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@article{Serwa:2019:10.1371/journal.ppat.1007956,
author = {Serwa, RA and Sekine, E and Brown, J and Teo, SHC and Tate, EW and O'Hare, P},
doi = {10.1371/journal.ppat.1007956},
journal = {PLoS Pathogens},
title = {Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry},
url = {http://dx.doi.org/10.1371/journal.ppat.1007956},
volume = {15},
year = {2019}
}
TY - JOUR
AB - We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together w
AU - Serwa,RA
AU - Sekine,E
AU - Brown,J
AU - Teo,SHC
AU - Tate,EW
AU - O'Hare,P
DO - 10.1371/journal.ppat.1007956
PY - 2019///
SN - 1553-7366
TI - Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry
T2 - PLoS Pathogens
UR - http://dx.doi.org/10.1371/journal.ppat.1007956
UR - https://www.ncbi.nlm.nih.gov/pubmed/31589653
UR - http://hdl.handle.net/10044/1/74102
VL - 15
ER -
Prof. Ed Tate
GSK Chair in Chemical Biology
Department of Chemistry
Molecular Sciences Research Hub, White City Campus,
82 Wood Lane, London, W12 0BZ
e.tate@imperial.ac.uk
Tel: +44 (0)20 759 + ext 43752 or 45821