ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency
- William P Halford1Email author,
- Carla Weisend1,
- Jennifer Grace1,
- Mark Soboleski2,
- Daniel JJ Carr3,
- John W Balliet4,
- Yumi Imai5,
- Todd P Margolis5 and
- Bryan M Gebhardt6
© Halford et al. 2006
Received: 26 April 2006
Accepted: 09 June 2006
Published: 09 June 2006
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.
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-α/β 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-α/β receptor and the downstream transcription factor, Stat 1.
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.
green fluorescent protein
herpes simplex virus
infected cell protein
long-short spanning transcript
protein kinase R
recombination-activated gene 2
- RL :
- Stat 1:
signal transducer and activator of transcription 1
Herpesviruses are double-stranded DNA viruses that establish life-long infections in their animal hosts, and which alternate between two programs of gene expression: i. productive replication, or ii. latent infection in which most of the viral genome is transcriptionally silent.
The long-repeated, RL, regions of the HSV-1 genome appear to regulate HSV-1's capacity to alternate between two programs of gene expression: productive replication or non-productive infection (Fig. 1A). Each copy of the RL region encodes the latency-associated transcript (LAT), the long-short spanning transcript (L/ST), infected cell protein 0 (ICP0), and infected cell protein 34.5 (ICP34.5) (Fig. 1A). Both the LAT and L/ST genes produce RNA transcripts that encode no known protein [2, 3]. A viral protein, ICP4, blocks the transcription of the LAT and L/ST genes during productive replication by binding the 5' end of each gene [3, 4]. The ICP0 and ICP34.5 genes, which lie on the opposite strand of DNA, promote HSV-1 replication. ICP0 is an E3 ubiquitin ligase that overcomes cellular repression of HSV-1 [5, 6]. ICP34.5 antagonizes protein kinase R (PKR)-induced shutoff of viral protein translation by inducing the dephosphorylation of the translation initiation factor, eIF-2α [7, 8].
The current model of HSV-1 gene regulation ascribes no significance to the genes in the HSV-1 latency-associated locus, and fails to explain why both RL-encoded proteins function as viral interferon (IFN) antagonists [9–11]. When IFNs bind their cognate receptors at the cell surface, the signal transducer and activator of transcription 1 (Stat 1) protein is phosphorylated and acts in concert with other transcription factors to induce IFN-stimulated gene expression, thus creating an antiviral state in the host cell [12, 13]. Wild-type HSV-1 is remarkably resistant to the antiviral state induced by activation of either IFN-α/β receptors or IFN-γ receptors . In contrast, HSV-1 ICP0- or ICP34.5- mutants are hypersensitive to the antiviral state induced by activation of IFN-α/β receptors in vitro [14–16]. Reciprocally, ICP0- and ICP34.5- viruses exhibit improved replication in IFN-α/β receptor-knockout mice [11, 17].
The opposing forces produced by IFN-inducible cellular repressors and the RL-encoded viral IFN antagonists, ICP0 and ICP34.5, may form two checkpoints that regulate whether or not HSV-1 completes its replication cycle in an infected cell in vivo. This hypothesis can be integrated into the current model of HSV-1 gene regulation via two modifications (Fig. 1C):
ICP0 and IFN-inducible cellular repressor(s) form an ON-OFF switch that controls whether or not viral IE mRNA synthesis occurs in an infected cell (Checkpoint 1).
ICP34.5 and an IFN-inducible cellular repressor, PKR, form an ON-OFF switch that controls whether or not viral L protein synthesis occurs in an infected cell (Checkpoint 2).
The OFF event at Checkpoint 1 is predicted to occur when ICP0 is not synthesized, and the host IFN response stably represses viral IE mRNA synthesis [14, 15]. The OFF event at Checkpoint 2 is predicted to occur when ICP34.5 is not synthesized, and the host IFN response acts through PKR to induce the shutoff of viral L protein synthesis [11, 18].
The proposed Checkpoint Model represents an attempt to explain how the genes in the HSV-1 latency-associated locus may influence the decision-making process that dictates whether the HSV-1 genome is expressed (productive replication) or repressed (quiescent infection) when HSV-1 enters a cell in vivo. The evidence that supports the model is circumstantial, and thus the accuracy of the model is questionable. For example, the Checkpoint Model assumes that the primary function of ICP0 lies in antagonizing IFN-inducible repression of HSV-1 in vivo (Fig. 1C). Several observations are consistent with, but do not prove, this hypothesis [14, 15, 17]. The current study was initiated to test two key predictions of the Checkpoint Model: i. ICP0- mutants should be susceptible to repression by the innate immune response in vivo, and ii. ICP0- mutants should replicate efficiently and be fully virulent in hosts that are IFN-unresponsive.
Given the extensive literature on HSV-1, no one manuscript can satisfactorily prove the accuracy of a new in vivo paradigm of HSV-1 gene regulation. On the other hand, the need for an improved in vivo model is clear. A model is needed which identifies the host and/or viral factors that can influence whether HSV-1 infection of a cell leads to productive replication or non-productive infection in vivo. The goals of the current study are to introduce the possibility that the cessation of HSV-1 replication in vivo may be regulated by an equilibrium between the host IFN response and viral IFN antagonists. The in vivo behavior of HSV-1 ICP0- mutants is described, which is inexplicable in terms of the current model of HSV-1 gene regulation, but which logically follows from the proposed Checkpoint Model (Fig. 1C). The evidence that supports this newly proposed model is discussed.
