes) sodium-bicarbinate
600 cm² tissue culture dish	Thermo Fisher Scientific	166508
Tris Buffered Saline (TBS), pH 7.4			137 mM NaCl, 5 mM KCl, 491 mM MgCl, 680 mM CaCl, 25.1 mM Tricine
HSV-1 stock			Stocks with titers greater than 1x109 PFU/mL work best
5´-Ethynyl-2´-deoxycytidine (EdC)	Sigma-Aldrich	T511307	Dissolve in DMSO to prepare 40 mM stock, aliquot, and store at -20 °C
Cell culture incubator
Biosafety cabinet
Heat blocks			65 °C and 95 °C