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. Author manuscript; available in PMC: 2006 Sep 1.
Published in final edited form as: J Gen Virol. 2005 Sep;86(Pt 9):2421–2432. doi: 10.1099/vir.0.80979-0

Interferon-β and interferon-γ synergize to block viral DNA and virion synthesis in herpes simplex virus-infected cells.

Amy T Pierce 1, Joanna DeSalvo 1, Timothy P Foster 2, Athena Kosinski 3, Sandra K Weller 3, William P Halford 4,*
PMCID: PMC1366490  NIHMSID: NIHMS4887  PMID: 16099899

SUMMARY

The capacity of herpes simplex virus type 1 (HSV-1) to replicate in vitro decreases tremendously when animal cell cultures are exposed to ligands of both the interferon (IFN)-α/β receptor and IFN-γ receptor prior to inoculation with low MOIs of HSV-1. However, the available evidence provides no insight into the possible mechanisms by which co-activation of the IFN-α/β- and IFN-γ-signaling pathways produces this effect. Therefore, it has not been possible to differentiate whether these observations represent an important in vitro model of host immunological suppression of HSV-1 infection, or an irrelevant laboratory phenomenon. Therefore, the current study was initiated to determine if co-activation of the host cell’s IFN-α/β and IFN-γ pathways either (a) induces death of HSV-1 infected cells such that viral replication is unable to occur or (b) disrupts one or more steps in the process of HSV-1 replication. To this end, multiple steps in HSV-1 infection were compared in populations of Vero cells infected with HSV-1 strain KOS (MOI=2.5) and exposed to ligands of the 1. IFN-α/β receptor, 2. IFN-γ receptor, or 3. both the IFN-α/β receptor and IFN-γ receptor. The results demonstrate that IFN-β and IFN-γ interact in a synergistic manner to block the efficient synthesis of viral DNA and nucleocapsid formation in HSV-1 infected cells, and do so without adversely affecting host cell viability. We infer that IFN-mediated suppression of HSV-1 replication may be a central mechanism by which the host immune system limits the spread of HSV-1 infection in vivo.

INTRODUCTION

Co-activation of the IFN-α/β- and IFN-γ-signaling pathways produces a block to herpes simplex virus type 1 (HSV-1) replication of a magnitude that is not attainable with IFN-α/β or IFN-γ alone, and this effect is not cell type-, species-, or virus strain-specific (Balish et al., 1992, Chen et al., 1994, Sainz & Halford, 2002). This observation has important ramifications because it suggests a previously unrecognized mechanism by which two cytokines of the immune system may cooperate to non-cytolytically control HSV-1 infection in vivo. Given that IFN-α/β and IFN-γ also act in concert to potently inhibit varicella-zoster virus (Desloges et al., 2005), human cytomegalovirus (Sainz et al., 2005), and the SARS virus (Sainz et al., 2004), the proposed interaction between the IFN-α/β- and IFN-γ- signaling pathways may be of general importance in host control of viral infections.

The available evidence indicates that an interaction between the IFN-α/β- and IFN-γsignaling pathways is functionally relevant in host control of HSV-1 infections. Recent comparisons in knockout mice demonstrate that mice that lack both IFN-α/β receptors and IFN-γ receptors (IFN-α/βR−/− and IFN-γR−/−) are profoundly impaired in their resistance to HSV-1 strain KOS infection, experience systemic viral spread, and die 4 to 6 days after footpad inoculation (Luker et al., 2003, Vollstedt et al., 2004). This phenotype is in stark contrast to single receptor knockout mice (IFN-α/βR−/− or IFN-γR−/−), which retain their capacity to limit HSV-1 spread and often survive infection with the KOS strain despite the absence of one of the two IFN receptors (Luker et al., 2003). While these in vivo studies establish that loss of both IFN receptors has a catastrophic effect on host defense against HSV-1, they provide only limited insight into how the IFN-α/β- and IFN-γ- pathways normally interact to control the spread of HSV-1 infection.

The nature of the block to HSV-1 replication produced by IFN-α/β and IFN-γ remains undefined. Does exposure to IFN-α/β and IFN-γ cause host cells to undergo apoptosis upon HSV-1 infection (Park et al., 2004, Takaoka et al., 2003)? The low multiplicity design of previous in vitro studies does not exclude this possibility (Balish et al., 1992, Chen et al., 1994, Sainz & Halford, 2002). Alternatively, IFN-β and IFN-γ may create a block to HSV-1 replication which does not compromise the viability of the host cell. If so, then at which step(s) between immediate-early (IE) mRNA transcription and virion egress does the inhibitory block occur? There are no published studies that address these questions.

The current study was initiated to refine our understanding of how co-activation of the IFN-α/β- and IFN-γ-signaling pathways prevents HSV-1 replication in vitro. Given the complexity of the HSV-1 replication cycle, we thought it unrealistic to sequentially test the scores of hypotheses that might explain the inhibitory effect. Rather, we felt a more directed approach was to first constrain the number of possible explanations by determining if IFN-β and IFN-γ are 1. cytotoxic to HSV-1 infected cells or 2. disrupt HSV-1 replication at one or more discernible steps relative to viral mutants or an inhibitor of viral DNA synthesis. Using this approach, the separate versus combined effects of IFN-β and IFN-γ on viral DNA, mRNA, protein, and virion accumulation were compared in Vero cells infected with 2.5 pfu/cell of HSV-1. The use of an MOI that infected nearly 100% of cells in the population assured that any differences in the measured parameters were the result of a defect in the first, and only, cycle of viral replication. We report that IFN-β and IFN-γ do not inhibit HSV-1 replication via a non-specific cytotoxic effect. Rather, IFN-β and IFN-γ render host cells non-permissive for viral DNA synthesis and nucleocapsid assembly, and thus non-cytolytically suppress HSV-1 replication in the vast majority of infected cells.

