Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process.
Synopsis
Regulation of subcellular organization and transport is essential for control of crucial biological processes. However, our knowledge often is hampered because these processes tend to be transient and difficult to study. Studies of how opportunistic microbes hijack cellular machinery have provided insights into various normal cell processes. For example, studies with intracellular microorganisms, such as Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, have significantly increased our understanding of the dynamic nature of the actin cytoskeleton. However, much less is known about subcellular organization and transport of cargo in the nucleus. The authors have discovered that alpha-herpesvirus infection of neurons leads to the transient formation of actin filaments in the nucleus. These filaments do not fill the nucleus, but rather associate with newly formed viral capsids. The nuclear actin filaments were initially identified in peripheral nervous system tissue using a new imaging technology, serial section scanning electron microscopy pioneered by Winfried Denk (a co-author). Their results suggest that nuclear actin filaments form as part of a general stress response to infection, but then are co-opted, perhaps to direct capsid transport to sites of budding along the nuclear envelope. This work illuminates a less well understood part of the viral life cycle and sets the stage for future work investigating control of how cargo is organized and moved in the nucleus.
