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Keywords:

  • herpes simplex virus type 1;
  • interferon;
  • protein kinase R;
  • US11

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

The herpes simplex virus type 1 (HSV-1) VRTK strain that was previously isolated in our laboratory as an acyclovir-resistant thymidine kinase (TK)-deficient mutant, is more sensitive to type 1 interferon than is the parent strain VR3. The properties of this mutant were investigated to clarify the mechanism for its hyper-sensitivity to interferon (IFN). It was found that: (i) IFN-pretreated cells, but not those treated with IFN after adsorption, are hyper-sensitive to IFN; (ii) the mutant cannot inhibit protein kinase R phosphorylation efficiently during the early stage of replication (2 hrs post-infection); (iii) expression of US11 in infected cells and its incorporation into the virion is reduced in the mutant compared to the wild type, despite the fact that a similar degree of DNA synthesis occurs during replication of both strains and; (iv) over-expression of wild-type viral TK has no effect on the phenotype of the VRTK strain, indicating that the phenotype is induced by a mutation(s) that does not involve the TK gene. These results suggested that the presence of US11 in the virion, but not that expressed after infection, plays an important role in the escape function of HSV-1 from the antiviral activity of type 1 IFN.

Abbreviations
ACV

acyclovir

DMEM

Dulbecco's modified Eagle's medium

dsRNA

double-strand RNA

E. coli

Escherichia coli

eIF-2α

eukaryotic initiation factor 2 subunit α

HSV-1

herpes simplex virus type 1

IC50

50% inhibitory concentration

IFI16

interferon inducible protein 16

IFN

interferon

IPTG

isopropyl β-D-thiogalactopyranoside

IRF

IFN regulatory factor

JAK

janus kinase

MEM

Eagle's minimum essential medium

MOI

multiplicity of infection

MxA

myxovirus (influenza) resistance protein A

OAS

oligoadenylate synthetase

PACT

PKR activating protein

pfu

plaque forming units

PKR

protein kinase R

PML

promyelocytic leukemia protein

qRT-PCR

quantitative reverse-transcribed real-time PCR

SOCS

suppressor of cytokine signaling

STAT

signal transducers and activators of transcription

TK

thymidine kinase

TK

thymidine kinase deficient

TTP

thymidine triphosphate

Interferons are secreted proteins that play an important role in innate immune defense. During viral infection, the antiviral function of IFNs operates through induction of antiviral proteins, such as OAS, PKR and MxA protein, via the JAK/STAT pathway [1]. Activated PKR, formed by binding to dsRNA, phosphorylates the alpha subunit of eIF2α, resulting in inhibition of translation. Many DNA and RNA viruses, such as influenza virus NS1, reovirus σ3, rotavirus NSP3, vaccinia virus E3L and Epstein–Barr virus SM protein, code dsRNA binding proteins and PKR inhibitors [2]. HSV-1 US11, a true late γ2 gene product, is also a PKR inhibitor and is known to inhibit activation of PKR by binding to dsRNA and PKR.

Herpes simplex virus type 1, a double-stranded DNA virus that belongs to the alphaherpesvirinae subfamily, is known to have little susceptibility to IFN [3]. Three virus polypeptides, ICP0, γ34.5 and US11, are reportedly involved in this resistance to IFN. ICP0 suppresses the anti-viral activity of IFN by inhibiting IRF3 and IRF7 functions [4-7] and γ34.5 aids recovery from inhibition of protein synthesis by de-phosphorylating PKR-phosphorylated eIF2 [8-14]. Recently, researchers have reported that degradation of nuclear IFI16 by ICP0 inhibits IRF3 signaling, and that ICP0 interacts with PML and induces its degradation [15-17]. In addition, that US11 reportedly inhibits phosphorylation of PKR by binding to dsRNA, the protein activator of the interferon-induced protein kinase (PACT) and PKR [18-24]. Recently, researchers have reported that US11 can also competitively inhibit activation of OAS by binding to dsRNA [25].