MATERIALS AND METHODS

Cells, viruses, and interferons

Vero cells, ICP4-complementing E5 cells (DeLuca et al., 1985), origin-binding protein (OBP)-complementing 2B.11 cells (Malik et al., 1992), and glycoprotein D (gD)-complementing V15-D1 cells (Warner et al., 1998) were propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS). Wild-type HSV-1 strain KOS (Smith, 1964) and the virus K26GFP (kindly provided by Prashant Desai, Johns Hopkins University), which expresses a chimeric VP26-GFP protein (Desai & Person, 1998), were propagated in Vero cells. The ICP4−virus n12 (kindly provided by Priscilla Schaffer, Harvard University; DeLuca et al., 1985), the OBP− virus hr94 (Malik et al., 1992), and the gD− virus KOS-gD6 (kindly provided by Patricia Spear, Northwestern University; Warner et al., 1998) were propagated in their corresponding complementing cell lines. Aliquots of recombinant human IFN-β and human IFN-γ (PBL Biomedical Laboratories) were stored at −80°C and were diluted to 200 U/ml just prior to use. Combinations of IFN-β and IFN-γ (100 U/ml each) were made by mixing 200 U/ml IFN-β with 200 U/ml IFN-γ in a 1:1 ratio. Throughout this study, IFN treatment and KOS infection were performed by a standardized protocol. Culture dishes were seeded with Vero cells and cells were treated 24 hours later with 200 U/ml IFN-β, 200 U/ml IFN-γ, or 100 U/ml each of IFN-β and IFN-γ. Sixteen hours later, Vero cells were inoculated with virus at an MOI of 2.5 pfu per cell. After allowing 45 minutes for adsorption, the inoculum was replaced with complete DMEM containing no IFN (vehicle), 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each IFN-β and IFN-γ, or 300 μM acyclovir.

Dot blot analysis of HSV-1 DNA yield

Vero cells were established at a density of 1 x 105 cells per well in 24-well plates, and were infected with KOS at an MOI of 2.5. Cultures were incubated in the presence of vehicle, 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir. Between 9 and 24 hours p.i., crude DNA lysates were harvested, probed for viral DNA, and the results enumerated as previously described (Halford et al., 2005).

Flow cytometry

i. Enumeration of morphologically normal cells

Vero cells were established at a density of 2 x 105 cells per well in 12-well plates, and were infected with KOS at an MOI of 2.5. Cultures were incubated in the presence of vehicle, 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir. Uninfected cells or KOS-infected cells were harvested by aspirating culture medium, rinsing with 1 ml PBS, dissociation in trypsin, resuspension in PBS + 10% FBS, and passing cells through a 50 μm mesh to remove cell debris. Cells were counted for a fixed period of time, 2 minutes, on a FACSCalibur (BD Biosciences, San Jose, CA). At each time point, the total number of cells in four cultures was determined using a hemacytometer, and compared to the number of cells counted by the flow cytometer. On average, the flow cytometer counted ~40% of cells in these cultures. Therefore, the total number of morphologically normal cells per culture was estimated by dividing the number of cells counted by the flow cytometer by the fraction of cells sampled (e.g., ~0.4).

ii. Two color analysis of viral protein expression in HSV-1-infected cells

Vero cells were established at a density of 4.5 x 106 cells per 100 mm dish, and were infected with KOS at an MOI of 2.5. At 9 hours p.i., Vero cells were dissociated with trypsin and prepared for immunofluorescent labeling by fixation in 2% formaldehyde + 2% sucrose, permeabilization in 90% methanol, passage through a 27 g needle, and resuspension in PBS + 0.5% FBS containing Fc-γ receptor blocking agents (i.e., 5 μg/ml each of human IgG, donkey IgG, and goat IgG). Cells were incubated for 1 hour with a 1:20,000 dilution of rabbit anti-HSV-1 (Dako Cytomation, Carpinteria, CA) and a 1:1000 dilution of mouse monoclonal antibodies against ICP0, ICP4, ICP6, gC, or gD (Rumbaugh Goodwin Institute, Plantation, FL). Cells were washed twice and incubated for 30 minutes with a 1:1000 dilution of phycoerythrin-labeled-donkey anti-rabbit IgG and a 1:350 dilution of FITC-labeled-goat anti-mouse IgG. Cells were washed twice and the fluorescent intensity of ~10,000 cells was measured in each sample (i.e., 10,000 events) using a FACSCalibur and CellQuest Pro software (BD Biosciences). For each viral protein, total protein yield was summated by multiplying the fraction of viral protein+ cells times the mean fluorescent intensity of viral protein staining, and this “fluorescent volume” was normalized relative to the lower limit of detection of the assay, which was defined as three times the background fluorescent volume associated with uninfected cells (Soboleski et al., 2005).

Measurement of HSV-1 virion yields

Vero cells were established at a density of 4.5 x 106 cells per dish in 100 mm dishes, and were infected with KOS at an MOI of 2.5. Cultures were incubated in the presence of vehicle, 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir. Cells were prepared for transmission electron microscopy at 18 hours p.i. as previously described (Foster et al., 2004, Foster et al., 2003).

