eIF4H nuclear relocalisation is coupled to the suppression of global protein synthesis.
(A) Cells were infected with HSV-2[186] according to the standard workflow and analysed (25 hr) for newly synthesised protein (green channel) or eIF4H localisation (red channel). Uninfected cells are at the topmost region of the panel with infection emanating from bottom left (as reflected by diagnostic NPD formation). White asterisks (DAPI, green channels) denote the cells in shutoff zone, demarcated by the dotted line. Diagonal arrows denote the same cells showing relocalisation of eIF4H to the nucleus in the shutoff zone while cells in the topmost region show cytoplasmic localisation. (B) For single step analysis, cells were synchronously infected with HSV-1[KOS] at MOI 10 and labeled at 2 hr p.i. for newly synthesised proteins (green) and stained in parallel for eIF4H (red). Diagonal arrows (in eIF4H panel) indicate the nuclei for mock-infected cells and infected cells respectively and the dramatic shift in relative eIF4H localisation in infected cells which are now actively synthesising proteins and exhibit NPD formation. (C) Magnified view of individual cells showing a mock-infected cell with typical eIF4H cytoplasmic localisation compared to high MOI infection showing cells in the shutoff phase (2nd row) or recovery phase with restored protein synthesis, nucleolar import, NPD formation (3rd row). eIF4H is relocalised to the nucleus during shutoff and retained there during translational recovery. (D, E) Infection as standard for localisation after low MOI infection and transmission (as in Fig 1b). Monolayers were infected with 50 pfu of ΔUL41-REP (D) or its mutant strain ΔUL41 (E) and labeled for protein synthesis (25 hr p.i.). Cells were then analysed for total protein synthesis and eIF4H localisation. The focal origin of infection is denoted in the green channel by a white semicircle with emanating arrows. For ΔUL41-REP, white asterisks (DAPI, green channels) denote cells in the shutoff zone, exhibiting pronounced translational suppression. These same cells are indicated for eIF4H localization (red channel). Asterisks are omitted in the merged channel. For the mutant ΔUL41, protein synthesis levels and localisation were observed together with progressive NPD formation, no shutoff zone was observed nor was there any relocalisation of eIF4H at the boundary of advancing infection.
