Abstract
We used the bioorthogonal protein precursor, homopropargylglycine (HPG) and chemical ligation to fluorescent capture agents, to define spatiotemporal regulation of global translation during herpes simplex virus (HSV) cell-to-cell spread at single cell resolution. Translational activity was spatially stratified during advancing infection, with distal uninfected cells showing normal levels of translation, surrounding zones at the earliest stages of infection with profound global shutoff. These cells further surround previously infected cells with restored translation close to levels in uninfected cells, reflecting a very early biphasic switch in translational control. While this process was dependent on the virion host shutoff (vhs) function, in certain cell types we also observed temporally altered efficiency of shutoff whereby during early transmission, naïve cells initially exhibited resistance to shutoff but as infection advanced, naïve target cells succumbed to more extensive translational suppression. This may reflect spatiotemporal variation in the balance of oscillating suppression-recovery phases. Our results also strongly indicate that a single particle of HSV-2, can promote pronounced global shutoff. We also demonstrate that the vhs interacting factor, eIF4H, an RNA helicase accessory factor, switches from cytoplasmic to nuclear localisation precisely correlating with the initial shutdown of translation. However translational recovery occurs despite sustained eIF4H nuclear accumulation, indicating a qualitative change in the translational apparatus before and after suppression. Modelling simulations of high multiplicity infection reveal limitations in assessing translational activity due to sampling frequency in population studies and how analysis at the single cell level overcomes such limitations. The work reveals new insight and a revised model of translational manipulation during advancing infection which has important implications both mechanistically and with regards to the physiological role of translational control during virus propagation. The work also demonstrates the potential of bioorthogonal chemistry for single cell analysis of cellular metabolic processes during advancing infections in other virus systems.
Highlights
Much of our understanding of the molecular mechanisms operating during virus infection comes from population studies
While protein synthesis has been generally studied by methods that investigate the average behaviour in cell populations, information at the individual cell level is critical for a true understanding of the processes governing infection
A schematic indicating the principle of HPG incorporation into proteins and ligation with azide-linked fluorophores is summarised in S1a Fig. Much accumulated data has demonstrated that HPG has no effect on global rates of protein synthesis nor protein degradation [42,43,44,45,46,47,48]
Summary
Much of our understanding of the molecular mechanisms operating during virus infection comes from population studies. The classic single-step virus growth cycle, the identification and characterisation of virus encoded transcripts and proteins, and the associated mechanisms governing temporal regulation of their production and turnover have been founded on population studies of infected cells in culture systems [1]. It is becoming clear in many fields that while analysis of the average behaviour in total infected cell populations is vital, information at the individual cell level is critical for a true understanding of the processes governing the outcomes of infection. The chemical pairs most routinely used are the azide- and alkyne moieties which are small, inert and can be introduced to a variety of precursors [28, 30,31,32,33]
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