For analysis of virion yields, cells were transferred to −80°C at 18 hours p.i. and virion yields were determined in cell lysates following purification through a series of two gradients. For each sample, fifteen fractions that spanned the 30% – 60% sucrose interface of the second gradient were identified using a refractometer, and virion yield in each fraction was measured by ELISA. Each fraction was diluted 1:10, 1:50, or 1:250 in PBS, and each dilution was used as coating antigen in two wells of a 96-well plate. After overnight incubation, virion fractions were discarded, wells were blocked with 0.5 % dry milk, and a 1:600 dilution of anti-HSV-1 conjugated to horseradish peroxidase (Dako Cytomation) was added per well. After 1 hour, excess antibody was rinsed away and bound antibody was measured with TMB Blue substrate (DakoCytomation) at 450 nm in a plate reader.

RESULTS

IFN-β and IFN-γ synergistically protect HSV-1 -infected cells from viral CPE

Flow cytometry was used to enumerate the number of morphologically normal cells per culture. The validity of the method was tested on Vero cells infected with MOIs of 0.001 to 10 of HSV-1 strain KOS for 36 hours. Flow cytometry confirmed that the fraction of normal cells decreased as viral MOI increased (Fig. 1A), and the results correlated well with the degree of CPE observed by light microscopy. Treatment of Vero cell monolayers with 200 U/ml IFN-β or IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir had no effect on cell morphology (Fig. 1B). However, treatments that contained IFN-β prevented proliferation to the same extent as vehicle-treated cells. By comparison, other compounds used to inhibit HSV-1 replication such as 10 μM MG132 (Poon et al., 2002) or 100 μM roscovitine (Schang et al., 1998) were overtly toxic to uninfected Vero cells within 24 hours (not shown).

Figure 1. IFN-β and IFN-γ protect HSV-1 infected cells from viral CPE. A.

Figure 1

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The percent of morphologically normal cells in HSV-1 infected cultures (relative to uninfected cultures) at 36 hours p.i. plotted as a function of viral MOI (n=2 per group). ‘Normal cells’ were defined as adherent cells that retained the forward and side-scatter properties of healthy, untreated Vero cells following dissociation with trypsin. B. Uninfected cells per culture that remained morphologically normal at times after treatment with 300 μM acyclovir (ACV), vehicle (VEH), 200 U/ml IFN-β, 200 U/ml IFN-γ, or 100 U/ml each of IFN-β and IFN-γ. C. Percent of KOS-infected cells (MOI=2.5) that remained morphologically normal at times after inoculation in the presence of ACV, VEH, IFN-β, IFN-γ, or IFN-β and IFN-γ.

Infection with HSV-1 strain KOS (MOI = 2.5) destroyed ~99% of vehicle-treated cells by 48 hours p.i. (Fig. 1C). Treatment with IFN-β or IFN-γ delayed the onset of cytopathic effect (CPE) in KOS-infected cultures and increased the fraction of normal cells recovered between 24 and 48 hours p.i. (Fig 1C). Acyclovir or a combination of IFN-β and IFN-γ provided the greatest protection, such that in these treatment groups ~30% of KOS-infected cells remained morphologically normal 48 hours p.i. (Fig. 1C). Therefore, treatment with IFN-β and/or IFN-γ was not cytotoxic to Vero cells, but rather delayed the onset of viral CPE in KOS-infected cells.

IFN-β and IFN-γ synergistically reduce the efficiency of HSV-1 DNA synthesis

The combined versus separate effects of IFN-β and IFN-γ on HSV-1 DNA synthesis were compared in Vero cells inoculated with HSV-1 strain KOS (MOI = 2.5). DNA samples harvested 9 to 24 hours p.i. were immobilized on a nylon membrane and hybridized to a probe specific for the HSV-1 US6 gene (Fig. 2A). Treatment with 200 U/ml IFN-β or IFN-γ alone delayed the detection of viral DNA synthesis by approximately 3 hours and slightly decreased the rate (slope) of viral DNA synthesis between 15 and 24 hours p.i. relative to vehicle-treated controls (Fig. 2A and B). Combined treatment with 100 U/ml each of IFN-β and IFN-γ delayed the detection of viral DNA synthesis by 6 hours, and greatly reduced the rate of HSV-1 DNA synthesis (Fig. 2A and B; p <0.001, as determined by one-way ANOVA and Tukey’s post hoc t-test). Unlike IFN-β and IFN-γ, which only reduced the efficiency of HSV-1 DNA synthesis, 300 μM acyclovir completely prevented the detection of HSV-1 DNA synthesis (Fig. 2A and 2B).

Figure 2. Effect of IFN-β and IFN-γ on viral DNA levels.

Figure 2

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A. Dotblot of DNA from uninfected (UI) cells and KOS-infected Vero cells treated with 300 μM acyclovir (ACV), vehicle (VEH), 200 U/ml IFN-β, 200 U/ml IFN-γ, or 100 U/ml each of IFN-β and IFN-γ. DNA was harvested 9 to 24 hours p.i with 2.5 pfu/cell of KOS and was hybridized to a US6-specific probe. B. Viral DNA yield in each group is plotted as a function of time. Viral DNA yields are scaled relative to the lower limit of detection of the assay, which was assigned a value of 1.

