Analysis of a fully infectious bio-orthogonally modified human virus reveals novel features of virus cell entry

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 revealing 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 or primary entering from secondary assembling, 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 with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, the work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions. Author Summary Bio-orthogonal chemistry and the application of non-canonical amino acid tagging (BONCAT) has opened opportunities for analysis of translational control, protein trafficking and modification in studies of infection and immunity. We expand on our earlier work, reporting the tractable, scalable production and analysis of a large structurally complex enveloped human virus that incorporates non-canonical amino acids into structural proteins from all parts of the virus particle. Thus in the complex translationally altered environment of a herpesvirus infected cell, non-canonical amino acid incorporation has no significant functional effect on the multiple cellular and viral functions required to assemble infectious virions. We further demonstrate the normal recruitment and stochiometries of all detected structural proteins, which combined with data showing unaltered virus yields and infectivity, indicates that the non-canonical residues in the various structural proteins have no effect on their subsequent ability to infect cells. Focusing on spatial analysis of virus entry, we provide examples and reveal novel insight into virus entry and processing. Together with previous reports using bio-orthogonal chemistry in studies of virus infection the potential applications of BMVs are considerable.