Fig 1.
Generation of luciferase reporter viruses bearing deletions of the core LAT promoter.
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 US5 locus. b) Genomic structures as analyzed by Southern blot hybridization. Restriction digest with PstI demonstrated all predicted restriction fragments, including the 203 bp LAT promoter. The 3.3-kb HpaI 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 in vitro growth curves of recombinant viruses and wildtype strain performed in BHK cells. e) Firefly luciferase reporter viruses express equivalent levels of luciferase during infection in vitro. 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.
Fig 2.
Luciferase reporter virus characterisation in vivo.
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).
Fig 3.
Luciferase expression is higher during LAT-negative HSV-1 infection in latent trigeminal ganglia.
a) Following injection of D-luciferin substrate, TGs were dissected at set times from each mouse and luciferase intensity was measured. Displayed are composite photographs of dissected TGs with luciferase signal represented by a colourimetric overlay. The luciferase signal overlay is relative to the displayed scale. b) Luciferase signal was quantified with Living Image software and normalised to relative HSV-1 DNA loads within the same TGs. 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. Statistically significant differences were observed between LAT-positive and LAT-negative viruses, with the exception of SC16CMVlucΔLAT-GFP-1 vs SC16CMVluc (SC16CMVlucΔLAT-GFP-1: P = 0.09 and 0.007 compared to SC16CMVluc and SC16CMVlucREV, respectively; SC16CMVlucΔLAT-GFP-2: P = 0.04 and 0.0002 compared to SC16CMVluc and SC16CMVlucREV, respectively; Kruskal-Wallis with Mann-Whitney post-tests).
Fig 4.
Latent HSV-1 genomes are highly enriched in facultative heterochromatin.
Chromatin aliquots isolated from latently infected mouse TG were precipitated with antibodies to H3ac or H3K27me3. The specificity of each antibody was assessed by quantifying the relative enrichment of sequences known to be associated with euchromatin (APRT) or facultative heterochromatin (upHoxa5). Symbols represent percentage enrichment of sequences from individual TGs (n = 16) and floating bars represent the means of these data following immunoprecipitation with a) anti-H3ac and b) anti-H3K27me3 antibodies. c) Fold enrichment of four virus sequences with facultative heterochromatin marker H3K27me3. Histogram displays the average of four independent experiments (+ SEM), each comprising groups of three pooled TGs per virus. Fold enrichment of H3K27me3 immunoprecipitation (IP) was calculated as: [Virus sequence IP ÷ (IP + Input)] ÷ [cellular APRT IP ÷ (IP + Input)].
Fig 5.
β-galactosidase reporter viruses reveal an increased frequency of gene expression in the absence of the LAT RNAs.
a) Genomic structures of SC16CMVlacZΔLAT-GFP and SC16CMVlacZREV. Both viruses harbour an HCMV MIEP-lacZ expression cassette within the non-essential HSV-1 US5 locus. b) Virus titres obtained from the whisker pads (WP) and trigeminal ganglia (TG) of C57BL/6, at 4 and 5 dpi. Symbols and error bars represent mean titre ± SEM from 5 mice per virus per time-point. c) LAT expression quantified by qRT-PCR from total TG pair cDNA. Each symbol represents HSV-1 major LAT intron RNA per 104 copies of cellular cyclophilin A in each TG pair (N = 5 mice per virus) and floating bars represent the median of these data. Significantly less major LAT RNA was detected in SC16CMVlacZΔLAT-GFP-infected TG compared to the revertant—P = 0.0079, Mann-Whitney U test. d) Photomicrograph of β-galactosidase-positive neurons during latent infection of C57BL/6 TG. e) Quantification of β-galactosidase-positive cells during latent infection with SC16CMVlacZΔLAT-GFP and SC16CMVlacZREV. Each symbol represents the number of positive cells per TG (N = 10) and floating bars represent the mean of these data. SC16CMVlacZΔLAT-GFP-infected TG possessed significantly more positive cells than the revertant—P < 0.0005, Student's t-test. f) Virus DNA loads quantified by qPCR. HSV-1 DNA was normalised to the cellular control, APRT. Each symbol represents normalised HSV-1 DNA per TG pair (N = 5) and floating bars represent the mean of these data.
Fig 6.
Luciferase expression decreases following long-term latent infection.
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 SC16CMVluc, SC16CMVlucΔLAT-GFP-1, SC16CMVlucΔLAT-GFP-2 and SC16CMVlucREV, respectively. A formal statistical comparison between viruses is not possible from these data.
Fig 7.
Analysis of latency at the single cell level reveals an increased frequency of reactivation during infection with LAT-negative HSV-1.
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 ex vivo cultures of neurons latently infected with SC16CMVCreΔLAT-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.
Table 1.
Primers and TaqMan probes used in this study.