IFN-β and IFN-γ synergistically reduce the accumulation of HSV-1 mRNAs

The combined versus separate effects of IFN-β and IFN-γ on HSV-1 mRNA synthesis were compared in Vero cells inoculated with HSV-1 strain KOS (MOI = 2.5). RNA samples harvested 9 hours after inoculation were compared by Northern blot analysis. In vehicle-treated cells, KOS infection reduced the amount of probe that hybridized to cellular G3PDH mRNA to 15% of that observed in uninfected Vero cells (Fig. 3). In KOS-infected cells treated with IFN-β or IFN-γ, G3PDH mRNA levels were ~50% of normal levels. However, G3PDH mRNA levels remained at 100% of normal levels in KOS-infected cells treated with IFN-β and IFN-γ or 300 μM acyclovir (Fig. 3). Northern blot analysis of viral IE or E mRNAs revealed that IFN-β or IFN-γ had little effect on the abundance of ICP4, ICP27, ICP0, or HSV DNA polymerase (pol) mRNAs in KOS-infected Vero cells (Fig. 3). However, treatment with IFN-β and IFN-γ, or 300 μM acyclovir, reduced the amount of probe that hybridized to ICP4, ICP27, ICP0, and pol mRNAs by 2- to 3-fold (Fig. 3). Treatment with IFN-β or IFN-γ alone caused a 1- to 3-fold reduction in the amount of probe that hybridized to viral L mRNAs that encode VP5, VP23, or gD (Fig. 3). Treatment with IFN-β and IFN-γ, or 300 μM acyclovir, caused a 5- to 10-fold reduction in the amount of probe that hybridized to these L viral mRNAs (Fig. 3). The degree to which IFN-β and IFN-γ reduced viral mRNA levels was equivalent to that achieved by 300 μM acyclovir. Thus, the acyclovir control suggested that a block to the amplification of the number of genomes available for mRNA synthesis might contribute significantly to the reduced viral mRNA levels observed in KOS-infected cells treated with IFN-β and IFN-γ.

Figure 3. Effect of IFN-β and IFN-γ on viral mRNA levels.

Figure 3

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Northern blot analysis of Vero cell RNA harvested 9 hours p.i. with nothing (UI=uninfected) or 2.5 pfu/cell of KOS and hybridized to radiolabeled probes specific for mRNAs that encode, from top to bottom, cellular G3PDH, viral ICP4, ICP27, ICP0, HSV-1 DNA polymerase (pol), VP5, VP23, or glycoprotein D (gD). KOS-infected Vero cells were treated with vehicle (VEH), 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir (ACV).

IFN-β and IFN-γ synergistically reduce the accumulation of HSV-1 proteins

Flow cytometry was used to compare the combined versus separate effects of IFN-β and IFN-γ on the expression of individual HSV-1 proteins in Vero cells inoculated with KOS (MOI = 2.5). Cells were double-labeled with mouse monoclonal antibodies against individual viral proteins (i.e., ICP0, ICP4, ICP6, or gD) and rabbit polyclonal antibody against HSV-1, which served as an internal control for the consistency of antibody labeling. Regarding the controls, KOS-infected cells were not labeled by irrelevant mouse antibodies or irrelevant rabbit antibodies. As expected, the ICP4− virus, n12, expressed high levels of ICP0 (Fig. 4A, 4B) and the first 251 amino acids of ICP4 (DeLuca & Schaffer, 1987) which contains the epitope that binds the anti-ICP4 antibody (Hubenthal-Voss et al., 1988) (Fig. 4C, 4D). The ICP4− virus expressed only low levels of ICP6 (Fig. 4E and 4F) and undetectable levels of gD (Fig. 4G, 4H). In contrast, OBP− and gD− viruses expressed high levels of these viral proteins with the exception that the gD− virus failed to express gD (Fig. 4G, 4H).

Figure 4. Effect of IFN-β and IFN-γ on viral protein levels.

Figure 4

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Viral protein expression in Vero cells which were uninfected (UI) or were harvested 9 hours after inoculation with 2.5 pfu/cell of KOS, n12 (ICP4−), hr94 (OBP−), or KOS-gD6 (gD−). The total abundance (A, C, E, and G) versus frequency of viral protein+ cells (B, D, F, and H) was analysed by flow cytometic measurement of the amount of ICP0, ICP4, ICP6, or glycoprotein D (gD) present in 7,000 – 9500 cells per culture (n=3 samples per group; 10,000 events per sample). KOS-infected cells were treated with vehicle (VEH), 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir (ACV). Protein abundance is represented as the mean ± sem above the lower limit of detection, which was assigned a value of 1 (n=3 per group). Frequency of viral protein+ cells is represented as the fraction of cells that were singly labeled by mouse monoclonal anti-ICP0, ICP4, ICP6, or gD only (green fluorescent label; green bar) versus cells that were doubly labeled with mouse monoclonal antibody and rabbit polyclonal anti-HSV-1 (yellow bar). Error bars indicate the average coefficient of variation for the two subpopulations.

