1 Department of Microbiology and Immunology, Tulane University Medical School, New Orleans, LA 70112, USA
2 Division of Biotechnology and Molecular Medicine, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA
3 Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030, USA
4 Department of Veterinary Molecular Biology, Montana State University, 960 Technology Boulevard, Bozeman, MT 59718, USA
Correspondence
William P. Halford
halford{at}montana.edu
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ABSTRACT |
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INTRODUCTION |
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The available evidence indicates that an interaction between the IFN-/
- and IFN-
-signalling pathways is functionally relevant in host control of HSV-1 infections. Recent comparisons in knockout mice demonstrated that mice that lacked both IFN-
/
receptors and IFN-
receptors (IFN-
/
R/ and IFN-
R/) were profoundly impaired in their resistance to HSV-1 strain KOS infection, experienced systemic viral spread and died 46 days after footpad inoculation (Luker et al., 2003
; Vollstedt et al., 2004
). This phenotype is in stark contrast to single-receptor knockout mice (IFN-
/
R/ or IFN-
R/), 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
). Whilst these in vivo studies have established that loss of both IFN receptors has a catastrophic effect on host defence against HSV-1, they provide only limited insight into how the IFN-
/
and IFN-
pathways normally interact to control the spread of HSV-1 infection.
The nature of the block in HSV-1 replication produced by IFN-/
and IFN-
remains undefined. Does exposure to IFN-
/
and IFN-
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-
and IFN-
may create a block in HSV-1 replication that 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-/
- and IFN-
-signalling pathways prevents HSV-1 replication in vitro. Given the complexity of the HSV-1 replication cycle, we thought it unrealistic to test the scores of hypotheses that might explain the inhibitory effect sequentially. Rather, we felt that a more directed approach was first to constrain the number of possible explanations by determining whether IFN-
and IFN-
are (i) cytotoxic to HSV-1 infected cells or (ii) disrupt HSV-1 replication at one or more discernible steps relative to viral mutants or an inhibitor of viral DNA synthesis. By using this approach, the separate versus combined effects of IFN-
and IFN-
on viral DNA, mRNA, protein and virion accumulation were compared in Vero cells infected with 2·5 p.f.u. per cell of HSV-1. The use of an m.o.i. that infected nearly 100 % of cells in the population ensured that any differences in the measured parameters were the result of a defect in the first, and only, cycle of virus replication. We report that IFN-
and IFN-
do not inhibit HSV-1 replication via a non-specific cytotoxic effect. Rather, IFN-
and IFN-
render host cells non-permissive for viral DNA synthesis and nucleocapsid assembly, and thus suppress HSV-1 replication in the vast majority of infected cells.
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METHODS |
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Dot-blot analysis of HSV-1 DNA yield.
Vero cells were established at a density of 1x105 cells per well in 24-well plates and infected with KOS at an m.o.i. of 2·5. Cultures were incubated in the presence of vehicle, 200 U IFN- ml1, 200 U IFN-
ml1, 100 U each of IFN-
and IFN-
ml1 or 300 µM acyclovir. Between 9 and 24 h post-infection (p.i.), crude DNA lysates were harvested and probed for viral DNA and the results were enumerated as described previously (Halford et al., 2005a
).
Flow cytometry
Enumeration of morphologically normal cells.
Vero cells were established at a density of 2x105 cells per well in 12-well plates and infected with KOS at an m.o.i. of 2·5. Cultures were incubated in the presence of vehicle, 200 U IFN- ml1, 200 U IFN-
ml1, 100 U each of IFN-
and IFN-
ml1 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 with 10 % FBS and passing cells through a 50 µm mesh to remove cell debris. Cells were counted for a fixed period of time (2 min) on a FACSCalibur (BD Biosciences). At each time point, the total number of cells in four cultures was determined by using a haemocytometer and compared with 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).
Two-colour analysis of viral protein expression in HSV-1-infected cells.
Vero cells were established at a density of 4·5x106 cells per 100 mm dish and infected with KOS at an m.o.i. of 2·5. At 9 h p.i., Vero cells were dissociated with trypsin and prepared for immunofluorescent labelling by fixation in 2 % formaldehyde with 2 % sucrose, permeabilization in 90 % methanol, passage through a 27-gauge needle and resuspension in PBS plus 0·5 % FBS containing Fc--receptor blocking agents (i.e. 5 µg each of human IgG, donkey IgG and goat IgG ml1). Cells were incubated for 1 h with a 1 : 20 000 dilution of rabbit anti-HSV-1 (Dako Cytomation) and a 1 : 1000 dilution of mouse mAb against ICP0, ICP4, ICP6, gC or gD (Rumbaugh Goodwin Institute). Cells were washed twice and incubated for 30 min with a 1 : 1000 dilution of phycoerythrin (PE)-labelled donkey anti-rabbit IgG and a 1 : 350 dilution of fluorescein isothiocyanate (FITC)-labelled 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) by using a FACSCalibur and CellQuest Pro software (BD Biosciences). ICP8 was detected by the same methodology, but cells were labelled singly by incubating for 1 h with a 1 : 20 000 dilution of rabbit polyclonal anti-ICP8 antibody (generously donated by William Ruyechan, State University of New York, Buffalo, USA). For each viral protein, total protein yield was summed by multiplying the fraction of viral protein-positive cells by 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 4·5x106 cells per dish in 100 mm dishes and infected with KOS at an m.o.i. of 2·5. Cultures were incubated in the presence of vehicle, 200 U IFN- ml1, 200 U IFN-
ml1, 100 U each of IFN-
and IFN-
ml1 or 300 µM acyclovir. Cells were prepared for transmission electron microscopy at 18 h p.i. as described previously (Foster et al., 2003
, 2004
).
