Date Published: July 20, 2018
Publisher: Public Library of Science
Author(s): Catherine Su Hui Teo, Peter O’Hare, James R. Smiley.
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.
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 . However, 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 also critical for a true understanding of the processes governing the outcomes of infection. Such analyses may support and refine conclusions from population studies, but can also yield results which are not accounted for in population studies and provide conceptually new mechanistic insight [2–5]. In this regard, while much effort has focussed on analysis of levels and variations in transcription patterns at the single cell level, we know much less with regard to protein synthesis. All viruses manipulate the host cell translational apparatus to promote the synthesis of their proteins and to supress cellular antiviral responses. At the same time, cells modulate both their qualitative translational output and their translational apparatus in the attempt to suppress virus replication [6–15]. Thus, overall infected cell protein synthesis results from a complex and temporally regulated interplay of multiple distinct translational objectives for the host and virus, in addition to selective controls on the abundance and localisation of individual protein species. However, global protein synthesis has been almost universally studied by population methods such as gel electrophoresis and autoradiography, Western blotting or mass spectrometry, potentially masking dynamic and diverse individual cell behaviour [16–21]. A complete understanding of infected cell protein metabolism requires a parallel approach to spatial aspects of protein synthesis and temporal alterations in these processes at the single cell level during the progression of infection. Traditional steady-state analysis using antibodies, or gene fusion to fluorescent proteins, provide powerful tools for the investigation of individual proteins [22–24]. However, global spatial analysis requires a different approach. Recent advances in bioorthogonal chemistry  have facilitated the development of new techniques based on the in vivo incorporation of metabolic precursors containing designed chemical end-groups. Subsequent highly specific covalent bond-forming reactions, commonly termed “click chemistry”, then link the macromolecular products incorporating these precursors to capture reagents via a dedicated, paired end-group [26–29]. 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, 30–33].