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. 2006 Jun 9;3:44.
doi: 10.1186/1743-422X-3-44.

ICP0 Antagonizes Stat 1-dependent Repression of Herpes Simplex Virus: Implications for the Regulation of Viral Latency

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Free PMC article

ICP0 Antagonizes Stat 1-dependent Repression of Herpes Simplex Virus: Implications for the Regulation of Viral Latency

William P Halford et al. Virol J. .
Free PMC article

Abstract

Background: The herpes simplex virus type 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase, which is encoded within the HSV-1 latency-associated locus. When ICP0 is not synthesized, the HSV-1 genome is acutely susceptible to cellular repression. Reciprocally, when ICP0 is synthesized, viral replication is efficiently initiated from virions or latent HSV-1 genomes. The current study was initiated to determine if ICP0's putative role as a viral interferon (IFN) antagonist may be relevant to the process by which ICP0 influences the balance between productive replication versus cellular repression of HSV-1.

Results: Wild-type (ICP0+) strains of HSV-1 produced lethal infections in scid or rag2-/- mice. The replication of ICP0- null viruses was rapidly repressed by the innate host response of scid or rag2-/- mice, and the infected animals remained healthy for months. In contrast, rag2-/- mice that lacked the IFN-alpha/beta receptor (rag2-/- ifnar-/-) or Stat 1 (rag2-/- stat1-/-) failed to repress ICP0- viral replication, resulting in uncontrolled viral spread and death. Thus, the replication of ICP0- viruses is potently repressed in vivo by an innate immune response that is dependent on the IFN-alpha/beta receptor and the downstream transcription factor, Stat 1.

Conclusion: ICP0's function as a viral IFN antagonist is necessary in vivo to prevent an innate, Stat 1-dependent host response from rapidly repressing productive HSV-1 replication. This antagonistic relationship between ICP0 and the host IFN response may be relevant in regulating whether the HSV-1 genome is expressed, or silenced, in virus-infected cells in vivo. These results may also be clinically relevant. IFN-sensitive ICP0- viruses are avirulent, establish long-term latent infections, and induce an adaptive immune response that is highly protective against lethal challenge with HSV-1. Therefore, ICP0- viruses appear to possess the desired safety and efficacy profile of a live vaccine against herpetic disease.

