Fig Nuclear accumulation of cellular proteins synthesised prior to infection.

<p>Uninfected Vero cells were pre-labeled with HPG (30 min pulse) followed by an infection with HSV-1 at an MOI of 10 for 4 hr (HSV-1) or mock infection (Mock). Cells were then fixed and stained for Hsc70, followed by click reactions. Cells containing Hsc70 foci were identified and the subnuclear localisation of newly synthesised proteins (green) and Hsc70 (red) are shown with diagonal arrowheads indicating the colocalisation of nuclear NPDs and Hsc70 foci as discussed in the text.</p

Schematic model of the spatial relationship between NPDs, PML domains and VICE domains and potential processing pathways of newly synthesised proteins.

<p>The dark red shading indicates PML domains in uninfected cells and their functioning with dynamic recruitment and dissociation of target proteins (arrows in and out). The transparent lighter red shading in HSV infected cells indicates their progressive structural and functional disruption. NPDs are formed de novo only after infection (A) or in model (B), the possibility of pre-existing functional NPDs is indicated. Like PML domains, these could represent sites of dynamic protein recruitment and onward transport. As such they would not accumulate bulk newly synthesised proteins, and are thus indicated with transparent, light green shading. Their functional disruption, or overload, in infected cells is indicated by the dark green shading, firstly as smaller domains recruiting selectively ICP22, and only later in infection recruiting additional proteins including components of the host cell protein quality control machinery and in particular Hsc70, the diagnostic marker of VICE domain formation. As discussed in the text, small arrows (NPD,a) indicate impaired forward transport from NPDs and crosses (NPD,b) indicating a more complete block of transport of client proteins after continual recruitment. NPD,c, indicates a more pronounced forward transport after recruitment of certain classes of client proteins. ICP22 (magenta sphere), but not Hsc70, are recruited to NPDs during early stage of infection. As infection progresses, bulk Hsc70 (yellow diamond), and polyubiquitinated species together with ICP22, accumulates at NPDs of larger size.</p

NPDs form adjacent to ICP0/PML domains.

<p>(A) Vero cells were infected and processed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005927#ppat.1005927.g008" target="_blank">Fig 8</a>, in this case for simultaneous localisation of newly synthesised proteins (i, green), PML (ii, red) and ICP0 (iii, blue). The higher magnification insert emphasises the juxtaposition of PML and ICP0 in relation to NPDs. (B) Typical examples of merged channels showing precise colocalisation of mutant ICP0 with PML (purple) and the juxtaposition with NPDs (green).</p

Biochemical analysis of newly synthesised proteins using HPG and click chemistry.

<p>(A) Schematic diagram illustrating comparative structures of methionine and HPG. The scheme indicates the in vivo incorporation of HPG into protein (solid black dots within a protein chain) and then the subsequent in vitro cycloaddition reaction to covalently cross link an azide fluorochrome-coupled capture reagent (coloured star) to HPG. (B) Mock-infected Vero cells were pulse-labeled using 1 mM HPG for 1 hr, lysed and subjected to click reactions using IRDye 800CW Azide Infrared Dye. Proteins were separated by SDS-PAGE and visualised by in-gel fluorescence using a LI-COR Odyssey Infrared Imaging System. Control experiments were carried out either in the absence of HPG (lane 5) or in the presence of 100 μg/ml of CHX (lane 4). Lanes 1–3 represent the total Coomassie blue staining protein profile and lanes 4–6 the in-gel fluorescence profile of the identical gel. (C) Cell viability of uninfected Vero cells (% live cells, in triplicate) was assessed by trypan blue exclusion. Control cultures were subject to either no methionine depletion and incubation in standard methionine-containing medium (Con; white bar) or methionine depletion with subsequent incubation in standard methionine-containing medium (Met; light grey bar); while HPG labelling was performed after methionine depletion with subsequent incubation in HPG -containing medium (30 min pulse). (D) In-gel fluorescence of newly synthesised proteins in total, cytosolic and nuclear fractions. Mock or HSV infected Vero cells (MOI 10) were pulse-labeled at the times indicated for 1 hr, lysed and fractionated prior to click reaction. Equal concentrations of proteins (20 μg, representing a 4-fold increased loading by cell equivalents for the nuclear fraction) were resolved by SDS-PAGE, and proteins visualised using a LI-COR Odyssey Infrared Imaging System scanned into the green channel. (E) The same gel was stained with Coomassie brilliant blue for total protein detection. Representative host cell proteins enriched within the cytoplasmic and nuclear fractions are labeled HC and HN respectively. (F) The same samples after separation by SDS-PAGE where transferred to a nitrocellulose membrane. Total steady-state levels of candidate viral proteins, ICP4, ICP0 and VP5 (red) were simultaneously detected using monoclonal antibodies, and newly synthesised proteins (green) were visualised on the blot.</p

ICP22 localises to NPDs and phase-dense nuclear bodies.