Acyclovir is a safe and potent anti-herpesvirus drug that has been widely used in the treatment of HSV infection. The majority of ACV-resistant HSV mutants are thymidine kinase deficient (TK) [26-30]. Leib et al. reported that the reduced replication of TK mutants in the corneas of wild-type mice is significantly greater in those of IFN receptor knockout mice [31]. Replication of TK mutant viruses is also readily detectable in the trigeminal ganglia of IFN receptor knock-out mice, but not in those of wild-type mice. Furthermore, the HSV-1 VRTK strain, isolated in our laboratory as an ACV-resistant mutant, is also a TK-deficient mutant with hyper-sensitivity to type 1 IFN [32-35]. Why the mutation in the enzyme for nucleoside metabolism influences sensitivity to IFN is unclear. Therefore, the mechanism for the hyper-sensitivity of the VRTK strain to IFN was studied here, with a focus on the function of US11 in mutant-infected cells.

1 MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

1.1 Cells and viruses

Telomerase-immortalized human fibroblasts, hTERT-BJ1 cells (Clontech Laboratories, Mountain View, CA, USA) were cultured in Dulbecco's MEM containing 10% FBS. Vero cells (an African green monkey kidney cell line) were cultivated in MEM supplemented with 5% calf serum. The human amnion cell line, FL, was routinely cultured in RPMI-1640 containing 10% FBS. The HSV-1 VR3 strain, obtained from the American Type Culture Collection (Manassas, VA, USA) and its TK-deficient mutant strain, VRTK, were used in this study [32-35].

1.2 Susceptibility of herpes simplex virus type 1 strains to α-interferon

Confluent hTERT-BJ1 cells in 48-well plastic plates were pretreated with natural-type IFN-α (Sumiferon; Dainippon Sumitomo Pharma, Osaka, Japan) for 16 hrs, infected with HSV-1 strains at a MOI of 3 pfu/cell and cultured for 16 hrs in the presence of IFN-α at the same concentration as that used for pretreatment. The titers of the progeny virus in the cultures were determined by plaque assay on Vero cells after three cycles of freezing and thawing of the infected cells.

1.3 Preparation of the cellular lysate

For detection of viral protein (ICP0, TK, VP16 and US11) expression and phosphorylation of PKR in the infected cells, cell lysates were prepared as follows. IFN-α pretreated hTERT-BJ1 cells in 24-well plastic plates were infected with HSV-1 strains at an MOI of 3 pfu/cell. After virus adsorption for 1 hr, the cells were washed with PBS and cultured in the presence or absence of IFN-α. At appropriate times post-infection, the culture medium was removed and the cells lysed in SDS–PAGE loading dye (50 mM Tris–HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 10% glycerol and 10% 2-mercaptoethanol).

1.4 Purification of herpes simplex virus type 1 virion

Confluent hTERT-BJ1 cells in sixteen 150 cm2 culture bottles were infected with HSV-1 at an MOI of approximately 3 pfu/cell and cultured for 16 hrs in the presence or absence of 0.1 µg/mL ACV. After incubation, the medium containing the cell-free virions was harvested and the cell debris removed by centrifugation at 3000 rpm for 10 mins. HSV-1 virions were purified by banding using 20–60% (w/w) sucrose gradient centrifugation at 20,000 rpm for 1 hr at 4°C, and then pelleted by centrifugation at 27,000 rpm for 1 hr at 4°C using an ultracentrifugator and a P28S swing rotor (Hitachi Koki, Tokyo, Japan). The pellets were resuspended in PBS and pelleted again by centrifugation at 27,000 rpm for 1 hr at 4°C to remove sucrose. Th epurified virions were suspended in 1 mL PBS and the virus titer determined by plaque assay on Vero cells.

1.5 Western blotting

For rabbit immunization, US11 polypeptide was prepared as follows. The full-length HSV-1 US11 gene (GenBank accession no. NC_001806) was amplified by PCR using the primer set US11-NdeI2 (5′-CCGGGGGTTGGGTCTGGCTCATATGGAG-3′) and US11-SalI2 (5′-GGGGTCGACAGACCCGCGAGCCGTACGTG-3′). The amplified US11 gene, which incorporates NdeI and SalI sites at the 5′ and 3′ ends, respectively, was digested with these restriction enzymes, and then cloned into pET26b (Novagen, Madison, WI, USA) to generate pET-US11. Escherichia coli strain BL21 (DE3) was transformed with pET-US11 and overexpression of a 6X His tag-US11 fusion protein was induced by adding IPTG to a final concentration of 1 mM.

