Distinctive roles for 2',5'-oligoadenylate synthetases and double-stranded RNA-dependent protein kinase R in the in vivo antiviral effect of an adenoviral vector expressing murine IFN-beta

Abstract

To evaluate the anti-HSV-1 mechanisms of murine IFN-beta in ocular infection, mice were transduced with an adenoviral vector expressing murine IFN-beta (Ad:IFN-beta). Ocular transduction with Ad:IFN-beta resulted in enhanced survival following infection with HSV-1. The protective effect was associated with a reduction in 1) viral titer, 2) viral gene expression, 3) IFN-gamma levels, and 4) the percentage of CD8(+) T lymphocyte and NK cell infiltration in infected tissue. Expression of IFN-beta resulted in an elevation of the IFN-induced antiviral gene 2',5'-oligoadenylate synthetase (OAS1a) but not dsRNA-dependent protein kinase R (PKR) in the cornea and trigeminal ganglion (TG). Mice deficient in the downstream effector molecule of the OAS pathway, RNase L, were no more sensitive to ocular HSV-1 compared with wild-type controls in the TG based on measurements of viral titer. However, the efficacy of Ad:IFN-beta was transiently lost in the eyes of RNase L mice. By comparison, PKR-deficient mice were more susceptible to ocular HSV-1 infection, and the antiviral efficacy following transduction with Ad:IFN-beta was significantly diminished in the eye and TG. These results suggest that PKR is central in controlling ocular HSV-1 infection in the absence of exogenous IFN, whereas the OAS pathway appears to respond to exogenous IFN, contributing to the establishment of an antiviral environment in a tissue-restricted manner.

Figure 1. Ad:GFP application to the cornea results in GFP expression in the epithelial layer of the cornea, as well as GFP expression in the TG

ICR mice were anesthetized and the Ad:GFP vector (1 × 10⁵ transducing units) was applied topically to scarified corneas. GFP expression was detected in the cornea at day 2 (A) as well as day 15 (data not shown) post transduction. Alternatively, Ad:GFP was injected into the corneal stroma resulting in localized expression detected at day 2 (B) as well as day 19 (data not shown) post transduction. (C) Flat mount of a cornea that was transduced 2 days before by direct application of Ad:GFP onto the scarified cornea. To detect any transgene expression in the TG, mice were transduced with the Ad:GFP vector by direct application of the vector onto scarified corneas. Three days later the TGs were extracted, collagenized and plated on laminin/collagen coated cover slips. Seven days later, cultures were fixed and stained for GFP (D) and neuron-specific enolase (E), and counterstained with DAPI. The combined image is shown in panel F.

Figure 2. The Ad:IFN-β construct enhances the cumulative survival of mice ocularly infected with HSV-1

The corneas of ICR mice were scarified and transduced with either Ad:IFN-β or Ad:Null vectors (1 × 10⁶ transducing units/eye) 2 (A) or 4 days (B) prior to infection with 150 pfu/eye HSV-1 (McKrae strain). Mortality was recorded for each group of animals. The squares represent Ad:IFN-β transduced mice, the circles represent Ad:Null transduced mice, and the triangles represent PBS-treated mice. This figure is a summary of three experiments (n = 5 mice/group per experiment). *p<0.5 comparing PBS- or Ad:Null-treated groups to Ad:IFN-β treated group at each indicated time point as determined using Mann Whitney rank order test.

Figure 3. Ad:IFN-β transduction of the cornea at the time of ocular HSV-1 infection enhances survival of mice

The corneas of ICR mice were scarified and transduced with either Ad:IFN-β or Ad:Null vectors (1 × 10⁶ transducing units/eye) at the time of or 24 hr prior to infection with 150 pfu/eye HSV-1 (McKrae strain). Mortality was recorded for each group of animals. The squares represent Ad:IFN-β transduction at the time of infection, the circles represent Ad:IFN-β transduction 24 hr prior to infection, the triangles represent the Ad:Null transduction at the time of infection, and the inverted triangles represent Ad:Null transduction 24 hr prior to infection. This figure is a summary of two experiments (n = 5–6 mice/group per experiment). *p<0.5 comparing Ad:Null to Ad:IFN-β groups transduced at the time of infection as determined using Mann Whitney rank order test.

Figure 4. Ad:IFN-β transduction of the cornea antagonizes viral replication in both the eye and the TG of HSV-1 infected mice

Female ICR mice (n=4 per group/experiment) were transduced (1 × 10⁶ transducing units/eye) with Ad:IFN-β (black column), Ad:Null (white column), or vehicle (PBS, dashed column). Two days later, the mice were infected with HSV-1 (150 pfu/eye). Mice were euthanized 3 or 6 days post infection, and the cornea (A) and the TG (B) were removed. The tissue was homogenized and the clarified homogenate (10,000 × g, 1 min) was quantified for infectious virus by plaque assay. Results are a summary of two independent experiments. Bars represent the log of the mean +/− SEM. *p<.05 comparing the Ad:IFN-β to the Ad:Null- or vehicle-treated mice.

Figure 5. Mice transduced with Ad:IFN-β express show a reduction in the expression of the ICP27 and TK transcript levels in both the eye and the TG

Female ICR mice (n=4 per group/experiment) were transduced (1 × 10⁶ transducing units) with Ad:IFN-β (dashed column), Ad:Null (black column), or vehicle (PBS, dotted column). Two days later, the mice were infected with HSV-1 (150 pfu/eye). Mice were euthanized 3 or 6 days post infection, and the cornea and the TG were removed. RNA was isolated and processed to cDNA. Real time PCR was performed to determine the relative gene expression in each sample of a representative panel of HSV-1 genes. Results are a summary of two independent experiments. Bars represent the mean +/− SEM. *p<.05 comparing the Ad:IFN-β to the Ad:Null- or vehicle-treated mice.

Figure 6. Ad:IFN-β transduction reduces lymphocyte infiltration in the TG of HSV-1 infected mice

Female C57BL/6 mice (n=4/group) were transduced with Ad Ad:IFN-β or Ad:Null vectors (1 × 10⁶ transducing units/eye). Two days later, the mice were infected with HSV-1 (600 pfu/eye). Mice were euthanized 7 days post infection and the TGs were removed. TGs were collagenized and single cell suspensions were stained with the fluorochrome conjugated anti-CD4, anti-CD8α, anti-DX5, and anti-CD3 antibodies for 30 min at 4°C. The cells were analyzed on a FACS Calibur using WinMDI data analysis software.

Figure 7. The absence of PKR reduces but does not eliminate the anti-viral action of the Ad:IFN-β vector against ocular HSV-1 infection while the absence of a functional OAS pathway transiently reduces the anti-viral efficacy of the Ad:IFN-β vector in the cornea

Wild type (WT) and mice deficient in RNase L (RL−/−) or PKR (PKR−/−) (n=4 per group/experiment) were transduced with Ad:IFN-β (dotted column) or Ad:Null vectors (black column) (1 × 10⁶ transducing units/eye). Two days later, the mice were infected with HSV-1 (600 pfu/eye). Mice were euthanized 3 or 6 days post infection, and the cornea (A, C) and the TG (B, D) were removed. The tissue was homogenized and the clarified homogenate (10,000 × g, 1 min) was quantified for infectious virus by plaque assay. Results are a summary of three independent experiments. Bars represent the log of the mean +/− SEM. *p<.05 comparing the Ad:IFN-β to the Ad:Null treated mice.