Vehicle-treated, KOS-infected cells expressed the IE proteins ICP0 and ICP4 at levels that were 80- and 130-times above the lower limit of detection (Fig. 4A, 4C). Treatment with 200 U/ml IFN-β or IFN-γ reduced ICP0 and ICP4 abundance by 1.1- to 1.8-fold, whereas 100 U/ml each of IFN-β and IFN-γ reduced ICP0 and ICP4 abundance by 2.5- and 3.3-fold, respectively (Fig. 4A, 4C). The majority of change in ICP0 and ICP4 abundance was not due to differences in protein level per cell (i.e., as measured by mean fluorescent intensity), but rather was due to the fact that IFN-β and IFN-γ decreased the frequency of KOS-infected cells that were ICP0+ or ICP4+ decreased by 70% and 60%, respectively (Fig. 4B, 4D). IFN-β and IFN-γ caused a 5.1-fold reduction in the abundance of the E protein ICP6 relative to vehicle-treated cells (Fig. 4E), which was largely attributable to a 75% decrease in the frequency of ICP6+ cells (Fig. 4F). Analysis of ICP8 expression (an E protein) revealed a similar pattern (not shown). IFN-β and IFN-γ caused a 7.0-fold reduction in the abundance of the leaky L protein gD relative to vehicle-treated, KOS-infected cells (Fig. 4G). This decrease was not only due to a 75% decrease in the frequency of gD+ cells (Fig. 4H), but was also compounded by a 45% reduction in gD protein per cell. IFN-β and IFN-γ caused a 27-fold reduction in the abundance of the true L protein gC relative to vehicle-treated, KOS-infected cells (not shown). This decrease was due to a 94% decrease in the frequency of gC+ cells, and a 40% reduction in gC protein per cell. Flow cytometric analysis suggested that IFN-β and IFN-γ acted at two levels to 1. prevent detectable viral IE and E protein synthesis in ~70% of KOS-infected cells (e.g., as measured by reduced frequency of ICP0+ cells) and 2. restricted viral replication in the remaining ~30% of KOS-infected cells in a manner that did not prevent viral IE and E protein synthesis.

The full inhibitory effect of IFN-β and IFN-γ is achieved before or during viral DNA synthesis

Vero cells were infected with HSV-1 strain KOS (MOI = 2.5) and were treated with vehicle, IFN-β, IFN-γ, IFN-β and IFN-γ, or 0.001 to 320 μM acyclovir. Cultures were harvested 18 hours p.i. to compare viral DNA yields (Fig. 5A and 5B) and viral titers (Fig. 5C). Relative to vehicle-treated cells, a combination of IFN-β and IFN-γ reduced HSV-1 DNA yields in KOS-infected cells by ~100-fold, which was equivalent to the reduction achieved by 32 μM acyclovir (Fig. 5A and 5B). The same combination of IFN-β and IFN-γ reduced infectious viral titers by ~1000-fold relative to vehicle-treated cells, and 32 μM acyclovir was found to reduce viral titers to precisely the same extent (Fig. 5C). Regression analysis of viral titers versus DNA yield confirmed that acyclovir, an acyclic guanosine analog (Elion, 1983), reduced HSV-1 titers in direct proportion to the drug’s inhibitory effect on viral DNA synthesis (closed circles in Fig. 5D). Likewise, it was observed that IFN-β alone, IFN-γ alone, or IFN-β and IFN-γ each reduced HSV-1 titers in direct proportion to their inhibitory effects on HSV-1 DNA synthesis (open symbols in Fig. 5D). Therefore, the results suggested that IFN-β and/or IFN-γ must achieve their full inhibitory effect on HSV-1 replication before or during the process of viral DNA synthesis.

Figure 5. IFN-β and IFN-γ inhibit HSV-1 replication before or during viral DNA synthesis.

Figure 5

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A. Dotblot of Vero cell DNA harvested 18 hours p.i. with 2.5 pfu/cell of KOS and hybridized to a US6-specific probe. KOS-infected cells were treated with vehicle (VEH), 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 0.3 to 320 μM acyclovir (ACV). B. Viral DNA yield as a function of ACV concentration (closed circles; n=2 per dose). Viral DNA yields observed in cells treated with vehicle, IFN-β, IFN-γ, or IFN-β and IFN-γ are shown on left side of graph (n=3 each). Dashed lines indicate viral DNA yield in cells treated with IFN-β and IFN-γ, and the ACV dose that reduces viral DNA to the same extent. C. Viral titer as a function of ACV concentration (closed circles; n=2 per dose). Viral titers recovered from cells treated with vehicle, IFN-β, IFN-γ, or IFN-β and IFN-γ are plotted on the left side of graph (n=3 each). Dashed lines indicate viral titer in cells treated with IFN-β and IFN-γ, and the ACV dose that reduces viral titer to the same extent. D. Correlation of viral DNA yield and viral titer recovered from infected cells treated with ACV (closed circles) versus cells treated with vehicle, IFN-β, IFN-γ, or IFN-β and IFN-γ. The solid line indicates the line of regression between viral titers and viral DNA yield in ACV treatments (r2=0.97).

IFN-β and IFN-γ synergistically inhibit HSV-1 virion synthesis

The combined versus separate effects of IFN-β and IFN-γ on HSV-1 virion synthesis were compared in Vero cells inoculated with HSV-1 strain KOS (MOI = 2.5) and harvested 18 hours p.i. Cellular sections were analyzed by transmission electron microscopy. No structures resembling nucleocapsids or enveloped virions were observed in uninfected cells or KOS-infected cells treated with 300 μM acyclovir (not shown). In KOS-infected cells treated with vehicle, enveloped virions were apparent on most cell surfaces and nucleocapsids were numerous within the nucleus (Fig. 6A). Treatment with IFN-β or IFN-γ alone reduced the nucleocapsid density per cell, but viral particles were still evident in >95% of cell sections (Fig. 6B, 6C). In contrast, treatment with IFN-β and IFN-γ inhibited virion formation and preserved the ultrastructural features of these cells (e.g., intact Golgi apparatus in Fig. 6D). Fewer than 3% of sections from cells treated with IFN-β and IFN-γ contained visible evidence of infection. In the few cells in which virus was observed, only 1 to 5 enveloped virions were observed in the cytoplasm or at the cell surface (Fig. 6D).