Failure to express ICP0 allows HSV-1 to be stably repressed in scid mice
Viruses and mice used in this study.
Genotype of virus
Phenotype of virus
CMV-GFP cassette between UL26 and UL27 genes
IFN-sensitive (Fig. 5B)
Immunological status of mouse
rag2 -/- g
IFN-γ receptor-null 
IFN-α/β receptor-null 
ifnar -/- ifngr -/-
IFN-α/β receptor-null + IFN-γ receptor-null 
Stat 1-null 
rag2 -/- stat1 -/-
lymphocyte-deficient + Stat 1-null
rag2 -/- ifnar -/-
lymphocyte-deficient + IFN-α/β receptor-null
Multiple experiments confirmed that the ICP0- virus n212 was avirulent in scid mice, whereas the wild-type KOS strain produced uniformly lethal infections in scid mice (Fig. 2B). Moreover, n212 appeared to rapidly exit the productive cycle of viral replication in scid mice based on i. low to undetectable levels of infectious virus in the tear film of scid mice between 3 and 70 days p.i., ii. undetectable levels of infectious virus in homogenates of eyes or trigeminal ganglia (TG) at 35 or 70 days p.i. (n = 10 tissues per time point), and iii. the fact that n212-infected scid mice remained indistinguishable from uninfected mice for more than 2 months p.i.
The in vivo repression of ICP0- viruses is Stat 1-dependent
In multiple experiments, the ICP0- viruses n212 and 0--GFP did not produce disease in strain 129 mice, rag2 -/- mice, and PML -/- mice, and 100% of the mice survived for 60 days p.i. (Fig. 3C). In contrast, n212 and 0--GFP produced lethal infections in 50 to 100% of stat1 -/- mice and in 100% of rag2 -/- stat1 -/- mice (Fig. 3C). Thus, the IFN-activated Stat 1 transcription factor was required for rag2 -/- mice to rapidly repress the replication of ICP0- viruses in vivo.
Rag2 -/- stat 1 -/- mice die of uncontrolled viral spread, not human handling
In vivo, 0--GFP replicated to high titers in the eyes of rag2 -/- stat1 -/- mice, acute swelling of periocular tissue occurred, and none of the mice survived beyond day 11 p.i. (Fig. 5C). In contrast, the ICP4- virus failed to replicate in rag2 -/- stat1 -/- mice or rag2 -/- mice, and all of the ICP4- virus-infected mice remained healthy for the 30-day test period (Fig. 5C and 5D). In rag2 -/- mice, which retained a functional Stat 1 pathway, the ocular replication of 0--GFP was rapidly repressed and 100% of 0--GFP-infected rag2 -/- mice remained healthy for the 30-day observation period (Fig. 5D). Thus, the pathogenesis of 0--GFP infection observed in rag2 -/- stat1 -/- mice appeared to be the result of unchecked viral replication, and was not the result of an unanticipated infection with the flora of the mice or their human handlers.
Stat 1 is necessary to restrict wild-type HSV-1 spread in vivo
IFN receptors are integral to the innate response that limits HSV-1 spread in vivo
Effect of interferon receptors versus Stat 1 on HSV-1 shedding and the survival of infected mice.
Viral titers per eye on day 3 p.i.c
1.8 ± 0.3
1.5 ± 0.4
18 ± 0.5
1.8 ± 0.5
2.8 ± 0.3
11 ± 1‡
ifnar -/- ifngr -/-
3.6 ± 0.1*
5.2 ± 0.1‡
4.3 ± 0.1*
8.1 ± 0.3‡
rag2 -/- stat1 -/-
4.0 ± 0.2*
8.6 ± 0.2‡
rag2 -/- ifnar -/-
4.2 ± 0.2*
7.9 ± 0.2‡
1.1 ± 0.6
0.7 ± 0.5
1.4 ± 0.4
2.2 ± 0.1
ifnar -/- ifngr -/-
3.4 ± 0.3*
11 ± 1‡
4.4 ± 0.1*
11 ± 1‡
rag2 -/- stat1 -/-
3.4 ± 0.2*
11 ± 1‡
rag2 -/- ifnar -/-
3.8 ± 0.2*
10 ± 1‡
Following inoculation with 2 × 105 pfu per eye of 0--GFP, similar levels of GFP reporter gene expression from the ICP0 gene (diagram in Fig. 4) were observed in the corneas of mice at 36 hours p.i. (Fig. 7B). By 60 and 84 hours p.i., GFP fluorescence decreased to nearly undetectable levels in the corneas of wild-type, rag2 -/-, and ifngr -/- mice (Fig. 7B). All mice with a defect in the ifnar or stat1 genes failed to limit 0--GFP spread, and GFP fluorescence was still evident throughout the cornea at 84 hours p.i. (Fig. 7B). Likewise, 0--GFP titers were an average 300 to 1000 times higher in the tear film of ifnar -/- ifngr -/-, stat1 -/-, rag2 -/- stat1 -/-, and rag2 -/- ifnar -/- mice relative to wild-type mice at 72 hours p.i. (Table 2). Most of the mice that shed titers of >1000 pfu per eye of 0--GFP on day 3 p.i. died of the infection. Rag2 -/- mice survived 0--GFP-infection for 60 days and exhibited no symptoms of disease. In contrast, rag2 -/- stat1 -/- mice and rag2 -/- ifnar -/- mice uniformly succumbed to 0--GFP infection (Table 2). Thus, the IFN-α/β receptor and downstream Stat 1 transcription factor are essential for the innate host response that represses 0--GFP replication in rag2 -/- mice.
