The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

open access articleHerpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir

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

The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored in adapted strains where pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication.Wellcome Trust Senior Research Fellowship (219447/Z/19/Z), Wellcome Trust Senior Research Fellowship (106207/Z/14/Z), Biotechnology and Biological Sciences Research Council Research Grant (BB/M021424/1), Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (098406/Z/12/B)

Histone modifications associated with herpes simplex virus type 1 genomes during quiescence and following ICP0-mediated de-repression

In the current study, it was shown that repressed virus genomes in quiescently infected MRC5 cells adopt a repressed histone-associated structure marked by the enrichment of deacetylated histones at a wide variety of herpes simplex virus type 1 (HSV-1) promoters. In addition, it was shown that genome de-repression, mediated by HSV-2 superinfection or delivery of ICP0 using a recombinant adenovirus vector, resulted in the enrichment of acetylated histones on HSV DNA. These data indicate that ICP0-mediated genome de-repression is intimately linked to enrichment of acetylated histones at virus promoters. The fold change in association of pan-acetylated histone H3 following Ad.TRE.ICP0-mediated de-repression consistently revealed promoter-specific variation, with the highest fold changes (>50-fold) being observed at the latency-associated transcript promoter and enhancer regions. Chromatin immunoprecipitation analyses using an antibody specific to the C terminus of histone H3 as a surrogate measure of nucleosome occupancy revealed little variability in the total loading of histone H3 at the various HSV promoters. This observation suggests that acetylation of histone H3 in response to ICP0 expression is not uniformly targeted across the HSV-1 genome during ICP0-mediated de-repression

A historical analysis of herpes simplex virus promoter activation in vivo reveals distinct populations of latently infected neurones

Herpes simplex virus type 1 (HSV-1) has the capacity to establish a life-long latent infection in sensory neurones and also to periodically reactivate from these cells. Since mutant viruses defective for immediate-early (IE) expression retain the capacity for latency establishment it is widely assumed that latency is the consequence of a block in IE gene expression. However, it is not clear whether viral gene expression can precede latency establishment following wild-type virus infection. In order to address this question we have utilized a reporter mouse model system to facilitate a historical analysis of viral promoter activation in vivo. This system utilizes recombinant viruses expressing Cre recombinase under the control of different viral promoters and the Cre reporter mouse strain ROSA26R. In this model, viral promoter-driven Cre recombinase mediates a permanent genetic change, resulting in reporter gene activation and permanent marking of latently infected cells. The analyses of HSV-1 recombinants containing human cytomegalovirus major immediate-early, ICP0, gC or latency-associated transcript promoters linked to Cre recombinase in this system have revealed the existence of a population of neurones that have experienced IE promoter activation prior to the establishment of latency

Near-native state imaging by cryo-soft-X-ray tomography of uninfected and herpes simplex virus (HSV)-1 infected mammalian cells