For analysis of virion yields, cells were transferred to 80 °C at 18 h p.i. and virion yields were determined in cell lysates following purification through a series of two gradients. For each sample, 15 fractions that spanned the 3060 % sucrose interface of the second gradient were identified by 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 % dried milk and a 1 : 600 dilution of anti-HSV-1 conjugated to horseradish peroxidase (Dako Cytomation) was added per well. After 1 h, excess antibody was rinsed away and bound antibody was measured with TMB Blue substrate (Dako Cytomation; A450) in a plate reader.
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RESULTS |
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IFN- and IFN-
synergistically reduce the efficiency of HSV-1 DNA synthesis
The combined versus separate effects of IFN- and IFN-
on HSV-1 DNA synthesis were compared in Vero cells inoculated with HSV-1 strain KOS (m.o.i. of 2·5). DNA samples harvested at 924 h p.i. were immobilized on a nylon membrane and hybridized to a probe specific for the HSV-1 US6 gene (Fig. 2
a). Treatment with 200 U IFN-
or IFN-
ml1 alone delayed the detection of viral DNA synthesis by approximately 3 h and slightly decreased the rate (slope) of viral DNA synthesis between 15 and 24 h p.i. relative to vehicle-treated controls (Fig. 2a and b
). Combined treatment with 100 U each of IFN-
and IFN-
ml1 delayed the detection of viral DNA synthesis by 6 h 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-
and IFN-
, which only reduced the efficiency of HSV-1 DNA synthesis, 300 µM acyclovir prevented the detection of HSV-1 DNA synthesis completely (Fig. 2a and b
).
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The full inhibitory effect of IFN- and IFN-
is achieved before or during viral DNA synthesis
Vero cells were infected with HSV-1 strain KOS (m.o.i. of 2·5) and treated with vehicle, IFN-, IFN-
, IFN-
and IFN-
, or 0·3320 µM acyclovir. Cultures were harvested at 18 h p.i. to compare viral DNA yields (Fig. 5
a and b) and viral titres (Fig. 5c
). Relative to vehicle-treated cells, a combination of IFN-
and IFN-
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 b
). The same combination of IFN-
and IFN-
reduced infectious viral titres by
1000-fold relative to vehicle-treated cells and 32 µM acyclovir was found to reduce viral titres to precisely the same extent (Fig. 5c
). Regression analysis of viral titres versus DNA yield confirmed that acyclovir, an acyclic guanosine analogue (Elion, 1983
), reduced HSV-1 titres in direct proportion to the drug's inhibitory effect on viral DNA synthesis (closed circles in Fig. 5d
). Likewise, it was observed that IFN-
alone, IFN-
alone or IFN-
and IFN-
each reduced HSV-1 titres in direct proportion to their inhibitory effects on HSV-1 DNA synthesis (open symbols in Fig. 5d
). Therefore, the results suggested that IFN-
and/or IFN-
must achieve their full inhibitory effect on HSV-1 replication before or during the process of viral DNA synthesis.
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DISCUSSION |
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Are the effects of IFN- and IFN-
on HSV-1 replication synergistic?
Pharmacological analysis of synergy focuses on differentiating whether an observed change in effect is due to (i) a cooperative interaction between two biologically active agents or (ii) the change in concentration that occurs whenever two agents are combined. Under a null hypothesis of dose additivity, IFN- and IFN-
would be assumed to have purely redundant effects, such that 100 U each of IFN-
and IFN-
ml1 should inhibit any step of HSV-1 replication to the average level of that achieved by 200 U IFN-
ml1 or 200 U IFN-
ml1 (Tallarida, 2001
; Tallarida et al., 1997
). The observed effects of IFN-
and IFN-
were compared with the predictions of the null hypothesis (Table 1
). In each case, IFN-
and IFN-
reduced the synthesis of viral proteins, viral DNA and virions to an extent far greater than that predicted by the null hypothesis. Thus, the block in HSV-1 replication that was produced by co-activation of the IFN-
/
and IFN-
pathways was quantitatively distinct from that which occurred when only one of these two signalling pathways was activated. The multiplicative nature of the interaction between the IFN-
/
and IFN-
pathways is considered in detail elsewhere (Halford et al., 2005a
).
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Inhibition of viral IE gene expression?