<p>Infected Vero cells were either untreated (standard media; control) or HPG pulse-labeled for 30 min at 2 hr p.i. (HPG), fixed and analysed by fluorescence (for newly synthesised proteins (green) and ICP22 (red) and by phase microscopy. Diagonal arrowheads indicate the colocalisation of NPDs with ICP22 as well as phase-dense nuclear domains. The inset shows the precise co-localisation of ICP22 with NPDs. The punctate localisation of ICP22, and its recruitment into phase dense bodies was independent of HPG pulse-labeling and also observed in the control infected cultures in the absence of HPG(Control).</p

NPDs juxtapose to and overlap with PML domains in HSV-1 ICP0[FXE] infected cells.

<p>Vero cells were infected with ICP0 RING-finger mutated HSV-1 strain FXE, pulse-labeled with HPG for 30 min at 4 hr p.i. and analysed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005927#ppat.1005927.g007" target="_blank">Fig 7</a>. Several typical fields are illustrated. The clear spatial relationship between PML domains and NPDs are indicated in the white squares. While many persisting PML domains were associated with NPDs, NPDs were in excess of PML domains. Quantification data of NPD/PML association as discussed in the text are presented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005927#ppat.1005927.s001" target="_blank">S1D–S1E Fig</a>.</p

Polyubiquitinated species are recruited to NPDs at later stages of infection.

<p>Vero cells were mock-infected or infected with HSV-1, pulse-labeled with HPG for 30 min at the times indicated, fixed and analysed for polyubiquitinated species (FK2 localisation, red) and newly synthesised protein (green). In mock infected cells HPG localised in a generally diffuse pattern with some nuclear la accumulation while polyubiquitinated species were found in a speckled diffuse nuclear pattern with variable numbers of discrete foci. NPD formation in infected cells is indicated by diagonal white arrowheads and early in infection NPDs show no colocalisation with FK2+ve foci. Conversely small vertical arrows indicate FK2+ve foci which show no obvious spatial relationship with NPDs. As described in the text (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005927#ppat.1005927.g010" target="_blank">Fig 10</a>) a subset of NPDs localised adjacent to, co-joining FK2+ foci (see inserts of the merged fields). Diagonal arrowheads later in infection show prominent co-localisation between NPDs and FK2+ve species, either as in example cell a, as co-joining asymmetric foci or frequently, as in example cell b, with virtually complete overlap FK2+ve species coating the exterior of the NPDs</p

NPDs do not generally colocalise with viral IE protein ICP0.

<p>Vero cells were infected with HSV-1, pulse-labeled for 30 min with HPG at 1 2 or 4 hr p.i., fixed and analysed for ICP0 (red) and newly synthesised protein (green). Arrows point to the red ICP0 foci on the merged image and are superimposed on the HPG channel to show that the NPDs do not precisely colocalise with ICP0 foci, but rather are frequently juxtaposed.</p

Spatial relationship between NPDs with SUMO and FK2-ubiquitin accumulation.

<p>Vero cells were infected and processed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005927#ppat.1005927.g008" target="_blank">Fig 8</a> for simultaneous detection of newly synthesised proteins (green) and total SUMO-modified (red) or ubiquitinated proteins (blue). Diagonal arrows denote example NPDs which have no obvious association with SUMO-containing foci. These latter foci do contain ubiquitinated species (horizontal arrows). For ease of inspection, the lower two fields show HPG versus SUMO alone, and HPG versus SUMO versus ubiquitin.</p

Imaging newly synthesised proteins by HPG incorporation and click chemistry.

<p>Uninfected Vero cells were pulse-labeled using 0.5 mM HPG for 30 min, fixed and subjected to click reaction using the Alexa Fluor 488-azide capture agent. Control experiments were performed either in the absence of HPG or using 100 μg/ml of CHX added prior to and during the pulse. Images were recorded as described in materials and methods.</p