This dataset consists of 3D tomographic data collected using cryo-soft-X-ray tomography (cryoSXT) and cryo wide field fluorescence microscopy data. These data were collected for a study designed to assess (a) if herpes simplex virus (HSV)-1 could be detected by cryoSXT and (b) how the morphology and organisation of cytoplasmic vesicles and mitochondria change during herpes simplex virus-1 (HSV-1) infection. Cryo-soft-X-ray tomography collection parameters === An UltraXRM-S/L220c X-ray microscope (Carl Zeiss Xray microscopy) was used to collect the datasets at Beamline B24 at Diamond Light Source. The raw data, known as tilt series, consist of a collation of images collected from the same field of view at different angles. These were collected within a maximum range of -70° and +70° degrees and at increments of 0.2° or 0.5° and with an exposure time of 0.5 seconds or 1 second. 500 eV X rays were focused with a zone plate objective capable of a nominal resolution of 25 nm or 40 nm. Full details on parameters used for individual tilt series can be found in the attached tomographic_collection_parameters.csv file. Tomograms were reconstructed in Imod version 4.9.2. For each field of view, a tilt series and a reconstructed tomogram are provided. Detection of HSV-1 by cryo-soft-X-ray tomography === Tomograms were collected from human foreskin fibroblast cells that had been immortalised with hTERT (HFF-hTERT). The ultrastructure of uninfected cells was compared with that of HSV-1-infected cells to determine if HSV-1 particles could be detected with cryoSXT. In this dataset, we note capsids in the nuclei, viral particles in the nuclear envelope and cytoplasm, and virions at the exposed cell surface and at cell junctions. Changes to cytoplasmic vesicles and mitochondria during HSV-1 infection === We used human osteosarcoma cells (U2OS) to study changes to cytoplasmic vesicles and mitochondria. A population of synchronously infected cells progress through infection at different rates and this could affect the state of these cellular compartments. To account for this, we used a recombinant of HSV-1, known as the timestamp virus, which contains two fusion proteins with different temporal expression. This allowed us to distinguish between early stages of infection (using the immediate early protein eYFP-ICP0) and late stages (using the late protein gC-mCherry). First we collected fluorescence data from infected cells to identify early-stage and late-stage cells for subsequent imaging by cryoSXT. To collect fluorescence data, we used a Zeiss AxioImager2 microscope with an achromatic 50× air objective (Zeiss LD EC Epiplan-Neofluar 50x/0.55 DIC M27; NA=0.55; free working distance=9.1 mm) with the following filters: Zeiss 46 HE YFP filter (Excitation 500±25 nm, Emission 535±30 nm) and the Zeiss 64 HE mPlum filter (Excitation 587±25 nm, Emission 647±70 nm). This fluorescence data are supplied here in the form of a map of the whole sample grid. Second, we imaged these early-stage and late-stage cells in addition to uninfected cells on the X-ray microscope as described above. Data were collected from three independent replicates

UL56 Is Essential for Herpes Simplex Virus-1 Virulence In Vivo but Is Dispensable for Induction of Host-Protective Immunity

Herpes simplex virus-1 (HSV-1) is common and can cause significant disease in humans. Unfortunately, efforts to develop effective vaccines against HSV-1 have so far failed. A detailed understanding of how the virus infects its host and how the host mounts potent immune responses against the virus may inform new vaccine approaches. Here, using a zosteriform mouse model, we examined how the HSV-1 gene UL56 affects the ability of the virus to cause morbidity and generate protective immunity. A UL56 deletion mutant, ΔUL56, was derived from the wild-type HSV-1 strain SC16, alongside a revertant strain in which UL56 was reintroduced in ΔUL56. In vitro, the three virus strains replicated in a similar manner; however, in vivo, only the wild type and the revertant strains caused shingles-like skin lesions and death. Mice previously infected with ΔUL56 became resistant to a lethal challenge with the wild-type SC16. The protective immunity induced by ΔUL56 was independent of IL-1, IL-33, and IL-36 signaling through IL-1RAP. Both skin and intramuscular ΔUL56 inoculation generated protective immunity against a lethal SC16 challenge. After 6 months, female mice remained resistant to infection, while male mice exhibited signs of declining protection. Our data demonstrate that UL56 is important for the ability of HSV-1 to spread within the infected host and that a ∆UL56 strain elicits an effective immune response against HSV-1 despite this loss of virulence. These findings may guide further HSV-1 vaccine development.</jats:p

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

Funder: Department of Pathology, University of CambridgeHerpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.This work was supported by a PhD studentship co-funded by Diamond Light Source and the Department of Pathology, University of Cambridge, to KLN, by a research fellowship from the Deutsche Forschungsgemeinschaft (SCHE 1672/2-1) to KMS, by the funding from the Engineering and Physical Sciences Research Council (EP/H018301/1) and the Medical Research Council (MR/K015850/1 and MR/K02292X/1) to CFK, by a Biotechnology and Biological Sciences Research Council Research Grant (BB/M021424/1) to CMC, and by a Sir Henry Dale Fellowship, jointly funded by the Wellcome Trust and the Royal Society (098406/Z/12/B) to SCG