Figures

Figure 1
Figure 1
Two alternative models of HSV-1 gene regulation. A. Genetic organization of the HSV-1 genome. The long-repeated (RL) and short-repeated (RS) regions of the HSV-1 genome regulate expression of 4 of 5 immediate-early (IE) genes (white arrows). The unique long (UL) and unique short (US) regions contain most of the early (E) and late (L) genes (yellow and red arrows). The 15 kb RL and RS regions include a 2 kb recombinogenic 'joint' sequence, the ICP34.5 gene (red arrow), and the LAT and L/ST genes which are repressed during productive replication (black arrows). B. The current model of HSV-1 gene regulation [1] describes a cascade of IE → E → L gene expression. C. The proposed Checkpoint model predicts that HSV-1 gene expression proceeds by the accepted cascade, but that viral gene expression can be blocked during viral IE mRNA synthesis if ICP0 is not synthesized (Checkpoint 1) or can be blocked during viral L protein synthesis if ICP34.5 is not synthesized (Checkpoint 2).
Figure 2
Figure 2
An ICP0- virus is avirulent in scid mice. A. Scid mice were inoculated with 2 × 105 pfu per eye of the ICP0- virus n212 (n = 6 mice). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time (open black symbols). The survival of n212-infected scid mice is plotted over time (red line). On day 70 p.i., n212-infected scid mice were challenged with 2 × 105 pfu per eye of wild-type HSV-1 strain KOS (subsequent viral titers are shown as open blue symbols). The dashed line indicates the lower limit of detection of the plaque assay used to determine viral titers. B. Survival of BALB/c mice versus scid mice infected with KOS or n212. Bars represent the mean ± sem of survival frequency of ICP0- virus-infected mice at day 60 p.i. (n = 5 experiments; Σn = 30 mice per group).
Figure 3
Figure 3
Loss of Stat 1 alleviates innate host repression of ICP0- viruses in vivo. A. Strain 129 mice, rag2-/- mice, PML-/- mice, or stat1-/- mice were inoculated with 2 × 105 pfu per eye of the ICP0- virus n212 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time. B. Strain 129 mice, rag2-/- mice, stat1-/- mice, or rag2-/- stat1-/- mice were inoculated with 2 × 105 pfu per eye of the ICP0- virus, 0--GFP (n = 4 mice per group). Dashed lines indicate the lower limit of detection of the plaque assay. C. Survival of strain 129 mice, rag2-/- mice, PML-/- mice, stat1-/- mice, or rag2-/- stat1-/- mice infected with the ICP0- viruses, n212 or 0--GFP. Bars represent the mean ± sem of survival frequency of ICP0- virus-infected mice at day 60 p.i. (n = 3 experiments; Σn = 14 mice per group).
Figure 4
Figure 4
The RL region. A. Genetic organization of the HSV-1 RL region. Numbers refer to base positions in the prototype HSV-1 genome, and arrows denote the LAT, L/ST, ICP34.5, and ICP0 primary transcripts. Reiterated DNA sequences in the RL region are denoted by small boxes containing vertical bars. The location of the DNA sequences to which ICP4 homodimers bind in the LAT and L/ST genes is denoted by pairs of black ovals at the 5' end of each gene. B. The ICP0 genes of wild-type HSV-1 and the ICP0- viruses n212 and 0--GFP. The mutation in n212 introduces a 14 bp linker sequence into codon 212 of the ICP0 open-reading frame, which terminates protein translation [53]. The insertion mutation in 0--GFP introduces an ~770 bp green-fluorescent protein (GFP) coding sequence in-frame with the ICP0 gene. The resulting mRNA is predicted to encode the N-terminal 104 amino acids of ICP0 fused to a 14 amino acid linker and 239 amino acids of C-terminal GFP.
Figure 5
Figure 5
Replication of ICP0- and ICP4- viruses in cell culture and immunodeficient mice. Vero cells were A. untreated or B. treated with 200 U per ml of IFN-β and were inoculated with 2.5 pfu per cell of wild-type HSV-1 (KOS), an ICP0- virus (0--GFP), or an ICP4- virus (n12). The mean ± sem of the logarithm of viral titers recovered from Vero cells is plotted over time (n = 4 per time point). C. Rag2-/- stat1-/- mice and D. rag2-/- mice were inoculated with 2 × 105 pfu per eye of the ICP0- virus 0--GFP or the ICP4- virus n12 (n = 4 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time (open black symbols). Dashed lines indicate the lower limit of detection of each plaque assay. The survival of 0--GFP-infected mice and ICP4- virus-infected mice is plotted over time (open red symbols).
Figure 6
Figure 6