The tagged US11 fusion protein was purified from the bacterial lysate using a Ni-column. Rabbits were immunized five times with the purified US11 fusion protein and the serum collected. Rabbit anti-TK serum was prepared as described previously [32]. Mouse anti-ICP0 and anti-VP5 antibodies were purchased from Virusys (Sykesville, MD, USA), goat anti-VP16 antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), rabbit anti-PKR and anti-phospho-PKR (Thr451) monoclonal antibodies from Epitomics (Burlingame, CA, USA) and anti-β-actin antibody from Sigma (St. Louis, MO, USA). Horse radish peroxydase-conjugated anti-rabbit, -mouse and -goat immunoglobulin antibodies were used as secondary antibodies. The protein bands were detected with an ECL plus kit (GE Healthcare UK, Buckinghamshire, UK) and an LAS-3000 lumino image analyzer (Fujifilm, Tokyo, Japan).

1.6 Plasmid construction

A 511 bp PCR product of the UL1 (glycoprotein L) gene from the HSV-1 KOS strain (ATCC VR-734) encompassing nucleotides 9658–10,168 was generated with the primers HSV1-seqF and HSV1-seqR [36]. This amplicon was cloned into pGEM-T easy plasmid (Promega, Madison, WI, USA). The resulting plasmid, pGEM-HSV1-real, was used as a standard to quantify HSV DNA. A pGEM-ICP0 plasmid was constructed for use as a standard for the quantification of ICP0 mRNA (GenBank accession no. NC_001806) by the same procedure as that described above; that is, a 575 bp PCR product of the ICP0 exon2 gene from the HSV-1 VR3 strain encompassing nucleotides 3093–3668 was generated with primers ICP0ex2F (5′-ATGTCTGGGTGTTTCCCTGCGACCG-3′) and ICP0ex2R (5′-TACCGCGGGGCGAACCGCTGATT-3′) and this amplicon was cloned into the pGEM-T easy plasmid to afford the pGEM-ICP0 plasmid. Construction of a US11-expressing plasmid for mammalian cells was performed as described below. A PCR product of the US11 gene from the HSV-1 VR3 strain was generated with the primers US11 pcDNA Eco (5′-GAATTCCCCCCCGTCGCTCTCGAGATG-3′) and US11 pGEX Rev (5′-GCGGCCGCTATACAGACCCGCGAGCCGTACG-3′). This amplicon was digested by EcoRI and NotI and then cloned into the pcDNA3.1 plasmid. Construction of the HSV-1 TK-expressing plasmid, pcDNA3.1(+)-HSV-1-TK, was performed as described previously [37].

1.7 Ectopic viral thymidine kinase and US11 expression

Eighty percent confluent FL cells in 24-well plastic plates were transfected with viral TK or the US11-expression plasmid using Fugene HD reagent (Promega). Two days after transfection, the cells were infected with the VRTK strain. At 10 hrs after virus infection, the culture medium was removed and the cells harvested for western blotting to measure TK and US11 expression. At 20 hrs post-infection, the culture medium was collected for recovery of cell-free virus to determine IFN susceptibility. Quantification of IFN susceptibility was performed as described above.

1.8 Real-time polymerase chain reaction

Quantification of the HSV-1 genome in the hTERT-BJ1 cells was carried out by real-time PCR using a TaqMan Universal PCR Master Mix kit and an ABI Prism 7700 instrument (Applied Biosystems, Foster City, CA, USA). Amplification of the HSV-1 genome was performed using the primer set 5′-GTGAAGGCTGGGTGTGTGAA-3′ and 5′-TGTAGGGCGACAGGATTTGG-3′ and the labeled probe, 5′FAM-TTTGACTATTCGCGCACCCGCC-TAMRA3′ and a standard curve was drawn using pGEM-HSV1-real as a template for each experiment.

Sample preparation was carried out as follows. Confluent hTERT-BJ1 cells in 24-well plastic plates pretreated with or without 1 unit/mL of IFN-α were infected with HSV-1 strains at an MOI of 3 pfu/cell and cultivated for 10 hrs in a medium containing 1 or 0 unit/mL of IFN-α. After incubation, the cells were washed once with PBS, and treated with proteinase K. Total DNA samples were prepared from the lysate by phenol–chloroform extraction and then suspended in deionized water. The amount of HSV-1 genome in the sample was quantified in triplicate by real-time PCR with a thermal cycling protocol consisting of initialization at 50°C for 2 mins, and then at 95°C for 10 mins, followed by 40 cycles of denaturation at 95°C for 15 s and annealing–extension at 55°C for 31 s.

