Abstract
Cell biological studies of a range of viruses have led to the discovery of virus-induced cellular compartments. In the case of herpes simplex virus type 1 (HSV-1), novel and discrete domains have been identified throughout the cytoplasm of infected cells, but their nature and purpose remain to be defined. The tegument protein UL46 has previously been observed to localise at specific cytoplasmic sites during infection with a number of HSV-1 strains. Here, a recombinant HSV-1 capable of expressing EGFP tagged UL46 (G46v) was developed and characterised, to be used as a tool for studying these HSV-1 induced cytoplasmic domains.
Mass spectrometry analysis of EGFP-UL46 complexes isolated from infected cell lysate revealed UL46 interacted with a range of cytoskeletal and ER-associated proteins. STED super-resolution microscopy further indicated a close association between cytoplasmic domains containing EGFP-UL46 and microtubules. Mobility studies showed two kinetic populations of UL46 reside within these domains, one that was stably associated, while the remainder were dynamically associated. These domains also appeared to contain separate sub-compartments, where different virus proteins were segregated. A number of proteins destined to be assembled into the tegument of the virion were observed to be targeted to these domains, including the minor tegument proteins ICP0 and ICP4, along with the major tegument protein UL49. Interestingly, mass spectrometry analysis of enriched EGFP-UL46 domains also revealed the presence of capsid and inner tegument proteins at these sites. Conversely, viral glycoprotein-studded membranes which form the virion envelope were not observed at these sites, and no viral glycoproteins were detected during mass spectrometry analysis. The formation of these domains was blocked when viral genome replication was inhibited, and during infection with a deletion virus defective in progeny capsid export to the cytoplasm (ΔUL34).
We propose that these virus-induced structures may represent sites through which capsids traffic to acquire tegument proteins along the pathway of virion assembly.