Figure 1.
Diagram of the latent HSV-1 genome.
Regions of long and short repeats (RL and RS) have been analyzed for H3K4me2 and acetyl H3 K9,K14 enrichment [20], [21], [23]. During latency, the ICP0 and ICP4 regions of the genome are under-enriched in euchromatin, relative to the LAT 5′exon. This juxtaposition of euchromatin and heterochromatin allude to the likelihood of segregation of chromatin domains during latency.
Figure 2.
HSV-1 genome equivalents between HSV-1 strains McKrae and KOS.
New Zealand White rabbits were inoculated in their corneas, following scarification, with viral titers of 200,000 pfu/eye for both McKrae and KOS strains of HSV-1. All infections were confirmed by slit-lamp examination. Equivalent latent infections between the two strains were confirmed by real-time PCR, measuring the relative quantity of HSV-1 genomes. All relative quantities of HSV-1 genomes were normalized to GAPDH quantities per sample (P>0.20). Relative quantities were calculated using a standard curve generated for each primer/probe set used.
Figure 3.
H3K4me2 enrichment during latency.
A.) H3K4me2 status of rabbit TG latently infected with wild type McKrae (n = 7). B.) H3K4me2 status of rabbit TG latently infected with wild type KOS (n = 7). Rabbits were infected by corneal scarification with 200,000 pfu/eye of virus. ChIP analysis were performed as previously described [20], [21], [23] with TaqMan real-time PCR used for the analysis of the LAT 5′exon, ICP0 promoter and ICP4 promoter. B/IN ratios for each target gene were normalized to the B/IN ratios of rabbit GAPDH (cellular control). Real time PCR was performed with TaqMan universal PCR master mix, No AmpErase uracil N-glycolase and target specific primers and a fluorescently labeled probe (see references within). All PCR reactions were performed in triplicate and the average C(t) values were used to determine the relative quantity of DNA in either the bound or input fractions by using the equation for a standard curve specific to the primer/probe set used. The mean values are represented by the graph and the ±SEM is presented for each region.
Table 1.
H3K4me2 enrichment values during latency in the McKrae and KOS infected rabbit TG.
Figure 4.
Changes in H3K4me2 enrichment and RNA abundance of the LAT 5′exon following transcorneal iontophoresis of epinephrine as a reactivation stressor in rabbits.
All samples were validated using a transcriptionally active (GAPDH) and a transcriptionally repressed (centromere) gene. Samples were precipitated with anti-H3K4me2 and analyzed by real-time PCR. B/IN ratios for each target gene were normalized to the B/IN ratios of rabbit GAPDH (cellular control). The mean values are represented by a horizontal bar. All assays were further validated to ensure equivalent HSV-1 genome copies were present using GADPH. One TG per experiment was used (n = 6–7) for all ChIP assays. LAT 5′exon (X14112.1-nucleotides 119326–119397) A.) H3K4me2 enrichment of the LAT 5′exon post-TCIE for the highly efficient reactivator McKrae: ChIP analyses were performed post-TCIE at 0.5, 1, 2 or 4 h (indicated on X-axis). B.) H3K4me2 enrichment of the LAT 5′exon post-TCIE for the poor reactivator KOS: ChIP analyses were performed post-TCIE at 0.5, 1, 2 or 4 h. C.) Relative change in LAT RNA of rabbits latent with McKrae following TCIE: RNA was isolated using TRIzol reagent according to the manufacturer specifications. One rabbit TG was used per sample, and 8–10 samples were used for each time point. RNA was transformed to cDNA, and analyzed by real time PCR in triplicate. Relative quantities of were normalized to rabbit GAPDH. (There is no significant change in GAPDH expression following iontophoresis in the rabbit P>0.10) The error bars represent the positive standard deviation from the mean. The graphs are depicted as fold change in the RNA relative to the 0 h time, where the 0 h time was set to equal a value of 1. D.) Relative change in LAT RNA of rabbits latent with KOS following TCIE.
Figure 5.
Changes in H3K4me2 enrichment of the ICP0 promoter following transcorneal iontophoresis of epinephrine as a reactivation stressor in rabbits.
ChIP analyses were performed post-TCIE at 0.5, 1, 2 or 4 h (indicated on X-axis). A.) H3K4me2 enrichment of the ICP0 promoter post-TCIE for McKrae Strain: B.) H3K4me2 enrichment of the ICP0 promoter post-TCIE for KOS strain: Through 4 h post-TCIE, we observed no significant change in the H3K4me2 enrichment of the ICP0 promoter following TCIE in either McKrae or KOS strains.
Figure 6.
Changes in H3K4me2 enrichment and RNA abundance of the ICP4 promoter following transcorneal iontophoresis of epinephrine as a reactivation stressor in rabbits.
All samples were validated using a transcriptionally active (GAPDH) and a transcriptionally repressed (centromere) gene. Samples were precipitated with anti-H3K4me2 and analyzed by real-time PCR. B/IN ratios for each target gene were normalized to the B/IN ratios of rabbit GAPDH (cellular control). The mean values are represented by a horizontal bar. All assays were further validated to ensure equivalent HSV-1 genome copies were present using GADPH. One TG per experiment was used (n = 6–7) for all ChIP assays. LAT 5′exon (X14112.1-nucleotides 119326–119397) A.) H3K4me2 enrichment of the ICP4 promoter post-TCIE for the highly efficient reactivator McKrae: ChIP analyses were performed post-TCIE at 0.5, 1, 2 or 4 h (indicated on X-axis). B.) H3K4me2 enrichment of the ICP4 promoter post-TCIE for the poor reactivator KOS: ChIP analyses were performed post-TCIE at 0.5, 1, 2 or 4 h. C.) Relative change in ICP4 transcript abundance in rabbits latent with McKrae following TCIE: RNA was isolated using TRIzol reagent according to the manufacturer specifications. One rabbit TG was used per sample, and 8–10 samples were used for each time point. RNA was transformed to cDNA, and analyzed by real time PCR in triplicate. Relative quantities of were normalized to rabbit GAPDH. (There is no significant change in GAPDH expression following iontophoresis in the rabbit P>0.10) The error bars represent the positive standard deviation from the mean. The graphs are depicted as fold change in the RNA relative to the 0 h time, where the 0 h time was set to equal a value of 1. D.) Relative change in ICP4 transcript abundance in rabbits latent with KOS following TCIE.