Stat 1 is not essential for the early synthesis of HSV-1 latency-associated transcripts
Abundance of LAT+ versus HSV antigen+ neurons in trigeminal ganglia infected with HSV-1 strain KOS.
LAT+ neurons b
HSV Ag+ neurons c
HSV Ag+ neurons
0.02 – 0.04% (12) d
7 – 15% (4399)
0.1 – 0.2% (45)
9 – 18% (5300)
0.02 – 0.04% (12)
10 – 20% (6122)
0.1 – 0.3% (82)
18 – 36% (10897)
0.03 – 0.06% (18)
10 – 20% (5978)
0.1 – 0.2% (48)
> 20% (TNTC) e
rag2 -/- stat1 -/-
0.02 – 0.04% (11)
8 – 17% (5005)
0.1 – 0.2% (59)
> 20% (TNTC)
During the acute infection, HSV Ag+ neurons were >100-fold more abundant in TG than LAT+ neurons (Table 3). On day 3 p.i., the frequency of HSV Ag+ neurons was equivalent in all groups of TG, and ~100 to 300 HSV Ag+ neurons were observed for every 1000 TG neurons analyzed. On day 5.5 p.i., HSV Ag+ neurons were twice as abundant in the TG of rag2 -/- mice relative to strain 129 mice (Table 3). On day 5.5 p.i., HSV Ag+ neurons were too numerous to count in the TG of stat1 -/- and rag2 -/- stat1 -/- mice and viral CPE severely compromised the integrity of the tissue. Thus, consistent with other results, the Stat 1 signaling pathway was essential to restrict the spread of wild-type HSV-1 from the site of inoculation to the TG.
An ICP0- virus establishes latent infections in the trigeminal ganglia of mice
Efficiency with which wild-type HSV-1 and ICP0- viruses reactivate from latently infected trigeminal ganglia.
Viral spread to L7 cellsd
Virus in TG on Day 14e
Viral genomes per TGf
2 × 105
100% (n = 15)
3.0 × 105
2 × 105
100% (n = 15)
1.3 × 105 *
2 × 105
100% (n = 25)
1.2 × 105 *
2 × 105
100% (n = 21)
3.5 × 105
rag2 -/- ifnar -/-
2 × 104
0% (n = 11)
ifnar -/- ifngr -/-
2 × 104
21% (n = 14)
2 × 104
50% (n = 50)
1.8 × 105
HSV-1 latently infected mice were sacrificed at day 40 p.i. to measure viral genome load per TG. KOS-latently infected strain 129 mice contained an average of 3.0 × 105 viral genomes per TG (Fig. 8C; Table 4). In strain 129 mice and ifngr -/- mice, the number of latent 0--GFP genomes per TG was ~40% of the wild-type level achieved by KOS. In ifnar -/- mice, the number of latent 0--GFP genomes per TG was ~115% of the wild-type level. In stat1 -/- mice (inoculated with a ten-fold lower dose of 0--GFP), the number of latent 0--GFP genomes per TG was ~60% of the wild-type level (Fig. 8C; Table 4). If one considers the primary data, 0--GFP established latent infections in 50% of ifnar -/- mice and stat1 -/- mice that met or exceeded the latent viral genome load per TG achieved by KOS in strain 129 mice (Fig. 8A).
An ICP0- virus reactivates inefficiently from latently infected trigeminal ganglia
The efficiency with which latent KOS (ICP0+) and 0--GFP (ICP0-) reactivated from TG explants was compared. TG were harvested from mice on days 38 and 39 p.i., heat stressed at 43°C for 3 hours, co-cultured with L7 cell monolayers for 14 days, and were homogenized on day 14 to test for the presence of infectious virus within each TG. Of the KOS-latently infected TG harvested from strain 129 mice, 11 of 16 TG explants produced infectious virus that spread to co-cultured L7 cells (Table 4), and 14 of 16 TG homogenates contained infectious KOS on day 14 post explant (Table 4). Of the 0--GFP-latently infected TG harvested from strain 129 mice, ifngr -/- mice, ifnar -/- mice, ifnar -/- ifngr -/- mice, or stat1 -/- mice, 0 of 98 TG explants produced infectious virus that spread to co-cultured L7 cells (Table 4). In contrast, infectious 0--GFP was detected in 2 of 16 ifnar -/- TG homogenates and 2 of 22 stat1 -/- TG homogenates on day 14 post explant (Table 4). Reactivation of 0--GFP was detected in none of the other TG homogenates on day 14 post explant (Table 4). Thus, consistent with previous results, even when wild-type HSV-1 and ICP0- viruses establish equivalent latent infections (e.g., ifnar -/- mice; Table 4), ICP0- viruses are severely impaired in their capacity to reactivate from latently infected TG explants [23, 24].
Prior infection with an avirulent ICP0- virus induces protective immunity against HSV-1
Equal numbers of rag2 -/- mice were included in this experiment. Inoculation of rag2 -/- mice with 0--GFP or the ICP4- virus produced an equivalent pattern of viral shedding as was observed in strain 129 mice between days 1 and 30 p.i. (Fig. 9C and 9D). On day 30 p.i., rag2 -/- mice were secondarily challenged with McKrae. McKrae replicated to high titers in rag2 -/- mice that were first inoculated with 0--GFP or the ICP4- virus (Fig. 9C and 9D). In ICP4- virus-infected rag2 -/- mice, McKrae infection progressed to fatal encephalitis by 5.7 ± 0.1 days post challenge (Fig. 9D). In 0--GFP-infected rag2 -/- mice, McKrae infection required 9.0 ± 0.5 days to progress to fatal disease (Fig. 9C), suggesting that the ongoing innate response to 0--GFP infection delayed the progression of McKrae infection to a fatal encephalitis. Therefore, while 0--GFP infection is avirulent in rag2 -/- mice, 0--GFP cannot induce protective immunity against HSV-1 in animals that lack a lymphocyte-driven adaptive immune response.