A Stat1-dependent host response restricts the spread of HSV-1 strain KOS-GFP into the central nervous system. Strain 129 mice, rag2-/- mice, stat1-/- mice, or rag2-/- stat1-/- mice were inoculated with 2 × 105 pfu per eye of HSV-1 strain KOS-GFP. A. The mean ± sem of the logarithm of viral titers recovered from homogenates of mouse eyes, TG, and hindbrain is plotted as a function of the time p.i. at which tissues were harvested (n = 5 per time point). Asterisks denote significant differences between stat1+/+ versus stat1-/- tissues (p < 0.001, as determined by two-way ANOVA). Dashed lines indicate the lower limit of detection of each plaque assay. B. GFP expression in tissues of KOS-GFP-infected mice. Representative photographs are shown of eyes harvested on day 3 p.i. (4× magnification, 250 ms exposure), TG harvested on day 5 p.i. (2× magnification, 500 ms exposure), and the ventral side of brains harvested on day 7 p.i. (2× magnification, 1000 ms exposure).
Figure 7
Figure 7
Loss of IFN-α/β receptors or Stat 1 impairs an innate host response that represses KOS-GFP and 0--GFP at the site of inoculation. Mice were inoculated with 2 × 105 pfu per eye of A. HSV-1 strain KOS-GFP, or B. the ICP0- virus, 0--GFP. GFP fluorescence was recorded in the right eyes of strain 129 mice, rag2-/- mice (lymphocyte-deficient), ifngr-/- mice (IFN-γ receptor-null), ifnar-/- mice (IFN-α/β receptor-null), ifnar-/- ifngr-/- mice, stat1-/- mice, rag2-/- stat1-/- mice, and rag2-/- ifnar-/- mice. Representative photographs are shown of GFP fluorescence in the virus-infected eye of one mouse per group photographed over time at 36, 60, and 84 hours p.i. (4× magnification; 39 ms exposure for KOS-GFP; 63 ms exposure for 0--GFP).
Figure 8
Figure 8
Measurement of KOS and 0--GFP viral genome loads in the trigeminal ganglia of HSV-1 latently infected mice. A. Dotblot of HSV-1 VP16 PCR products. Each "dot" contains VP16 PCR product amplified from the TG DNA of a single mouse, and the n-values indicate numbers of mice per group. TG harvested from uninfected (UI) mice served as negative controls for the PCR. TG harvested from mice dying of encephalitis (Day 9 p.i.) belonged to one of the following groups: rag2-/- ifnar-/- mice, ifnar-/- ifngr-/- mice, or stat1-/- mice inoculated with 2 × 104 pfu per eye of 0--GFP. TG harvested from mice that were latently infected with HSV-1 (Day 40 p.i.) belonged to one of the following groups: strain 129 mice inoculated with 2 × 105 pfu per eye of KOS; strain 129 mice, ifngr-/- mice, or ifnar-/- mice inoculated with 2 × 105 pfu per eye of 0--GFP; or stat1-/- mice inoculated with 2 × 104 pfu per eye of 0--GFP. The standard curve on the right consists of PCR products amplified from a two-fold dilution series of VP16 plasmid DNA. B. The ratio of yields of VP16 to competitor PCR product yields (competitor dotblot not shown) was used to estimate viral genome copy number per PCR. The logarithm of viral genomes per TG, y, was plotted as a function of the mean logarithm of the ratio of VP16 PCR product yield: competitor PCR product yield, x, amplified from duplicate PCRs of each dilution of VP16 plasmid (error bars indicate the standard deviation between duplicate PCRs). The relationship between viral genome load and PCR product yields was described by the equation, y = 0.2556•x3 + 0.1055•x2 + 1.2079•x + 5.9309 (r2 = 0.99). The number of HSV-1 genomes per TG in each sample was derived from fitting the data shown in panel A to the standard curve shown in panel B. C. Number of HSV-1 genomes per TG in mice that were uninfected or were latently infected with KOS or 0--GFP. The dashed line indicates the lower limit of detection of the PCR assay. Asterisks denote significant differences in viral genome load per TG relative to strain 129 mice latently infected with KOS (p < 0.05, two-way t-test).
Figure 9
Figure 9
An ICP0- virus induces a protective immune response against HSV-1. Strain 129 mice were inoculated with 2 × 105 pfu per eye of the A. ICP0- virus 0--GFP or B. the ICP4- virus n12 (n = 6 mice per group). Rag2-/- mice were inoculated with 2 × 105 pfu per eye of the C. ICP0- virus 0--GFP or D. the ICP4- virus n12 (n = 6 mice per group). The mean ± sem of the logarithm of viral titers recovered from mouse eyes is plotted over time (black symbols). The survival of 0--GFP-infected mice and ICP4- virus-infected mice is plotted over time (red symbols). On day 30 p.i., 0--GFP-infected and ICP4- virus-infected mice were secondarily challenged with 2 × 105 pfu per eye of HSV-1 strain McKrae. Viral titers recovered from the eyes of strain 129 mice or rag2-/- mice after secondary challenge with McKrae is plotted over time (blue symbols). Dashed lines indicate the lower limit of detection of the plaque assay.

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