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

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

Generation of luciferase reporter viruses bearing deletions of the core LAT promoter.

<p>a) Genomic structures of SC16CMVluc and SC16CMVlucREV (LAT-positive recombinants), and SC16CMVlucΔLAT-GFP-1 and SC16CMVlucΔLAT-GFP-2 (LAT-negative recombinants). All four viruses harbour an HCMV MIEP-firefly luciferase expression cassette within the non-essential HSV-1 U_S5 locus. b) Genomic structures as analyzed by Southern blot hybridization. Restriction digest with <i>Pst</i>I demonstrated all predicted restriction fragments, including the 203 bp LAT promoter. The 3.3-kb <i>Hpa</i>I fragment encoded within pPSTD1 was utilized as a probe. c) LAT expression was quantified by qRT-PCR from total TG cDNA. Primers for the major LAT intron and cyclophilin RNA were used. Histograms represent the mean (± SEM) numbers of major LAT RNA copies per 10⁵ copies of cyclophilin RNA from triplicate PCRs. d) Low (0.01) moi <i>in vitro</i> growth curves of recombinant viruses and wildtype strain performed in BHK cells. e) Firefly luciferase reporter viruses express equivalent levels of luciferase during infection <i>in vitro</i>. BHK cells were infected at an moi of 0.1 and harvested for luciferase assay at 3, 4, 8, 24, 31 and 49 hours post-infection. Symbols represent mean luciferase activity from three biological replicates. Error bars represent SEM.</p

Analysis of latency at the single cell level reveals an increased frequency of reactivation during infection with LAT-negative HSV-1.

<p>a) Genomic structures of the Ai6 ZsGreen transgenic locus before and after HSV-1 Cre-mediated excision of the lox-STOP-lox cassette. The constitutively active CAG promoter drives expression of the locus. Following excision of the lox-STOP-lox cassette, ZsGreen mRNA is stabilised by the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). b) Fluorescence photomicrograph of an intact latently infected Ai6 neuron in MRC5 culture. c) Fluorescence photomicrograph of a dying latently infected Ai6 neuron in MRC5 culture. d) Neuron fragmentation was recorded each day during an eight day culture on MRC5 cells as an indicator of cell survival in culture. e) Photomicrograph of uninfected MRC5. f) Photomicrograph of MRC5 displaying HSV-1 cytopathic effect. g,h) Fluorescence photomicrographs of latently infected neurons undergoing reactivation. MRC5 CPE is visible in close proximity to the cultured neurons (arrows). i) Cumulative reactivation observed from <i>ex vivo</i> cultures of neurons latently infected with SC16CMV<u>Cre</u>ΔLAT-GFP and revertant virus, as assessed by CPE in the MRC5 feeder layer (n = 6 mice per virus). Symbols represent average percentage reactivation per day and error bars represent ± SEM. * represents P < 0.05; Student's T-test. j) Single cell HSV-1 DNA loads were distributed equivalently between LAT-positive and LAT-negative virus recombinants. Each symbol represents the HSV-1 genome copy in an individual fluorescent neuron. Floating bars represent median copies per cell in each virus group.</p

Luciferase reporter virus characterisation in vivo.

<p>a) Virus titres obtained from the whisker pads of C57BL/6, 4 dpi. Each symbol represents titres from an individual mouse and the floating bar represents the mean. b) Virus titres obtained from pairs of TG of C57BL/6, 4 dpi. Each symbol represents titres from an individual mouse and the floating bar represents the mean. c) Representative image of luciferase signal (colourimetric overlay) in live mice 2 days post-infection. d) Luciferase signal observed from reporter virus infection of C57BL/6 animals during acute infection. Each symbol represents mean luciferase signal (± SEM) (n = 4 mice 2 dpi, n = 7–8 on days 4–8 post infection). e) Luciferase signal observed from dissected TG homogenates during acute infection. Each symbol represents mean luciferase signal (± SEM) (n = 8 individual TGs).</p

Luciferase expression decreases following long-term latent infection.

<p>a) Luciferase signal was quantified with Living Image software and normalised to relative HSV-1 DNA loads within the same TGs. Data acquisition was performed separately on 30 and 120 dpi. Each symbol represents normalised luciferase signal from an individual TG. Floating bars represent the median signal of each virus group. A signal of 1x10⁴ ps^-1cm^-2sr^-1 represents a background threshold of detection. A significant reduction in luciferase signal was observed for both LAT-negative viruses (P < 0.01). b) The fold-decrease in median luciferase signal between day 30 and 120 post-infection for SC16CMV<u>luc</u>, SC16CMV<u>luc</u>ΔLAT-GFP-1, SC16CMV<u>luc</u>ΔLAT-GFP-2 and SC16CMV<u>luc</u>REV, respectively. A formal statistical comparison between viruses is not possible from these data.</p