IFN receptors and Stat 1 are integral to innate host control of HSV-1 infection
Loss of IFN-α/β receptors in ifnar -/- mice compromised innate resistance to HSV-1, but the defect was not as profound as observed in stat1 -/- mice. Thus, on day 3 p.i., ifnar -/- mice shed 30- to 100-times less infectious virus per eye than stat1 -/- mice (Table 2). Likewise, ifnar -/- mice uniformly survived ICP0- viral infections with little disease (Table 2), whereas 50% of stat1 -/- mice died of 0--GFP infection and all of the survivors were frankly diseased. The capacity of rag2 -/- mice to repress 0--GFP replication was clearly dependent on the IFN-α/β receptor and Stat 1, because 0--GFP produced uniformly fatal infections in rag2 -/- ifnar -/- mice and rag2 -/- stat1 -/- mice. Mice that lacked IFN-α/β receptors and IFN-γ receptors failed to suppress the replication of wild-type or ICP0- viruses at the site of inoculation. Because the extent of the defect was similar to that observed in stat1 -/- mice, the combined activities of IFN-α/β and IFN-γ appear to activate the Stat 1-dependent host response that limits HSV-1 spread in vivo.
The innate resistance of host structural cells to HSV-1 appears to be compromised in the absence of Stat 1 or IFN-α/β receptors, presumably because these cells fail to mount an antiviral state (Fig. 7). However, this simple explanation does not account for all of the observations presented herein. For example, HSV-1 produced a rapid and fatal disease in ifnar -/- ifngr -/- mice, which as described by Luker, et al. , appeared to be a viral hepatitis. HSV-1 infection rarely causes hepatitis in humans , and liver infection is not a prominent feature in animal models. Moreover, stat1 -/- mice infected with wild-type HSV-1 died of encephalitis two to three days later. This divergence in the outcomes of infection in ifnar -/- ifngr -/- mice and stat1 -/- mice draws attention to an important caveat of studies in knockout mice: despite the targeted nature of the genetic mutations, loss of a single protein may have pleiotropic effects. For example, basic immune functions such as hematopoiesis or leukocyte activation may be altered by combinations of these mutations. Alternatively, the non-permissiveness of leukocytes for HSV-1 replication may be dependent on the combined activities of IFN-α/β receptors and IFN-γ receptors [26, 27]. Thus, the fulminant liver infection that develops in HSV-1-infected ifnar -/- ifngr -/- mice may be due to uncontrolled HSV-1 replication in at least one leukocyte subset, which would confer upon HSV-1 the unusual property of blood-borne spread to the liver. Given such possibilities, the results of the current study must be interpreted with a certain degree of caution.
Stat 1 is not essential for the early synthesis of HSV-1 latency-associated transcripts
A subset of neurons express LAT RNAs, but not viral proteins, when HSV-1 first enters the TG on day 3 after ocular inoculation . Comparison of the numbers of LAT+ neurons in stat1 +/+ versus stat1 -/- mice indicated that this phenomenon is not Stat 1-dependent (Table 3). Thus, the Checkpoint Model fails to explain how HSV-1 establishes latent infections in the subpopulation of stat1 -/- neurons that are LAT+ on days 3 and 5.5 p.i. (Table 3). Although LAT+ neurons were certainly present in stat1 -/- mice, HSV Ag+ neurons were 100- to 500-fold more abundant between days 3 and 5.5 p.i. (Table 3). On day 5.5 p.i., stat1 -/- and rag2 -/- stat1 -/- mice were diseased, their TG were beginning to hemorrhage, and the animals were within 24 hours of death. Clinically, herpesviral latency refers to the absence of infectious virus and disease in a host, and requires the repression of viral replication in the entire population of virus-infected cells in the body. Thus, although a small subset of neurons represses HSV-1 replication in a Stat 1-independent manner, we conclude that both Stat 1 and lymphocytes are required for a latent HSV-1 infection to be established at the clinically relevant level of the host organism.
ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus
ICP0- viruses are rapidly repressed in scid or rag2 -/- mice. The failure of ICP0- viruses to sustain replication in lymphocyte-deficient mice is not due to a leaky adaptive immune response, nor is this a non-specific phenotype of any HSV-1 mutant. For example, VP16- and thymidine kinase- mutants of HSV-1 establish slowly progressing infections that are fatal in scid mice [28, 29]. Rather, it appears that an innate host response that is dependent upon the IFN-α/β receptor and Stat 1 potently represses the replication of ICP0- viruses in scid or rag2 -/- mice.
It is generally believed that ICP0 functions as an activator in the balance between HSV-1 latency and reactivation, and that ICP0 acts to prevent host cells from repressing HSV-1 replication in vitro [5, 6, 30]. However, a credible explanation has been lacking to explain how synthesis of ICP0, or lack thereof, can globally effect whether or not HSV-1 initiates productive viral replication. The results of the current study suggest a potentially simple explanation: when ICP0 is synthesized, the protein overcomes Stat 1-dependent cellular repression of HSV-1 such that the virus can complete its replication cycle; when ICP0 is not synthesized, Stat 1-dependent cellular repression silences the HSV-1 genome.
