Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Summary

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Abstract

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

Introduction

Herpes simplex virus 1 (HSV-1) causes a wide range of mild to severe herpetic diseases including herpes labialis, genital herpes, stromal keratitis, and encephalitis. Once infected, the virus establishes a lifelong latent infection in ganglia neurons. Occasionally, the virus can be reactivated by various reasons such as fever, stress, and immune suppression¹, leading to recurrent herpes infection. Infected cell protein 0 (ICP0) is a key viral regulator crucial for both lytic and latent HSV-1 infection. It transactivates downstream virus genes via counteracting the host intrinsic/innate antiviral defenses^2,<....

Protocol

1. Cell Seeding and Virus Infection

1. 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.
2. 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-infected cells for 1 h at 37 ....

Results

To understand the molecular basis and biological functions of ICP0 trafficking during HSV-1 infection, we use an immunofluorescent microscopy method to analyze ICP0 subcellular distribution at different infection phases. Figure 1 shows the representative cells with distinctive ICP0 localization as the infection progresses. To quantify the nuclear-to-cytoplasmic translocation of ICP0, we analyze ICP0 distribution relative to the nucleus by categorizing infecte.......

Discussion

This protocol has been used to study the nuclear-to-cytoplasmic translocation of HSV-1 ICP0. ICP0 undergoes subcellular trafficking during HSV-1 infection (Figure 1). Likely, ICP0 interacts with various cell pathways to carry out different functions at different locations. This enables ICP0 to fine tune its multiple functions in the tug-of-war with human host¹³. However, how ICP0 coordinates the multiple functions in a spatial-temporal manner has not been well studied.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 )	Dr. Haidong Gu lab
Rabiit Anti-ICP0 antibody	Dr. Haidong Gu lab
PML (PG-M3)-Mouse monoclonal IgG	santa Cruz Biotechnology	SC-966
Alexa Fluor 594-goat anti-rabbit IgG	invitrogen	A11012
Alexa Fluor 488-goat anti-mouse IgG	invitrogen	A11001
Vectashield Mouting medium with DAPI	Vector laboratories	H-1200
Pasteur pipette	Fisher Brand	13-678-20D
Nail Polish	Sally Hansen
Equipment
Confocal Microscope	Leica SP8
Confocal Software	Leica LAS X Application suite
Excel software	Microsoft Excel
HERAcell 150i CO2 incubator	Thermo Scientific	Order code 51026282