1.9 Quantitative reverse-transcribed real-time polymerase chain reaction assay

Expression of the ICP0 gene in HSV-1 infected cells was evaluated by qRT-PCR. Confluent hTERT-BJ1 cells in 24-well plastic plates pretreated with or without 1 unit/mL of IFN-α were infected with HSV-1 strains at an MOI of 3 pfu/cell and cultured in the presence or absence of IFN-α. At 0, 1, 2 and 4 hrs post-infection, the cells were harvested and mRNA extracted using a Micro-FastTrack 2.0 mRNA Isolation Kit (Invitrogen, Carlsbad, CA, USA). To generate cDNA from the purified mRNA, a reverse transcript reaction was carried out using SuperScript III Reverse Transcriptase (Invitrogen) and a random hexamer primer. The copy number of the ICP0 cDNA was quantified in triplicate with real-time PCR using the primer set, 5′-CATGAAAACCTGGATGCAATTG-3′ and 5′-CCCACTATCAGGTACACCAGCTT-3′ and the labeled probe 5′FAM-CAACACCTGCCCGCTGTGCAA-TAMRA3′, with the pGEM-ICP0 plasmid as a standard.

2 RESULTS

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

2.1 Susceptibility of herpes simplex virus type 1 strains to α-interferon

To examine the susceptibility of HSV-1 strains to IFN-α, the replication of wild-type VR3 and its TK mutant, VRTK, strains in IFN-α-treated hTERT-BJ1 cells was comparatively analyzed by yield reduction assay at one-step growth. Because potent anti-HSV-1 activity was observed in the IFN-α-pretreated, but not the non-pretreated cells; therefore, pretreatment was used in all experiments in this study (Fig. 1a). The yield of the TK mutant strain, VRTK, in the IFN-α-treated cells was reduced in a dose-dependent manner (Fig. 1b). The IC50 values of IFN-α were 1.59 and 0.23 unit/mL for the VR3 and VRTK strains, respectively, indicating the VRTK strain is more sensitive to IFN-α than is the parental VR3 strain.

image

Figure 1. Susceptibility of HSV-1 strains to IFN-α in hTERT-BJ1 cells. (a) IC50 values of the wild-type (VR3) and its TK mutant (VRTK) strains for IFN-α. Data are given as the means ± SD of three independent experiments. “Pretreatment” indicates the presence of IFN-α both before and after virus infection and “after infection” indicates the presence of IFN-α after virus infection alone. (b) Yield of HSV-1 strains in IFN-α-pretreated hTERT-BJ1 cells relative to that in control cultures. Error bars represent the SD. P values calculated by the Student's t-test are as follows: *, 0.0019; **, 0.0052; ***, 0.0134. ●, VR3 strain; ▪, VRTK strain.

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2.2 Phosphorylation of protein kinase R in herpes simplex virus type 1-infected cells

The VRTK strain was more sensitive to IFN-pretreatment than was the parent VR3 strain. Type 1 IFN is known to induce the antiviral protein PKR, and induced-PKR is activated by binding to dsRNA, resulting in inhibition of translation. Therefore, whether HSV-1 infection enhances PKR phosphorylation in IFN-pretreated cells was investigated. At 2 hrs post-infection, there was a twofold increase in PKR phosphorylation in VRTK-infected cells compared to at 0 hr, but not in VR3-infected cells (Fig. 2). This finding shows that VRTK does not prevent PKR phosphorylation: this phenomenon might account for its high susceptibility to IFN.

image

Figure 2. Quantitative analysis of PKR phosphorylation in HSV-1-infected cells. The total amount of PKR and phosphorylated PKR in the VR3 and VRTK strains were detected by western blotting using anti-PKR and anti-p-PKR (Thr-451), respectively. IFN-α treatment at 1 unit/mL was started immediately after virus adsorption (shown as IFN+ in the figure). The relative values of phosphorylated PKR normalized to total PKR (relative PPKR/PKR ratio) are shown in the figure. The value of phosphorylated PKR normalized to total PKR at 0 hr postinfection was set at 1.