Cells infected with an ICP4 virus expressed five times as much ICP0 as KOS-infected cells treated with IFN- and IFN-
. However, ICP4 virus-infected cells expressed low levels of ICP6 and undetectable levels of gD. In contrast, KOS-infected cells treated with IFN-
and IFN-
exhibited a more gradual attenuation of E and L protein synthesis (i.e. ICP6, ICP8, gC and gD). Relative to the ICP4 virus, viral E and L proteins were far too abundant in IFN-treated, KOS-infected cells for repression of viral IE protein synthesis to be the sole mechanism by which IFN-
and IFN-
inhibit HSV-1 replication.
Inhibition of viral DNA synthesis?
IFN- and IFN-
did not block viral DNA synthesis in KOS-infected cells with the efficiency of 300 µM acyclovir. However, IFN-
and IFN-
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 in viral DNA synthesis. Relative to these controls, viral IE and E protein expression was far too restricted in IFN-treated, KOS-infected cells for repression of viral DNA synthesis to be the sole mechanism by which IFN-
and IFN-
inhibited HSV-1 replication.
IFN- and IFN-
do not inhibit HSV-1 at the same step in every infected cell.
Flow-cytometric analysis of viral protein expression revealed that IFN- and IFN-
must inhibit KOS replication at at least two distinct points. The first point of inhibition produced by IFN-
and IFN-
reduced the frequency of KOS-infected cells that expressed detectable levels of ICP0 by 70 % relative to vehicle-treated cells (Fig. 4
). Because IFN-
and IFN-
do not preclude viral entry (Sainz & Halford, 2002
), we inferred that IFN-
and IFN-
repressed viral ICP0 gene expression successfully in
70 % of KOS-infected cells. Absence of ICP0 renders viral IE genes highly susceptible to repression by IFN-
/
(Härle et al., 2002
; Mossman et al., 2000
). Thus, whether or not ICP0 is synthesized may create two divergent outcomes of HSV-1 infection. Under this hypothesis, one would predict that, if IFN-
and IFN-
repress the ICP0 gene in
70 % of KOS-infected cells, then all viral IE and E gene expression will be repressed in this subpopulation of cells (Mossman et al., 2000
). However, if IFN-
and IFN-
fail to repress the ICP0 gene in
30 % of KOS-infected cells, the resulting ICP0 protein that is synthesized will destabilize IFN-induced repression of viral IE genes (Härle et al., 2002
) and thus allow virus 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- and IFN-
lies at the level of viral DNA synthesis. The 50- to 100-fold reduction in viral DNA synthesis that was produced by IFN-
and IFN-
would not be predicted based on the fact that
25 % of KOS-infected cells (relative to vehicle-treated controls) expressed normal levels of the E proteins ICP6 and ICP8 at 9 h p.i. The underlying mechanism that accounts for the block in viral DNA synthesis induced by IFN-
and IFN-
remains to be elucidated and will certainly be the focus of future investigation. Given the hypersensitivity of HSV-1 ICP34·5 mutants to IFN-
/
(Cerveny et al., 2003
; Mossman & Smiley, 2002
), one intriguing possibility is that expression of the other major viral IFN antagonist, ICP34·5, is repressed synergistically by IFN-
and IFN-
such that HSV-1 is rendered highly susceptible to a protein kinase-induced shutoff of L viral protein translation (Leib et al., 2000
). The validity of this specific hypothesis remains to be tested.
Effect on downstream steps of virus replication.
IFN- and IFN-
did 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-
, IFN-
or IFN-
and IFN-
reduced infectious viral titres to precisely the same extent as each IFN treatment. Given that acyclovir acts solely to inhibit HSV-1 DNA synthesis, these data suggest strongly that the full inhibitory effect of IFN-
and/or IFN-
is achieved before or during the process of HSV-1 DNA synthesis.
Relevance of the experimental design
In nature, animals are first infected with a virus and then respond by secreting cytokines such as IFN-/
or IFN-
. Given the natural order of events, what natural process is being modelled when Vero cells are pre-treated with IFN-
and IFN-
for 16 h 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 virus 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 approximately ten subsequent cycles of virus replication proceed during a typical 7 day course of acute infection in vivo (Halford et al., 2005b
; Luker et al., 2003
; Vollstedt et al., 2004
). Thus, the natural process being modelled when Vero cells are pre-treated with IFN-
and IFN-
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-/
and IFN-
signalling pathways produces a multiplicative inhibition of HSV-1 replication (Halford et al., 2005a
). 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-
-expressing CD8+ T cells at sites of latent HSV-1 infection (Khanna et al., 2003
; Theil et al., 2003
), these results suggest that IFN-
secretion may contribute to the capacity of CD8+ T cells to protect HSV-1-infected neurons from viral CPE (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-
/
and IFN-
signalling pathways (i) does not compromise host-cell viability, (ii) prevents detectable viral IE and E protein synthesis in two-thirds of HSV-1-infected cells, (iii) blocks viral DNA synthesis in the remaining one-third of HSV-1-infected cells and (iv) effectively prevents the synthesis of new infectious virions.
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ACKNOWLEDGEMENTS |
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Received 18 February 2005;
accepted 10 June 2005.