Department of Ophthalmology, School of Medicine, University of California at Irvine, Irvine, CA 92697, USA
Correspondence
Guey-Chuen Perng
gperng{at}uci.edu
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ABSTRACT |
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INTRODUCTION |
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Replication of HSV-1 in tissue culture is temporally orchestrated and tightly regulated (Roizman & Sears, 1996). Initiation of replication requires several immediate-early (IE) genes to be expressed sequentially and simultaneously. The HSV-1 IE gene ICP0 is a promiscuous activator of viral gene expression and is required for efficient initiation of lytic infection in tissue culture (Everett, 2000
; Hagglund & Roizman, 2004
), particularly at low m.o.i. values (Everett et al., 2004
; Sacks & Schaffer, 1987
; Stow & Stow, 1986
). In addition, ICP0 may be important in reactivation from latency (Halford & Schaffer, 2001
; Leib et al., 1989
). It has been suggested that ICP0 regulates the balance between lytic and latent HSV-1 infection (Loiacono et al., 2003
). The function of the protein itself has been extensively studied in vitro, and an increasingly detailed picture of the interactions of ICP0 with cellular proteins and its biochemical functions is emerging (Boutell & Everett, 2003
; Boutell et al., 2002
; Everett, 2000
; Everett et al., 1998
; Gu & Roizman, 2003
; Hagglund & Roizman, 2004
; Jackson & DeLuca, 2003
; Kawaguchi et al., 1997
, 2001
; Parkinson et al., 1999
). ICP0 can counteract the host antiviral response by disarming the interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes. The RING finger domains of ICP0 are essential for this activity (Lin et al., 2004
; Mossman et al., 2000
).
Ocular infections of the cornea with HSV-1 can induce herpetic stromal keratitis (HSK). HSK is the most common infectious cause of corneal opacity leading to blindness. HSK is characterized by tissue destruction, oedema, opacification, corneal scarring and neovascularization (Pavan-Langston, 2000; Shimeld et al., 2001
). Interactions between the recurrent virus and the pre-existing immune response are believed to be the main cause of corneal scarring (Deshpande et al., 2004
; Streilein et al., 1997
). The current knowledge of HSK immunopathology is mainly derived from murine models, although spontaneous reactivation is a rare event in mice (Feldman et al., 2002
). In the HSK mouse model, CD4+ T cells play a pivotal role in the HSK immunopathological response (Gangappa et al., 2000
). It has been hypothesized that HSK may be due either to HSV-1 inducing auto-reactive T cells (Avery et al., 1995
; Zhao et al., 1998
) or to bystander damage by infiltrating CD4+ T cells (Deshpande et al., 2001
). However, it is generally thought that HSK is the result of an immune response to one or more viral proteins. Surprisingly, no specific HSV antigens have been recovered from HSK corneas. Thus, it remains unclear which, if any, viral protein(s) is involved in HSK.
We report here that when HSV-1-infected rabbit corneal buttons were soaked in fixative or buffer, ICP0 could be consistently detected in the soaking solution. ICP0 was also consistently detected in virus-free tears from infected rabbits. In contrast, no other viral antigens were detected. This suggests that ICP0 is water soluble and either rapidly diffuses out of, or is actively secreted from, cells in the cornea.
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METHODS |
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Rabbits.
Eight- to 10-week-old New Zealand White male rabbits (Irish Farms) were used. Rabbits were treated in accordance with ARVO (Association for Research in Vision and Ophthalmology), AALAC (American Association for Laboratory Animal Care) and National Institute of Health guidelines.
Rabbit model of HSV infection.
Rabbits were bilaterally infected without scarification or anaesthesia by placing, as eye drops, 2x105 p.f.u. virus into the conjunctiva cul-de-sac, closing the eyes and rubbing the lid gently against the eye for 30 s as previously described (Perng et al., 1994). At this dose of HSV-1 McKrae, virtually all of the surviving rabbits harbour a latent HSV infection in both trigeminal ganglia. Latency is assumed to have been completely established by 28 days post-infection (p.i.).
