An Immunofluorescence Technique for Viral Protein Localization in Infected Cells

In This Article

Abstract

Source: Samrat, S. K. et al., Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy. J. Vis. Exp. (2018)

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 infection.
On the next day, remove the growth medium and infect the cells with viruses in Medium-199 at a range of 4–10 pfu/cell. Incubate virus....

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Results

PML and ICP0 colocalization analysis, time-lapse microscopy diagram, viral protein interaction study.
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 a.......