Given ICP0's capacity to function as a viral IFN antagonist in vitro [9, 10, 14, 15], we infer that ICP0 most likely functions in virus-infected cells to prevent an IFN-inducible block to viral replication (Checkpoint 1, Fig. 1C). Although PML has been proposed to serve a critical role in repressing ICP0- viruses , our findings in PML -/- mice do not support a direct role for PML in the process by which host cells repress ICP0- viruses in vivo. Therefore, it remains to be determined how ICP0 antagonizes Stat 1-dependent repression of HSV-1.
Implications for the regulation of viral latency
The available evidence suggests that the 9.3 kb RL region, which contains the ICP0 gene, plays a pivotal role in regulating HSV-1 latency (Fig. 4). The evidence that supports this point is summarized: 1. Synthesis of ICP0 promotes HSV-1 reactivation in latently infected TG neurons [24, 31]; 2. The antisense LAT gene is the only viral gene transcribed during latency and produces stable 1.5 and 2.0 kb introns that accumulate in neurons [32, 33]; 3. Synthesis of ICP34.5 is necessary for HSV-1 to complete its replication cycle in neurons [34–36]; and 4. De-repression of the antisense L/ST gene produces an HSV-1 mutant that overexpresses L/ST RNA and is replication-impaired in neurons despite an intact ICP34.5 open-reading frame .
Failure to synthesize the RL-encoded viral IFN antagonists, ICP0 or ICP34.5, is a possible mechanism by which HSV-1 may cease productive replication in vivo (Fig. 1C). Failure to synthesize ICP0 renders HSV-1 vulnerable to repression by the host IFN response. Likewise, failure to synthesize ICP34.5 renders HSV-1 vulnerable to innate immune repression in scid mice , and this repression is dependent on IFN-α/β receptors and PKR . If synthesis of ICP0 and ICP34.5 is necessary to promote HSV-1 replication in vivo, then it is possible that the LAT and L/ST genes provide HSV-1 with an efficient mechanism to pro-actively halt the synthesis of ICP0 or ICP34.5, such that HSV-1 can rapidly cease productive replication in vivo.
This hypothesis may seem counterintuitive. Why would HSV-1 carry two genes that promote the IFN-induced repression of the viral genome? Therefore, it is worth noting that herpesviruses are presented with two choices at the height of an immune response: 1. cytolytic destruction of the host cell by CD8+ T cells, or 2. cessation of viral antigen synthesis before CD8+ T cells can destroy the host cell. Given the arrangement of genes in the RL region (Fig. 4), it is possible that the LAT and/or L/ST genes may serve to ensure the timely shutoff of HSV-1 replication and antigen synthesis when the local immune response exceeds a critical threshold in an HSV-1 infected tissue. This hypothesis may be relevant in explaining: i. how viral antigen synthesis decreases from high to undetectable levels within 8 hours after T cells infiltrate HSV-1 infected ganglia ; ii. how HSV-1 infected neurons avoid cytolytic destruction by T cells [39, 40]; and iii. why neurons infected with LAT- viruses undergo apoptosis (cytolysis?) more frequently than neurons infected with LAT+ viruses [41–43]. Given the dearth of evidence that the LAT or L/ST genes actually antagonize the synthesis of ICP0 and ICP34.5, further work will be required to address these hypotheses.
HSV-1 becomes hypersensitive to IFN-inducible repression in the absence of ICP0 or ICP34.5 . Given the extensive similarity between HSV-1 and HSV-2, the results suggest a logical approach to develop a live, attenuated vaccine against genital herpes. When introduced at the peripheral epithelium, ICP0- viruses are incapable of producing disease in lymphocyte-deficient scid or rag2 -/- mice. Despite their avirulence, ICP0- viruses establish latent infections in the ganglia that innervate the site of inoculation (Fig. 8) and elicit an adaptive immune response that protects immunocompetent mice against virulent HSV-1 (Fig. 9). In contrast, a replication-defective ICP4- virus fails to elicit any semblance of protective immunity.
The advantage of a live, attenuated HSV vaccine would be two-fold: 1. the vaccine strain would be antigenically identical to wild-type HSV with the exception of a single mutated open-reading frame (e.g., ICP0), and 2. the latent infection established by such a virus might boost the adaptive immune response over time through periodic synthesis of viral antigen at the single cell level [44, 45]. Failure to sustain a broad-based immune response over time likely accounts for the failure of subunit vaccines or replication-defective viruses to protect against genital herpes [46, 47]. While a live HSV vaccine is often considered unsafe because the vaccine strain would establish a latent infection in the recipient, such claims ignore the natural history of HSV infections [48, 49]. About 4 billion people currently harbor latent HSV-1 and/or HSV-2 in their nervous system, and ~3 billion of these infections are asymptomatic. Further investigation will be required to determine if ICP0- strains of HSV-2 can be used to achieve an appropriate balance between safety and antigenicity in developing a live, attenuated vaccine against genital herpes.
In recent years, it has become increasingly evident that the host immune response plays a more direct role in the maintenance of HSV-1 latency than previously envisioned [50, 51]. The results of the current study add to this body of evidence, and suggest that the antagonistic actions of host IFNs and the viral IFN antagonist ICP0 may help HSV-1 "choose" between one of its two programs of gene expression, productive replication or latent infection. The proposed Checkpoint Model (Fig. 1C) represents an initial attempt to explain these observations, and will require extensive testing to determine its validity.