Beginning on day 31 p.i., tear film specimens were collected daily from each eye for 26 days as previously described (Perng et al., 1994), using a nylon-tipped swab. The swab was then placed in 0·5 ml tissue culture medium and squeezed, and the inoculated medium was used for plaque assays as previously described (Perng et al., 1994
).
Collection of tears for the detection of viral proteins.
Tears were collected on indicated days using Whatman no. 41 Schirmer strips placed in the inferior lid margin of the eye for 2 min. The strips were then placed in microcentrifuge tubes containing 100 µl PBS with protease inhibitor and strongly vortexed for 5 min. The liquid in the tube was spun through a 0·22 µm filter for 10 min at 14 000 r.p.m. (Beckman Microfuge 22R centrifuge) to remove cell debris and virus particles. The supernatant was lyophilized and stored at 80 °C until all the samples were collected. The dried samples were each resuspended in 20 µl SDS-PAGE sample buffer, boiled for 5 min and briefly spun. They were then subjected to SDS-PAGE, transferred to PVDF membrane and probed with the indicated antibody.
Monitoring corneal scarring.
Recurrent ocular disease was monitored from day 30 to day 58 p.i. Briefly, corneal scarring was scored as being present or absent and was visualized by the unaided eye. A drop of 1 % fluorescein was topically applied to the eye, photographed and confirmed by slit lamp biomicroscopy at the time of sacrifice.
Collection of corneal buttons.
An 8 mm trephine (similar to a cork borer) was used to cut out and collect corneal buttons from the corneas of euthanized rabbits.
Soluble corneal extract preparation.
Corneas were trephined and immediately immersed in 100 µl cold PBS buffer with a cocktail of protease inhibitors (Roche Diagnostics). The sample was kept on ice for 2 h and the supernatant was transferred to a new tube. The protein concentration was measured, adjusted and subjected to SDS-PAGE and Western blot analysis.
Antibodies.
All HSV-1 monoclonal antibodies (mAbs) were purchased from Virusys Corporation except for the ICP27 mAb, which was a gift from Dr Sandri-Goldin (Department of Microbiology and Molecular Genetics, University of California at Irvine, USA). Goat anti-HSV-1 polyclonal antibody was from Biodesign. Goat anti-human albumin antibody was from BiosPacific. GAPDH mAb was from Research Diagnostics. Secondary antibodies conjugated to horseradish peroxidase were from Chemicon International.
Immunoblotting.
Protein samples in gel sample buffer [2 % (v/v) SDS, 50 mM Tris pH 6·8, 3 % (v/v) sucrose, 5 % 2-mercaptoethanol, 0·1 % bromophenol blue] were subjected to electrophoresis in 10 % bisacrylamide gels, transferred to PVDF membranes, blocked for 2 h with 5 % (v/v) non-fat dry milk in PBS and reacted overnight at 4 °C with the appropriate primary antibody diluted in PBS with 1 % BSA and 0·05 % Tween 20 (PBS/BSA/Tween). The appropriate secondary antibody conjugated to horseradish peroxidase was diluted 1 : 5000 in PBS/BSA/Tween and reacted with the membrane for 1 h at room temperature. The blot was rinsed in PBS/BSA/Tween and antibody bound to the blots was detected using SuperSignal West Pico Chemiluminescent substrate (Pierce) and visualized by autoradiography. For reprobing the membrane, blots were stripped by incubating with stripping buffer (PBS containing 7 µl 2-mercaptoethanol ml1 and 2 % SDS) for 30 min at room temperature with constant agitation and then reprobed.
Histology and immunohistochemistry.
Trephined corneas were fixed in 4 % paraformaldehyde in PBS for a minimum of 24 h, embedded in paraffin and sectioned as previously described (Barsam et al., 2005). Sections (5 µm) were stained with haematoxylin and eosin (H&E) and morphology was examined by light microscopy. Serial adjacent sections were stained with HSV-1 polyclonal antibody or specific HSV-1 mAb using the Vectastain ABC kit (Vector Laboratories). Antigens were retrieved by antigen unmasking solution (Vector Laboratories) according to the manufacturer's protocol. The mAb-stained sections were not counterstained in order to increase the clarity of the stain.