Figure 6. Effect of IFN-β and IFN-γ on virion formation in HSV-1 infected cells.

Figure 6

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Electron micrographs of Vero cells harvested 18 hours p.i. with 2.5 pfu/cell of KOS, and treated with A. vehicle, B. 200 U/ml IFN-β, C. 200 U/ml IFN-γ, or D. 100 U/ml each of IFN-β and IFN-γ. The letters n, c, and e denote nucleus, cytoplasm, and extracellular space. Arrows denote areas containing virus particles.

The combined versus separate effects of IFN-β and IFN-γ on HSV-1 virion yield were enumerated. At 18 hours p.i., cells from each treatment group were freeze-thawed and the released virions were purified over a series of two discontinuous sucrose gradients. Fractions from the interface between the 30% and 60% layers of the second gradient were used as coating antigens in an ELISA, and the capacity of each fraction to capture polyclonal anti-HSV-1 antibody was measured (Fig. 7A). The results of two independent experiments demonstrated that the yield of HSV-1 virions in vehicle-treated cells was ~225 times greater than the background of the assay (Fig. 7B). The yield of HSV-1 virions obtained from cells treated with IFN-β or IFN-γ was 29% and 46% of that recovered from vehicle-treated cells, respectively (Fig. 7B). In contrast, in cells treated with IFN-β and IFN-γ, HSV-1 virion yield was only 2% of that recovered from vehicle-treated cells (Fig. 7B). Therefore, combinations of IFN-β and IFN-γ repress HSV-1 virion synthesis to a much greater extent than can be achieved with either IFN-β or IFN-γ alone.

Figure 7. Effect of IFN-β and IFN-γ on virion yield.

Figure 7

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A. Virion yield in sucrose gradient fractions, as determined by ELISA, plotted as a function of the density of each fraction. Virions were purified from Vero cells harvested 18 hours p.i. with 2.5 pfu/cell of KOS, and which were treated with either vehicle, 200 U/ml IFN-β, 200 U/ml IFN-γ, 100 U/ml each of IFN-β and IFN-γ, or 300 μM acyclovir (ACV). Uninfected cells processed in parallel were used to define the background of the ELISA. B. Virion yield from cells treated with vehicle, IFN-β, IFN-γ, or IFN-β and IFN-γ, as determined from two experiments (mean ± sd).

DISCUSSION

IFN-β and IFN-γ do not compromise cell viability

Combinations of IFN-β and IFN-γ were not toxic to Vero cells, nor did these cytokines prime Vero cells to undergo virus-induced apoptosis. To the contrary, IFN-β and IFN-γ delayed the onset of viral CPE in HSV-1-infected cultures as measured by the preservation of 1. cellular morphology, 2. cellular G3PDH mRNA levels, and 3. ultrastructural morphology. Therefore, we infer that combinations of IFN-β and IFN-γ non-cytolytically suppress HSV-1 replication via the disruption of productive viral replication.

Are the effects of IFN-β and IFN-γ on HSV-1 replication synergistic?

Pharmacological analysis of synergy focuses on differentiating whether an observed change in effect is due to a 1. cooperative interaction between two biologically active agents or 2. the change in concentration that occurs whenever two agents are combined. Under a null hypothesis of dose additivity, IFN-β and IFN-γ would be assumed to have purely redundant effects, such that 100 U/ml each of IFN-β and IFN-γ should inhibit any step of HSV-1 replication to the average of that achieved by 200 U/ml IFN-β or 200 U/ml IFN-γ (Tallarida, 2001, Tallarida et al., 1997). The observed effects of IFN-β and IFN-γ were compared to the predictions of the null hypothesis (Table 1). In each case, IFN-β and IFN-γ reduced the synthesis of viral proteins, viral DNA, and virions to an extent far greater than predicted by the null hypothesis (Table 1). Thus, the block to HSV-1 replication that is produced by co-activation of the IFN-α/β and IFN-γ pathways is quantitatively distinct from that which occurs when only one of these two signaling pathways is activated. The multiplicative nature of the interaction between the IFN-α/β and IFN-γ pathways is considered in detail elsewhere (Halford et al., 2005).

Table 1.

IFN-β and IFN-γ synergistically inhibit multiple steps in HSV-1 replication.