Despite its limitations, the Checkpoint Model offers a useful conceptual framework from which to begin considering whether these observations are really unique to HSV-1. Most persistent viruses possess the capacity to alternate between rapid, slow, and/or non-existent modes of productive replication. Therefore, it is worth considering that perhaps other persistent viruses also encode "activators" such as ICP0 whose synthesis (or lack thereof) regulates the rate of productive replication in vivo by virtue of antagonizing a host repressor that has been present since the dawn of life: the innate host response to viral infections.
Cells and viruses
Vero cells and L7 cells  were propagated in Dulbecco's Modified Eagle's medium (DMEM) containing 0.15% HCO3 - supplemented with 5% fetal bovine serum (FBS), penicillin G (100 U/ml), and streptomycin (100 mg/ml), hereafter referred to as "complete DMEM." Wild-type HSV-1 strains KOS, KOS-GFP, and McKrae were propagated in Vero cells cultured in complete DMEM. The ICP0- virus n212 [53, 54] contains a 14 bp insertion (CTAGACTAGTCTAG) in codon 212 of the ICP0 gene of HSV-1 strain KOS, which inserts stop codons into all three open-reading frames of the ICP0-encoding DNA strand (Fig. 4). The n212 (ICP0-) virus was propagated and titered in ICP0-complementing L7 cells . The ICP4- virus n12 contains a 16 bp insertion (GGCTAGTTAACTAGCC) in codon 262 of the ICP4 gene of HSV-1 strain KOS, which inserts stop codons into all three open-reading frames of the ICP4-encoding DNA strand. The n12 (ICP4-) virus was propagated and titered in E5 cells  (generously provided by Priscilla Schaffer, Harvard University).
KOS-GFP is a recombinant virus derived from HSV-1 strain KOS that expresses green fluorescent protein (GFP) from a CMV promoter-GFP gene cassette inserted in the intergenic region at the 3' ends of the UL26 and UL27 genes, which converge from opposite strands of DNA . To avoid disrupting the 3' untranslated region of either the UL26 or UL27 gene, the intergenic region (Kpn I – Fsp I; 52,733 – 53,150) was duplicated and the CMV-GFP cassette is inserted between these duplicated sequences. No known open-reading frames are disrupted by this 2.0 kbp insertion into the HSV-1 genome.
The ICP0- virus 0--GFP was constructed from a chimeric ICP0-GFP gene , in which the GFP open-reading frame was inserted into a Xho I restriction site in codon 105 of the ICP0 gene (Fig. 4). This mutation was transferred into HSV-1 strain KOS by homologous recombination. Southern blot analysis demonstrated that 0--GFP possessed the desired mutation in both copies of the RL region (Fig. 4). Northern blot analysis confirmed that 0--GFP expresses an ICP0 mRNA that migrates at ~3.1 kb as opposed to the wild-type 2.4 kb ICP0 mRNA transcribed from KOS. 0--GFP is phenotypically identical to the well-established ICP0- null virus n212 [53, 54], based on the following observations: 1. Both n212 and 0--GFP are repressed with identical kinetics in rag2 -/- mice; 2. Both n212 and 0--GFP are virulent in stat1 -/- mice; 3. Both n212 and 0--GFP are hypersensitive to IFN-α/β in vitro ; 4. Only 1% of n212 and 0--GFP virions form plaques on Vero cells [54, 58]; 5. ICP0, but not ICP4 or VP16, provided in trans from adenovirus vectors allow n212 and 0--GFP to efficiently form plaques on Vero cells , and the efficiency of plaque formation is the same as observed on ICP0-complementing L7 cells [14, 31, 52].
HSV-1 infection of mice
Female mice of the strains specified in Table 1 were inoculated with HSV-1 at 6- to 10-weeks of age and were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals. BALB/c mice, BALB/c scid mice, and IFN-γ receptor null (ifngr -/-) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Strain 129 mice, rag2 -/- mice, stat1 -/- mice, and rag2 -/- stat1 -/- mice were obtained from Taconic Farms (Germantown, NY). PML-/- mice were obtained from the National Cancer Institute (Frederick, MD). IFN-α/β receptor-knockout (ifnar -/-) mice and ifnar -/- ifngr -/- mice were obtained from B & K Universal Ltd. (East Yorkshire, United Kingdom). Rag2 -/- ifnar -/- mice were generously provided by Nicole Meissner and Allen Harmsen (Montana State University, Bozeman). Prior to viral inoculation, mice were anesthetized by i.p. administration of xylazine (7 mg/kg) and ketamine (100 mg/kg). Mice were inoculated by scarifying the cornea with a 26-gauge needle and by placing 4 μl complete DMEM containing 2 × 105 pfu of virus on each eye.
Viral titers in the ocular tear film of mice were determined at times after inoculation by swabbing the ocular surface of both eyes with a cotton-tipped applicator, and transferring the tip into 0.4 ml complete DMEM. Viral titers were determined by a 96-well plate plaque assay on the appropriate cell line cultured in complete DMEM containing 0.5% methlycellulose (i.e., Vero cells for wild-type HSV-1, L7 cells for ICP0- mutants, and E5 cells for ICP4- mutants).
Titers of infectious virus in homogenates of whole eyes, trigeminal ganglia (TG), or hindbrain were determined by homogenizing tissues in 0.5 ml complete DMEM with a Pro 200 homogenizer (Pro Scientific, Oxford, CT), removing cell debris via centrifugation, and titering 10-fold serial dilutions of clarified supernatant on 24-well plates containing Vero or L7 cells. For all plaque assays, virus-infected cells were cultured in complete DMEM containing 0.5% methlycellulose for two to three days before staining with 20% methanol and 0.5% crystal violet.
