Abstract
Herpesviruses are large and complex viruses that have a long history of coevolution with their host species. One important factor in the virus–host interaction is the alteration of intracellular morphology during viral replication with critical implications for viral assembly. However, the details of this remodeling event are not well understood, in part because insufficient tools are available to deconstruct this highly heterogeneous process. To provide an accurate and reliable method of investigating the spatiotemporal dynamics of virus-induced changes to cellular architecture, we constructed a dual-fluorescent reporter virus that enabled us to classify four distinct stages in the infection cycle of herpes simplex virus-1 at the single cell level. This timestamping method can accurately track the infection cycle across a wide range of multiplicities of infection. We used high-resolution fluorescence microscopy analysis of cellular structures in live and fixed cells in concert with our reporter virus to generate a detailed and chronological overview of the spatial and temporal reorganization during viral replication. The highly orchestrated and striking relocation of many organelles around the compartments of secondary envelopment during transition from early to late gene expression suggests that the reshaping of these compartments is essential for virus assembly. We furthermore find that accumulation of HSV-1 capsids in the cytoplasm is accompanied by fragmentation of the Golgi apparatus with potential impact on the late steps of viral assembly. We anticipate that in the future similar tools can be systematically applied for the systems-level analysis of intracellular morphology during replication of other viruses.
Highlights
Herpesviruses are large DNA viruses that are typified by their ability to establish both lytic and latent infection cycles
infected cell protein (ICP0) is one of five immediate early proteins in herpes simplex virus-1 (HSV-1), and it begins to be expressed as soon as the viral genome is deposited inside the nucleus of the host cell
Single-step growth curve analysis demonstrated nearly identical replication kinetics for the timestamp reporter and wild-type HSV-1 (Fig. S1), suggesting fusion of fluorescent proteins to ICP0, and glycoprotein C (gC) does not impair pathways involved in the replication cycle of HSV-1
Summary
For a direct visual readout of the replication state on the single cell level, we developed a fluorescent reporter virus (Fig. 1A). These observations indicate that Rab vesicles cluster at gC-rich compartments (red arrows in Fig. 3C) and that Rab is taken up by those compartments that contain the viral glycoprotein gC. Expansion microscopy [19] involves physical, isotropic swelling of fixed samples that can provide up to 64- to 80-fold increased sample volume, effectively providing super resolution imaging data using conventional fluorescence microscopes (Fig. S5) Using this technique, we demonstrate that mitochondria and viral assembly compartments are intimately intertwined, with the mitochondria interlacing the gC-enriched membranes (Fig. 4B). This number is dramatically increased in cells with fragmented Golgi compartment (corresponding to stage 4) with an average number of 51 capsids per cell
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