Fold-Reduction:
Measure of HSV-1 replication IFN-β f (200 U/ml) IFN-γ g (200 U/ml) IFN-β + IFN-γ h (100 U/ml each) Predicted dose-additive effect i H0: P - O = 0 j
ICP0 (IE) a 1.1 ± 0.1 1.5 ± 0.0 2.5 ± 0.2 1.3 ± 0.1 p < 0.01
ICP4 (IE) a 1.8 ± 0.2 1.7 ± 0.2 3.3 ± 0.2 1.8 ± 0.2 p < 0.001
ICP6 (E) a 2.2 ± 0.2 1.8 ± 0.1 5.1 ± 0.3 2.0 ± 0.2 p < 0.01
ICP8 (E) b 2.5 ± 0.2 2.5 ± 0.2 6.6 ± 0.5 2.5 ± 0.1 p < 0.05
gC (L) a 5.1 ± 0.2 2.1 ± 0.1 27 ± 5 3.5 ± 0.1 p < 0.05
gD (L) a 2.4 ± 0.3 2.1 ± 0.1 7.0 ± 0.4 2.3 ± 0.2 p < 0.01
VP26 (L) c 17 ± 1 21 ± 1 48 ± 5 19 ± 1 p < 0.05
viral DNA yield d 8.9 ± 0.1 6.6 ± 0.2 106 ± 6 7.8 ± 0.1 p < 0.001
virion yield e 3.5 ± 0.8 2.2 ± 0.3 45 ± 5 2.9 ± 1.5 N/A k
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a

Mean ± sem of fold-reduction in ICP0, ICP4, ICP6, gC, or gD proteins at 9 hours p.i. (n=3 per group). Based on flow cytometric analysis of each protein using mouse monoclonal antibodies as the primary label and FITC-labeled goat-anti mouse IgG as the secondary label. These conclusions are based on the data presented in Figure 4 with the exception of the gC data which is not shown. The kinetic class of each viral protein is indicated in parentheses (Immediate-Early, Early, or Late).

b

Mean ± sem of fold-reduction in ICP8 protein at 9 hours p.i. (n=3 per group). Based on flow cytometric analysis using rabbit polyclonal anti-ICP8 as the primary label and PE-labeled donkey-anti rabbit IgG as the secondary label (data not shown).

c

Mean ± sem of fold-reduction in expression of a VP26-GFP chimeric protein at 12 hours p.i. (n=3 per group). Based on flow cytometric analysis of GFP fluorescence in cells infected with the virus K26GFP, which expresses a chimeric VP26-GFP protein (data not shown). The quantitative reliability of GFP as a reporter protein is established elsewhere (Soboleski et al., 2005).

d

Mean ± sem of fold-reduction in relative DNA yield at 18 hours p.i. (n=3 per group). Based on results presented in Figure 5.

e

Mean ± sd of fold-reduction in virion yield at 18 hours p.i. (n=2 per group). Based on results presented in Figure 7.

f

Fold-reduction observed in KOS-infected cells treated with 200 U/ml IFN-β relative to vehicle-treated controls.

g

Fold-reduction observed in KOS-infected cells treated with 200 U/ml IFN-γ relative to vehicle-treated controls.

h

Observed fold-reduction in KOS-infected cells treated with 100 U/ml each of IFN-β and IFN-γ relative to vehicle-treated controls.

i

Predicted fold-reduction achieved by IFN-β and IFN-γ relative to vehicle-treated controls, based on a null hypothesis that nothing more than a dose-additive effect occurs when IFN-β and IFN-γ are combined.

j

Probability that nothing more than a dose-additive effect occurs when IFN-β and IFN-γ are combined. Determined by a paired t-test of the null hypothesis that Predicted - Observed fold-reduction is equal to 0.

k

Not applicable. The number of independent tests (n=2) is insufficient for statistical comparison of the summated value of total virion yield by the paired t-test used in this table. It should be noted that an appropriate statistical analysis of the 30 gradient fractions tested per group (e.g., two-way ANOVA) would indicate that the null hypothesis, H0: P - O = 0, should be rejected.

Where do IFN-β and IFN-γ act to inhibit HSV-1 replication?

Viral mutants or acyclovir were used to help break down the complex process of HSV-1 replication into discernible phases. The phenotypes that follow from failure to exit the IE phase of gene expression were defined by the ICP4− virus, n12 (DeLuca et al., 1985). The consequences of a block to viral DNA synthesis were defined by the OBP− virus hr94 (Malik et al., 1992) or treatment of KOS-infected cells with 300 μM acyclovir (Elion, 1983).

i. Inhibition of viral IE gene expression?

Cells infected with an ICP4− virus expressed 5 times as much ICP0 as KOS-infected cells treated with IFN-β and IFN-γ. However, ICP4− virus-infected cells expressed low levels of ICP6 and undetectable levels of gD. In contrast, KOS-infected cells treated with IFN-β and IFN-γ exhibited a more gradual attenuation of E and L protein synthesis (i.e, ICP6, ICP8, gC, and gD). Relative to the ICP4− virus, the abundance of viral E and L proteins was too high to be consistent with a hypothesis that repression of viral IE protein synthesis is the sole mechanism by which IFN-β and IFN-γ inhibit HSV-1 replication.

ii. Inhibition of viral DNA synthesis?

IFN-β and IFN-γ did not block viral DNA synthesis in KOS-infected cells with the efficiency of 300 μM acyclovir. However, IFN-β and IFN-γ caused a greater reduction in viral IE and E protein synthesis than acyclovir treatment of KOS-infected cells. Likewise, an OBP− virus expressed high levels of ICP0, ICP4, ICP6, and ICP8, despite a complete block to viral DNA synthesis. Relative to these controls, the reduction in viral IE and E protein synthesis was too great to be consistent with a hypothesis that repression of viral DNA synthesis is the sole mechanism by which IFN-β and IFN-γ inhibit HSV-1 replication.

iii. IFN-β and IFN-γ do not inhibit HSV-1 at the same step in every infected cell