GFP fluorescence in eyes, TG, and brains of mice infected with KOS-GFP was visualized on a Nikon TE2000 inverted fluorescent microscope (Nikon Instruments, Lewisville, TX) using a Jenoptik ProgRes C10+ Digital Camera (JenOptik Laser, Jena, Germany). Images were collected at 2× or 4× magnification using identical exposure conditions within a given comparison group, and composite images of the brain were created by stitching together photographs that covered the ventral surface of the brain using the graphics editor, Paint Shop Pro (Jasc Software, Eden Prairie, MN). GFP fluorescence in the eyes of living mice was obtained by placing anaesthetized mice on a petri dish on the stage of the microscope.
Analysis of HSV-1 replication in vitro
Cultures of Vero cells were established in 12-well plates at a density of 2 × 105 cells per well and were cultured in complete DMEM. Cells were treated 24 hours later by replacing the culture medium with complete DMEM containing no IFN or 200 U/ml IFN-β (PBL Biomedical Laboratories, Piscataway, NJ). Sixteen hours later, Vero cells were inoculated with 2.5 pfu per cell of wild-type HSV-1 strain KOS, 0--GFP, or the ICP4- virus n12. After allowing 45 minutes for adsorption, the inoculum was replaced with complete DMEM containing no IFN or 200 U/ml IFN-β. Cultures were harvested 4.5, 9.0, 13.5, or 18.0 hours after inoculation by transfer to a -80°C freezer. Upon thawing, viral titers were determined by plaque assay on appropriate indicator cells.
Dual labeling of trigeminal ganglion sections for LAT RNA and viral proteins
Mice were euthanized and perfused with 0.1 M phosphate-buffered saline (PBS) followed by 4% paraformaldehyde. The six TG from each group of mice were immersed in 4% paraformaldehyde for 1 hour, equilibrated with 30% sucrose, embedded in Tissue-Tek® O.C.T. compound (Sakura Finetechnical, Tokyo, Japan), and frozen in liquid nitrogen. Each block of tissue containing the six TG from one group of mice was cut into ~160 sections of 7 μM thickness. Slides were processed to label 1. LAT+ neurons with rhodamine, and 2. HSV antigen+ neurons with fluorescenin, as follows. LAT riboprobes, specific for bases 119629–119975 of HSV-1, were prepared at 37°C with digoxigenin RNA Labeling Mix (Roche, Mannheim, Germany). LAT riboprobe synthesis reactions were treated with DNase I and filtered through a Sephadex G-50 column prior to use. Tissue sections were incubated in a pre-hybridization buffer, and then hybridized to LAT-specific riboprobes by an overnight 45°C incubation. Tissue was washed with 2× SSC, treated with 20 μg/ml of RNase A in 2× SSC at 37°C, followed by serial washes in 0.5× SSC and 0.1× SSC. Tissue sections were equilibrated with 0.1 M PBS, and were sequentially incubated with 1. rhodamine-labeled anti-digoxigenin Fab fragments, 2. 3% normal rabbit serum, and 3. fluorescein-labeled rabbit polyclonal anti-HSV antibodies (DAKO Cytomation, Carpinteria, CA) diluted 1:100 in 1% normal rabbit serum. Fluorescent-labeled tissue sections were then washed with 0.1 M PBS, and mounted under cover slips.
Analysis of HSV-1 reactivation in trigeminal ganglion explants
Latently infected mice were sacrificed on days 38 and 39 p.i., TG were aseptically removed, and each TG was placed in one well of a 24-well plate containing 1 ml of complete DMEM. Once TG were harvested for an entire 24-well plate, the TG were heat stressed by transfer to a 43°C, 5% CO2 incubator for 3 hours. After heat stress, explants were transferred to a 37°C, 5% CO2 incubator. Twenty-four hours later, TG and cell culture medium were transferred to 24-well plates seeded 6 h earlier with 5 × 104 L7 cells per well in a volume of 0.5 ml of complete DMEM. On days 6 and 10 after explantation, TG were transferred to a 24-well plate containing freshly seeded L7 cells. After 14 days in culture, TG explants were homogenized in 500 μl of complete DMEM with a Pro 200 homogenizer (Pro Scientific) and TG homogenates were transferred to a -80°C freezer. Freeze-thawed TG homogenates were centrifuged to remove tissue debris, and 200 μl of clarified supernatant was used to inoculated each well of a 12-well plate of L7 cells (1 × 105 cells per well). After allowing 45 minutes for viral adsorption, the viral inoculum was aspirated, the cell monolayer was rinsed with 1 ml complete DMEM, and the rinse solution was replaced with 1 ml complete DMEM. Monolayers of L7 cells treated with TG homogenates were observed for six days for the development of viral cytopathic effects.
Measurement of HSV-1 DNA load in latently infected mouse trigeminal ganglion
The left and right TG from each mouse were placed into a single 1.5 ml microfuge tube, and transferred to -80°C until the time of DNA extraction. DNA was isolated by a standard phenol: chloroform extraction procedure , and the number of HSV-1 genomes per TG was determined by a competitive PCR assay, which is described as follows.
