A fluorescent reporter system enables spatiotemporal analysis of host cell modification during herpes simplex virus-1 replication

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.

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection

“…2B). Stage 4 is defined by the dispersal of gC-mCherry throughout the cytoplasm and, given the colocalization of gC with Golgi markers 58K protein and beta-1,4-galactosyltransferase 1, this dispersal is thought to arise via fragmentation of the Golgi complex 16 . The reduced number of U2OS cells in stage 4 may suggest a delay in, or resistance to, Golgi complex fragmentation.…”

“…The copyright holder for this this version posted October 11, 2021. ; https://doi.org/10.1101/2021.10.11.463900 doi: bioRxiv preprint virus, expressing fluorescent chimeras of the early protein ICP0 and the late protein gC to distinguish between early and late stages of infection 16 . Eight cellular compartments were compared between uninfected and timestamp virus-infected human TERT-immortalized human foreskin fibroblast (HFF-hTERT) cells, with high-resolution spatial data collected using structured illumination microscopy (SIM) and expansion microscopy.…”

“…The ultrastructure of the samples was protected in a near-native state using a non-disruptive cryopreservation protocol 26 . Our study focused on the ultrastructural changes that accompany HSV-1 infection of human osteosarcoma U2OS cells, allowing comparison with previous reports from infection of HFF-hTERT cells 16 . Although a few differences were observed between the extent of Golgi fragmentation and the subcellular distribution of ICP0, we determined that remodelling of cytoplasmic vesicles and mitochondria was largely similar between these cultured cells.…”

Herpes simplex virus 1 inhibits phosphorylation of RNA polymerase II CTD serine-7

“…The mutations are non-phospho-accepting alanine substitutions in place of serines 2 or 7 (S2A and S7A, respectively) or the phospho-serine mimic glutamic acid at serine 7 (S7E). After 24h, αamanitin was added to degrade endogenous RPB1 for an additional 24h, followed by infection with fluorescent HSV-1 expressing eYFP-ICP0 as an immediate-early gene marker and gC-mCherry as a late gene marker (55). As shown in Fig 5, detectable levels of IE and late genes could be visualized in cells with replaced WT CTD at 24h p.i., albeit at substantially lower levels than amanitin-untreated cells, but not with empty pcDNA3 vector control.…”

pUL21 is a viral phosphatase adaptor that promotes herpes simplex virus replication and spread

“…Herpes simplex virus (HSV)-1 is a highly-prevalent human pathogen that establishes life-long latent infection, with reactivation of virus from peripheral neurons manifesting as orofacial or genital lesions or (occasionally) life-threatening viral encephalitis (Jørgensen et al, 2017;Looker et al, 2015;Nicoll et al, 2012). Like all herpesviruses, HSV-1 dramatically remodels the intracellular environment of infected cells to promote the production and dissemination of progeny virus particles (Das et al, 2007;Scherer et al, 2020). After receiving their genomic cargo in the nucleus, HSV-1 capsids traverse the nuclear envelope via sequential envelopment and de-envelopment steps catalysed primarily by the viral nuclear egress complex (NEC), comprising pUL31 and pUL34 (Bigalke and Heldwein, 2016).…”