Abstract
We used pulse-labeling with the methionine analogue homopropargylglycine (HPG) to investigate spatiotemporal aspects of protein synthesis during herpes simplex virus (HSV) infection. In vivo incorporation of HPG enables subsequent selective coupling of fluorochrome-capture reagents to newly synthesised proteins. We demonstrate that HPG labeling had no effect on cell viability, on accumulation of test early or late viral proteins, or on overall virus yields. HPG pulse-labeling followed by SDS-PAGE analysis confirmed incorporation into newly synthesised proteins, while parallel processing by in situ cycloaddition revealed new insight into spatiotemporal aspects of protein localisation during infection. A striking feature was the rapid accumulation of newly synthesised proteins not only in a general nuclear pattern but additionally in newly forming sub-compartments represented by small discrete foci. These newly synthesised protein domains (NPDs) were similar in size and morphology to PML domains but were more numerous, and whereas PML domains were progressively disrupted, NPDs were progressively induced and persisted. Immediate-early proteins ICP4 and ICP0 were excluded from NPDs, but using an ICP0 mutant defective in PML disruption, we show a clear spatial relationship between NPDs and PML domains with NPDs frequently forming immediately adjacent and co-joining persisting PML domains. Further analysis of location of the chaperone Hsc70 demonstrated that while NPDs formed early in infection without overt Hsc70 recruitment, later in infection Hsc70 showed pronounced recruitment frequently in a coat-like fashion around NPDs. Moreover, while ICP4 and ICP0 were excluded from NPDs, ICP22 showed selective recruitment. Our data indicate that NPDs represent early recruitment of host and viral de novo translated protein to distinct structural entities which are precursors to the previously described VICE domains involved in protein quality control in the nucleus, and reveal new features from which we propose spatially linked platforms of newly synthesised protein processing after nuclear import.
Highlights
The manipulation of cellular metabolic processes during virus infection promotes or tempers virus production and determines the outcome of infection at the cellular level and e.g., acute versus long-term persistence, latency, reactivation and transmission [1]
We provide new insight into protein metabolism in herpes simplex virus infected cells which is not approachable by standard methods
We report the formation of novel subnuclear domains termed newly synthesised protein domains (NPDs) with a spatial link to pre-existing nuclear PML domains and to previously described domains involved in protein quality control
Summary
The manipulation of cellular metabolic processes during virus infection promotes or tempers virus production and determines the outcome of infection at the cellular level and e.g., acute versus long-term persistence, latency, reactivation and transmission [1]. Recent advances in global proteomic approaches and mass spectrometry methods have provided broad insight into the synthesis, modification and degradation of viral and host proteins as infection progresses [3,4,5,6,7,8] These studies reveal alterations of cellular pathways including for example, the remodeling of glycolytic and metabolic pathways [9], inflammatory and innate immune response factors [6,10] or nucleotide and RNA processing pathways [11]. One method to visualise total nascent protein synthesis relies on the incorporation of puromycin, an aminonucleoside antibiotic, either using a fluorescent derivative of puromycin [15] or by the detection of polypeptide-puromycin conjugates using anti-puromycin antibodies [16] This approach has yielded insight in the spatial analysis of cellular protein synthesis and modulation during bacterial [17] and viral infection [18]. There are disadvantages for spatial analysis of nascent proteins including low signal-noise ratios, qualitative differences with expected patterns [15,19,20] and importantly that puromycin is a tRNA mimetic that terminates translation, perturbing the system and eliminating the possibility of spatiotemporal analysis of fully translated proteins in e.g. pulse-chase experiments
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