The oligonucleotide primers used in the competitive PCR assay, VP16-a (5'-GGACTCGTATTCCAGCTTCAC-3') and VP16-b (5'-CGTCCTCGCCGTCTAAGTG-3'), amplified a 260-bp fragment of the HSV-1 VP16 gene. To provide an internal control for each PCR assay, a VP16 competitor template was generated by the method of Siebert and Larrick . In brief, an irrelevant sequence from pUC18 was amplified with the primers VP16 mimic-a (5'-GGACTCGTATTCCAGCTTCACGGAGGACCGAAGGAG-3') and VP16 Mimic-b (5'-CGTCCTCGCCGTCTAAGTGCCAGTGCTGCAATGA), which amplify a 361-bp PCR product whose 5' ends are identical in sequence to the VP16-a and VP16-b primers. The VP16 competitor was cloned into the pCR II vector (Invitrogen Corp., Carlsbad, CA), and the resulting plasmid was used as a competitor template in the competitive PCR assay. DNA for the standard curve was obtained by diluting the plasmid pCRII: VP16 into a solution of TE buffer containing 33 ng/μl of salmon sperm DNA as carrier DNA. The most concentrated standard contained 45 fg of plasmid DNA per μl (10,000 copies per μl), and 13-serial twofold dilutions were made using TE buffer and salmon sperm DNA as the diluent.
PCR assays were conducted as follows: A solution containing 1× Taq buffer, 50 μM of each dNTP, 0.25 μM of each VP16 primer, 5% glycerol, and ~1500 copies of VP16 competitor template per 50 μl reaction was prepared. Forty-two microliters of this master mix were placed in 0.65 ml tubes and overlaid with mineral oil, and 100 ng of TG DNA (3 μl), DNA standards, or negative control DNA sample was added to each tube. The tubes were heated to 90°C in a thermal cycler, and 2.5 U of Taq polymerase diluted in 5 μl of Taq buffer was added to each sample. PCR samples were incubated for 35 cycles of 94°C for 1 minute 15 seconds, 59.5°C for 1 minute 15 seconds, and 72°C for 40 seconds.
Measurement of VP16 gene and competitor PCR product yields was performed by a modification of the dot blot procedure of Hill et al. . From each amplified PCR sample, 15 μl was diluted in 500 μl of a 0.4 M NaOH solution, transferred to a 0.6 ml microfuge tube, heated to 90°C for 10 minutes, snap-cooled on ice, and blotted on Zeta Probe GT nylon membrane (BioRad Laboratories, Hercules, CA) in an 8 × 12 dotblot pattern using a Convertible™ vacuum filtration manifold (Whatman-Biometra, Gröningen, Germany). Two identical dotblots of each set of PCR samples were produced and crosslinked with 0.2 J/cm2 in a UV crosslinker (Spectronics Corporation, Westbury, NY). One dotblot was hybridized to a radiolabeled oligonucleotide specific for VP16 (5'-GTCGTCGTCCGGGAGATCGAGCAGGCCCTC-3'), and the second duplicate dotblot was hybridized to a radiolabeled oligonucleotide specific for the competitor PCR product (5'-CGCTCGTCGTTTGGTATGGCTTCATTCAGC-3'). Both oligonucleotide probes were end-labeled with [α-32P] dATP using terminal deoxynucleotidyl transferase (Promega Corporation, Madison, WI). Each probe was allowed 16 h to hybridize to a membrane at 42°C in a solution containing 20 ng/ml labeled probe, 7% SDS, 120 mM NaH2PO4, and 250 mM NaCl. Excess probe was removed from membranes by sequential rinses in 0.1× standard saline citrate (SSC) containing 0.1% SDS and membranes were exposed to phosphor screens, which were scanned on a Cyclone PhosphorImager (Perkin Elmer Life Sciences, Boston, MA). The amount of radolabeleled probe hybridized to each dotblot was determined using OptiQuant v4.0 software (Perkin Elmer Life Sciences).
A two-fold dilution series of VP16 plasmid DNA defined the relationship between the copy number of VP16 genes in each PCR (x) and the logarithm of the ratio of VP16 to competitor PCR product yields (y) amplified from each DNA sample. The standard curve was described by the equation x = arctanh (y), but for convenience Microsoft Excel's trendline-fitting feature was used to rapidly define a third order polynomial equation (x = ay3 + by2 + cy + d) that approximates the sigmoid shape described by this equation (Fig. 8B). To estimate the copy number of viral genomes per TG, the number of HSV-1 genomes per PCR was multiplied times 150. This derivation is based on the fact that an average of 15 μg of DNA is extracted from each TG pair using an extraction procedure that recovers 50% of the total DNA. Thus, ~15 μg of DNA was present in a single TG, and the input of 0.1 μg of TG DNA per PCR contains ~1/150th (0.1 μg out of 15 μg) of the total number of HSV-1 genomes present in a single TG.
Analysis of numerical data was performed with the software packages Microsoft Excel and Modstat (Modern Microcomputers, Mechanicsville, VA). All viral titers were transformed by adding a value of 1 to the number of plaque-forming units detected per sample, such that all data could be analyzed on a logarithmic scale. The significance of differences between multiple groups was evaluated by one-way analysis of variance followed by a post hoc t-test. The significance of differences between strain 129 mice, rag2 -/- mice, stat1 -/- mice, versus rag2 -/- stat1 -/- mice was evaluated by two-way analysis of variance. The goodness of fit of the standard curve used in the competitive PCR assay was determined by regression analysis using the method of least squares.
This work was supported by grants from the National Institutes of Health (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 thank Francis Carbone, Roger Everett, Michele Hardy, and Chris Preston for their thoughtful and critical evaluation of this manuscript, and thank Justin Patrick for outstanding technical assistance.
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