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RESULTS |
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Since some water-soluble corneal proteins can diffuse out of the cornea (Piatigorsky, 2001), we decided to look for potential viral proteins in the fixative. The fixative was dialysed against a large volume of PBS buffer overnight at 4 °C. The solution inside the dialysis bag was collected and precipitated with acetone, and the precipitate was collected by centrifugation, vacuum dried and suspended in SDS-PAGE sample buffer. Western blotting was performed as described in Methods using the above well-characterized commercial HSV-1 mAbs. The HSV-1 ICP0 mAb consistently detected a specific band (Fig. 1
), whereas no bands were observed with the other mAbs (not shown). All of the mAbs detected the appropriate HSV-1 protein by Western blotting of acutely infected RS cell lysates and by immunohistochemistry of acutely infected RS cells (Fig. 2
a and b). These results suggest that ICP0 might have rapidly diffused into the fixative.
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ICP0 detection in rabbit tears
To determine whether ICP0 could also be readily detected in the tears of rabbits infected with HSV-1, tears were collected daily as described in Methods between days 3 and 10 p.i. from each of the 10 rabbits described above. The tear films were centrifuged through a 0·22 µm membrane spin column to remove cellular debris and virus particles, and processed for Western blotting. ICP0 was readily detected in the tears from all 20 eyes (tears collected from four eyes are shown in Fig. 5). In 17 eyes, ICP0 was detected on at least one day between days 7 and 9 p.i. In one eye, ICP0 was detected from day 3 to day 9 p.i. In two eyes, ICP0 was detected only on day 5 p.i. We were unable to detect any other HSV-1 proteins in any of the tear samples using the other HSV-1-specific mAbs. This suggests that detection of ICP0 was not the result of virus particles in the samples and that other viral proteins did not diffuse out of the cornea as efficiently as ICP0. Albumin is a major protein in tears and was used as a loading standard. Membranes were reprobed with anti-albumin antibody. All tear samples contained albumin (Fig. 5
).
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The staining pattern seen with anti-gB and -ICP4 mAbs was similar to that seen with HSV-1 polyclonal antibody, with the exception that gB-positive staining appeared to be distributed closer to the epithelial layer, while ICP4-positive staining appeared to be deeper in the stromal layer. In contrast, sections stained with ICP0 mAb showed a different pattern (Fig. 6). ICP0-positive staining on the epithelial layer was not readily observed until day 5 p.i. On days 7 and 10 p.i., spatial distribution of ICP0 staining was visible in the stromal lamellae. By day 14 p.i., the staining was very weak and diffuse in the stromal layer.
ICP0 detection in the soaking buffer of scarred corneas
To readdress the issue of HSV-1 proteins in HSK corneas, we performed a more detailed study than that described in the first paragraph of Results above. Ten rabbits were ocularly infected in both eyes with wild-type HSV-1 McKrae. Beginning on day 31 p.i., eye swabs were collected daily from both eyes of all five surviving rabbits and the amount of recurrent virus shedding was determined by plaque assay (Table 1). A high spontaneous reactivation rate (23 %) was observed and six of ten eyes developed corneal scarring (Table 1
, CS). This is consistent with a report that eyes with a high HSV reactivation rate are more likely to develop HSK (Liesegang, 2001
). Interestingly, a high burst of virus shedding (>500 p.f.u.) occurred prior to the appearance of corneal scarring (Table 1
) and virus shedding was limited after the appearance of corneal scarring. Corneal scarring is known to interfere with the detection of spontaneous reactivation in rabbit tears (Perng et al., 2000b
).