Flow cytometric analysis of viral protein expression revealed that IFN-β and IFN-γ must inhibit KOS replication at least two distinct points. The first point of inhibition produced by IFN-β and IFN-γ reduced the frequency of KOS-infected cells that expressed detectable levels of ICP0 by 70% relative to vehicle-treated cells (Fig. 4). Because IFN-β and IFN-γ do not preclude viral entry (Sainz & Halford, 2002), we infer that IFN-β and IFN-γ successfully repress viral ICP0 gene expression in ~70% of KOS-infected cells. Absence of ICP0 renders all of the viral IE genes highly susceptible to repression by IFN-α/β (Harle et al., 2002, Mossman et al., 2000). Therefore, whether or not ICP0 is successfully synthesized may create two divergent outcomes of HSV-1 infection. Under this hypothesis, one would predict that if IFN-β and IFN-γ successfully repress the ICP0 gene in ~70% of KOS-infected cells, all viral IE gene expression will be stably repressed by the IFN treatment (Mossman et al., 2000). However, if IFN-β and IFN-γ fail to repress the ICP0 gene in ~30% of KOS-infected cells, ICP0 protein will be synthesized and will destabilize IFN-induced repression of the viral IE genes (Harle et al., 2002), thus allowing viral replication to proceed beyond this first restriction point. Although this hypothesis is highly consistent with the results, its validity remains to be tested.

The second discernible point of inhibition produced by IFN-β and IFN-γ lies at the level of viral DNA synthesis. The 50- to 100-fold reduction in viral DNA synthesis that is produced by IFN-β and IFN-γ would not be predicted based on the fact that ~25% of KOS-infected cells (relative to vehicle controls) express normal levels of the E proteins ICP6 and ICP8 at 9 hours p.i. The underlying mechanism that accounts for the block to viral DNA synthesis induced by IFN-β and IFN-γ remains to be elucidated, and will certainly be the focus of future investigation. Given the hypersensitivity of HSV-1 ICP34.5− mutants to IFN-α/β (Cerveny et al., 2003, Mossman & Smiley, 2002), one intriguing possibility is that expression of the other major viral IFN antagonist, ICP34.5, is synergistically repressed by IFN-β and IFN-γ such that HSV-1 is rendered highly susceptible to a PKR-induced shutoff of L viral protein translation. The validity of this specific hypothesis remains to be tested.

iv. Effect on downstream steps of viral replication?

IFN-β and IFN-γ do not appear to inhibit phases of HSV-1 replication beyond HSV-1 DNA synthesis, such as virion maturation or egress. Doses of acyclovir that inhibited viral DNA synthesis to the same extent as IFN-β, IFN-γ, or IFN-β and IFN-γ reduced infectious viral titers to precisely the same extent as each IFN treatment. Given that acyclovir acts solely to inhibit HSV-1 DNA synthesis, these data strongly suggest that the full inhibitory effect of IFN-β and/or IFN-γ is achieved before or during the process of HSV-1 DNA synthesis.

Relevance of experimental design?

In nature, animals are first infected with a virus and then respond by secreting cytokines such as IFN-α/β or IFN-γ. Given the natural order of events, what natural process is being modeled when Vero cells are pre-treated with IFN-β and IFN-γ for 16 hours prior to infection? At the time at which HSV-1 first infects an animal, IFNs are absent in host tissues and the first productive cycle of viral replication proceeds unhindered in vivo. However, the available evidence suggests that host IFNs play a pivotal role in restricting the efficiency with which each of the ~10 subsequent cycles of viral replication proceed during a typical 7-day course of acute infection in vivo (Halford et al., 2005, Luker et al., 2003, Vollstedt et al., 2004). Thus, the natural process being modeled when Vero cells are pre-treated with IFN-β and IFN-γ relates to the pivotal role that host IFNs play in restricting HSV-1 spread within infected tissues in vivo.

Conclusion

Co-activation of the IFN-α/β and IFN-γ signaling pathways produces a multiplicative inhibition of HSV-1 replication (Halford et al., 2005). Studies in IFN receptor knockout mice corroborate the functional relevance of this interaction in vivo (Luker et al., 2003, Vollstedt et al., 2004). Given the persistence of IFN-γ-expressing CD8+ T cells at sites of latent HSV-1 infection (Khanna et al., 2003, Theil et al., 2003), these results suggest that IFN-γ secretion may contribute to the capacity of CD8+ T cells to protect HSV-1 infected neurons from viral cytopathic effects (Simmons & Tscharke, 1992). Although the current study offers only limited insight into the real mechanism of IFN-induced inhibition of HSV-1 replication, the results provide an empirical basis to focus on the relevant possibilities. We conclude that any viable hypothesis must be constrained by the facts that co-activation of the IFN-α/β and IFN-γ signaling pathways 1. does not compromise host cell viability, 2. prevents detectable viral IE and E protein synthesis in two-thirds of HSV-1 infected cells, 3. blocks viral DNA synthesis in the remaining one-third of HSV-1 infected cells, and 4. effectively prevents the synthesis of new infectious virions.

Supplementary Material

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Acknowledgments

This work was supported by grants from the National Institute of Allergy and Infectious Disease (R01 AI51414) and the National Center for Research Resources (P20 RR-020185-01). Dr. William Halford is supported by a National Science Foundation EPSCoR grant to Montana State University (EPS 0346458) and the Montana Agricultural Experiment Station. The authors extend their thanks to Carla Weisend for excellent technical assistance and to Bryan Gebhardt and Joel Graff for critical evaluation of this manuscript.

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