This video demonstrates a method for monitoring infected cell protein 0, ICP0 trafficking in herpes simplex virus-1 infection. Post-de novo synthesis, ICP0 translocates to the nucleus, later moving to the cytoplasm during infection progression. Immunofluorescence microscopy reveals and analyzes the protein's subcellular localization, offering insights into its trafficking across infection phases.
Protocol
1. Cell Seeding and Virus Infection At 20–24 h before the virus infection, seed 5 x 104 of human embryonic lung (HEL) fibroblast cells or other cells to be examined on a 4-well 11 mm staggered slide in growth medium (Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS)). Incubate the cells at 37 °C with 5% carbon dioxide (CO2). NOTE: Each well should have 70-80% cell confluency at the time of i…
Representative Results
Figure 1: Dynamic trafficking of ICP0 during HSV-1 infection. HEL cells grown on 4-well slides were infected with prototype HSV-1 (strain F) at 10 pfu/cell. At 1, 5, and 9 h post infection (hpi), cells were fixed, permeabilized, and reacted to rabbit anti-ICP0 and mouse anti-PML primary antibodies, and then reacted to Alexa 594-conjugated anti-rabbit and Alexa 488-cojugated anti-mouse second…
Divulgazioni
The authors have nothing to disclose.
Materials
Cells and viruses
Human Embryonic Lung fibroblasts (HEL Cells)
Dr. Thomas E. Shenk (Princeton University)
HEL cells were grown in DMEM supplemented with 10% FBS
HSV-1 viral Stock (Strain F)
Dr. Bernard Roizman Lab
Medium
Dulbecco's modified Eagle's medium (DMEM)
Invitrogen
11965-092
Fetal Bovine Serum (FBS)
Sigma
F0926-500ml
Medium-199 (10X)
Gibco
11825-015
Reagents
4- well 11 mm staggered slide
Cel-Line/Thermofisher Scientific
30-149H-BLACK
16% Paraformaldehyde solution(w/v) Methanol free
Thermo Scientific
28908
Triton X-100
Fisher reagents
BP151-1C0
Bovine Serum Abumin (BSA)
Calbiochem
CAS 9048-46-8
Horse Serum
Sigma
H1270
Phosphate Buffered Saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, pH7.4)
Dr. Haidong Gu lab
NaCl
Fisher Bioreagent
BP358-212
KH2PO4
Fisher Bioreagent
BP362-500
KCl
Fisher Scientific
BP366-500
Na2HPO4
Fisher Bioreagent
BP332-500
Blocking buffer (PBS with 1% BSA and 5% Horse serum )