Spatiotemporal esolution of global protein synthesis during herpes simplex virus infection using bioorthogonal precursors and click chemistry
File(s)
Author(s)
Teo, Su Hui Catherine
Type
Thesis
Abstract
Herpes simplex virus (HSV) modulates the host cell’s proteome and transcriptome during infection to fulfil the needs of the virus for productive replication and transmission. Using bioorthogonal precursors and click chemistry, I have examined spatiotemporal aspects of global protein synthesis during single step replication and cell-to-cell transmission with results revealing new insights into the complex spatial interplay between translational control processes, protein localisation and transcription during HSV infection. For the first time, translational suppression and recovery is visualised at the single cell level, reflecting a very early biphasic switch in translational control. The biphasic switch is dependent on the RNase activity of HSV virion host shutoff protein (vhs), and vhs also mediates eIF4H nuclear translocation coupled to the initial suppression. During translational recovery, my results also show rapid accumulation of newly synthesised proteins in novel subnuclear domains termed newly synthesised protein domains (NPDs). ICP22 is specifically required for NPD formation and shows selective recruitment to these domains. Additionally, a host protein, SSRP1, is also displaced from its normal localisation in the nucleolus and selectively recruited to NPDs very early in infection, dependent on ICP22 expression. Furthermore, spatial analysis of newly transcribed RNA also reveals that early after infection transcripts accumulate in irregular structures termed RNA islands which form distinct populations. While a fraction of RNA islands localises juxtaposed with a subpopulation of NPDs, the majority of this newly synthesised RNA shows precise colocalisation in distinct domains which also recruits a cellular RNA helicase, p68. These later RNA domains adjoin foci containing the immediate-early transcriptional regulator ICP4 in a specifically organised manner. In addition to profound qualitative changes observed during translational recovery, quantitative proteomics and identification of the newly synthesised nuclear proteome using HPG pulsed-SILAC with LC- MS/MS indicates coordinated mobilisation of distinct cellular pathways, in particular eIF2 signalling pathway and RNA post-transcriptional modification. These results reveal new features of protein metabolism during infection, and involvement of ICP22 in newly synthesised protein processing pathways.
Version
Open Access
Date Issued
2018-05
Date Awarded
2018-09
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
O'Hare, Peter
Sancho Medina, Ana
Sponsor
Imperial College London
Grant Number
F22415
Publisher Department
Department of Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)