Abstract
Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid–liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid–liquid phase-separated condensates.
Highlights
DNA virus viral replication compartments (VRCs) have liquid-like properties and that their formation and maintenance is driven by viral IDPs (vIDPs) that can phase-separate
We used the VSL2 algorithm provided by the online platform predictor of natural disordered regions (PONDR) to evaluate the overall percentage disorder of 71 Herpes Simplex Virus Type-1 (HSV-1) proteins in silico and compared them with known viral and human intrinsically disordered protein (IDP) that have been shown to phase-separate in cells (Figure 1a and Tables S1–S3)
Confirming the previous findings of McSwiggen et al [13], we found that all HSV-1 IE proteins are generally highly disordered, rendering this class of viral proteins ideal candidate vIDPs
Summary
Replication of DNA viruses, such as herpesvirus, parvovirus, papillomavirus, or adenovirus, occurs in distinct inclusions within the nucleus These inclusions are called viral replication compartments (VRCs) and constitute membrane-less, spherical domains where viral transcription, viral DNA (vDNA) replication, virion assembly, and genome packaging occur (reviewed in [1,2,3,4]). From DNA viruses, most RNA viruses replicate in the cytoplasm of susceptible cells, where they form membrane-bound or membrane-less viral assemblies Such membrane-less viral compartments in the cytoplasm are called viral factories or viroplasms and, to DNA viruses, constitute distinct domains where genome replication and packaging occur (reviewed in [5]). The formation of VSV factories is driven by the condensation of the viral phosphoprotein (P) together with the nucleocapsid protein (N) and the multifunctional large protein (L) via liquid–liquid phase separation (LLPS) [6]
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