The use of fluorescence microscopy to study the association between herpesviruses and intrinsic resistance factors

Abstract

Intrinsic antiviral resistance is a branch of antiviral defence that involves constitutively expressed cellular proteins that act within individual infected cells. In recent years it has been discovered that components of cellular nuclear structures known as ND10 or PML nuclear bodies contribute to intrinsic resistance against a variety of viruses, notably of the herpesvirus family. Several ND10 components are rapidly recruited to sites that are closely associated with herpes simplex virus type 1 (HSV-1) genomes during the earliest stages of infection, and this property correlates with the efficiency of ND10 mediated restriction of HSV-1 replication. Similar but distinct recruitment of certain DNA damage response proteins also occurs during infection. These recruitment events are inhibited in a normal wild type HSV-1 infection by the viral regulatory protein ICP0. HSV‑1 mutants that do not express ICP0 are highly susceptible to repression through intrinsic resistance factors, but they replicate more efficiently in cells depleted of certain ND10 proteins or in which ND10 component recruitment is inefficient. This article presents the background to this recruitment phenomenon and summaries how it is conveniently studied by fluorescence microscopy.

Keywords: Herpes Simplex Virus type 1; ICP0; ND10; PML nuclear bodies; SUMO; intrinsic antiviral resistance.

Figure 1

Examples of recruitment of ND10 components to sites associated with HSV-1 genomes. (A) A view of an edge of a developing plaque. The centre of the plaque is down and left of the lower left corner of the image. The cells were infected with an ICP0‑deficient virus expressing EYFP-linked ICP4 (green) then developing plaques were examined 24 h later. PML was detected using anti-PML monoclonal antibody 5E10 (red). The numbered cells show different stages of recruitment as infection develops: cell 1—asymmetric distribution of PML can occur before a cell has detectable ICP4 expression (compare with the random distribution of PML foci in the cell to the right of cell 3 and in the bottom row of panel 3); cell 2—asymmetric distribution of PML with very small ICP4 foci; cell 3—multiple small ICP4 and PML foci; cell 4—larger ICP4 foci, with overlapping strong PML foci; cell 5—a cell in the mid stages of infection with developing replication compartments associated with small remaining foci of PML. (B) More detailed examples of ICP4 association with PML (top row), hDaxx (in the same cell, middle row) in comparison with an uninfected cell (bottom row). (C) Use of a cell line depleted for endogenous PML and reconstructed with expression of EYFP-linked PML.VI (green), an isoform that lacks the SIM. The cells were infected with ICP0 null mutant HSV-1 and stained for ICP4 expression (monoclonal antibody 58S, red) and hDaxx (rabbit polyclonal antibody from Upstate, blue). Note efficient recruitment of hDaxx, despite the lack of any recruitment of PML.VI. (D) Recruitment of the DNA damage response protein 53BP1 (blue) to regions in close proximity to ICP4 foci (EYFP-ICP4, green in cells infected with ICP0 null mutant virus expressing EYFP-linked ICP4. PML (red) is recruited to ICP4 associated foci that are distinct from those of 53BP1. The scale bars show 10 μm in A, C and D, 5 μm in B.