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DISCUSSION |
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We found here that ICP0 also diffused into the cornea soaking buffer from acutely infected corneas. In addition, ICP0 was also readily detectable in fixed corneal sections during acute infection. This suggests that (i) there is much more ICP0 present during acute infection than during HSK; (ii) some or all of the ICP0 is located in different compartments of the cornea during acute infection compared with HSK; (iii) the structure of ICP0 differs during HSK, resulting in increased diffusion out of the cornea; and/or (iv) the HSK cornea allows ICP0 to diffuse out more rapidly. These possibilities are not mutually exclusive.
In contrast to the diffusion of ICP0, none of the other HSV-1 proteins examined during acute infection was readily detected in the soaking buffer, although they were readily detected in corneal sections. The lack of these other HSV-1 proteins in the fixative and/or soaking buffer from HSK corneal buttons and in sections of the HSK corneal buttons therefore suggests that they are significantly less abundant than ICP0 in HSK cornea. Taken together, these results suggest that ICP0 may be the most abundant, if not the only, HSV-1 protein present in HSK corneas. Thus, if HSK is due to an immune response to an HSV-1 antigen, ICP0 is a likely candidate. However, this does not rule out the possibility that small amounts of other viral proteins below levels detectable by Western blotting may be able to trigger an immune-based aetiology of HSK.
Consistent with ICP0 diffusing into the soaking buffer, ICP0 was also found in cell-free and virus-free tears of acutely infected rabbits. Interestingly, the peak of ICP0 in tears (days 710 p.i.) was delayed compared with the peak of infectious virus in tears (day 5 p.i.) and the peak of ICP0 expression in corneal sections (days 57 p.i.). This suggests that in vivo it takes a few days for ICP0 to diffuse into the tears. The temporal disconnection between peak virus replication and the peak of ICP0 in tears also suggests that ICP0 is stable and can be retained for a significant time after virus can no longer be detected. This may explain how ICP0 is present in HSK cornea in the absence of detectable virus or other viral proteins.
The McKrae strain of HSV-1 does not usually cause high rates of recurrent corneal scarring in the rabbit model. Our experience is that typically <2 % of eyes develop corneal scarring. However, in this study, six of ten eyes developed corneal scarring. We believe that this may have been the result of an unusually high spontaneous reactivation rate in this batch of animals (23 % compared with a typical rate of 10 %). This may have been due to one or more unusual environmental factors in our new vivarium facility, including elevated temperatures and 24 h lighting (rather than 12 h on, 12 h off).
Multiple forms of ICP0 occur because of extensive post-translational processing, and this may contribute to the biological properties of ICP0 (Ackermann et al., 1984). In addition, different phosphorylation patterns of ICP0 occur in infected cells (Davido et al., 2005
). Whether one or several forms of phosphorylated ICP0 occur in the corneal soaking buffer is not known. How ICP0 might contribute to HSK immune-related disease is also unclear. However, if ICP0 remains stable in the cornea for long periods of time after acute and recurrent infection, it may be a key antigen recognized by the immune response leading to HSK. In addition, ICP0 plays an important role in blocking the antiviral effects of interferon (Leib et al., 1999
). Thus, ICP0 may act as an antagonistic factor against the innate immune response. The extended presence of ICP0 might therefore facilitate virus replication during reactivation from latency, resulting in increased HSK.
In summary, our findings suggest that ICP0 is present in HSK corneas, but has not previously been detected because it rapidly diffuses out of corneal buttons into the fixative. In contrast, other viral proteins were not detected in either HSK corneas or fixative. This suggests that following acute infection and/or reactivation of HSV-1 from latency, ICP0 has a long-term presence in the cornea. ICP0 is therefore a candidate for a viral antigen involved in HSK. In addition, ICP0 is particularly important for virus replication following low m.o.i. values (Everett et al., 2004; Hagglund & Roizman, 2004
) and this may be particularly relevant for acute or recurrent corneal infections (Kaye et al., 2000
; Ling et al., 2003
). Thus, the presence of ICP0 in tears may play a role in stimulating replication of small amounts of HSV-1 that return to the eye following reactivation.
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ACKNOWLEDGEMENTS |
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Received 7 June 2005;
accepted 22 July 2005.
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