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2.3 Differences between herpes simplex virus type 1 strains in viral gene expression

Because ICP0 is one of the factors reportedly necessary for resistance to the IFN antiviral system [5-7, 15, 31, 38, 39], it is possible that the difference in IFN susceptibility between the VR3 and VRTK strains is caused by differences in their degree of expression of the ICP0 polypeptide. Therefore, ICP0 expression was studied by western blotting (Fig. 3a) and ICP0 mRNA quantified by qRT-PCR (Fig. 3b). In the absence of IFN-α, expression of ICP0 protein in TK mutant-infected cells was slightly reduced or delayed compared to that in wild type-infected cells. However, in the presence of IFN-α, there was a more striking difference in expression of ICP0 protein between the wild-type and mutant strains (Fig. 3a). Synthesis of ICP0 mRNA by the mutant virus was reduced to about 46% of that of the wild-type virus at 2 hrs post-infection (Fig. 3b). The differences in ICP0 mRNA synthesis corresponded to those in ICP0 polypeptide expression. These results suggested that one reason for the IFN sensitive phenotype of VRTK is reduced ICP0 expression.

image

Figure 3. Expression of ICP0 polypeptide and quantification of ICP0 mRNA in HSV-1-infected cells. (a) Amount of ICP0 polypeptide in VR3- and VRTK mutant-infected cells. (b) Amounts of ICP0 mRNA in VR3- and VRTK mutant-infected cells. Samples were taken at the indicated times from infected cells, the amounts of ICP0 polypeptide analyzed by western blotting and the copy numbers of ICP0 mRNA determined by reverse-transcribed RT-PCR. β actin was also visualized as an internal control. Data are given as the means ± SD of three independent experiments. Error bars represent the SD. *, P value calculated by Student's t-test 0.0615; ○, samples taken from the VR3-infected cells cultured in the presence of IFN-α; ●, samples from VRTK mutant-infected cells; IFN, cultured in the absence of IFN-α; IFN+, cultured in the presence of 1 unit/mL IFN-α both before and after infection.

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US11 inhibits PKR activation via virus infection-induced phosphorylation. In order to clarify whether differences between VR3 and VRTK in degree of PKR-phosphorylation are induced by differences in their amounts of US11 gene expression, a polyclonal rabbit serum against the US11 protein was produced and the viral proteins quantified by immunoblotting. To attain this objective, the US11 gene was cloned in frame with the gene encoding the 6X His Tag as detailed in the Materials and Methods Section. 6X His tag-US11 fusion protein was expressed in E. coli, and the affinity-purified fusion protein then used to immunize a rabbit for production of polyclonal antiserum (Fig. 4a). To test the specificity of the antiserum, western blotting was carried out. When the anti-serum was reacted with the virus-infected cell lysates, only one band of 24 KDa corresponding to US11, which was not observed in the mock-infected cells, was detected (Fig. 4B). Using this anti-serum, US11 in HSV-1 infected cells was evaluated. Expression of US11 was strongly affected by both the mutation in the TK gene and IFN treatment (Fig. 4c). This difference was not observed in any of γ1 genes, the UL48 gene coding VP16 or in the presence or absence of IFN-α at 10 hrs post-infection (Fig. 4c). The TK polypeptide was also expressed in equal amounts in wild-type virus-infected cells both with and without IFN-treatment, whereas it was not observed in the VRTK-infected cells due to significantly reduced expression, as described previously [33]. The results of the immunoblot analysis of viral proteins suggest that reduced expression of US11 is also involved in the hyper-sensitivity of TK mutants to IFN-α.

image

Figure 4. Expression of TK, VP16 and US11 in HSV-1 infected cells. (a) 6X His tag-US11 fusion protein purified from the E. coli lysate by Ni-column. The arrow head indicates the 6X His tag-US11. (b) Western blotting analysis of the specificity of the constructed anti-US11 antiserum. Mock- or HSV-1 VR3 strain-infected cell lysates were reacted with the anti-US11 antiserum constructed in this study. HSV-1-infected cell lysates were recovered at the indicated times post-infection and mock-infected cell lysates at 10 hrs post-infection. The arrow indicates US11. MW, molecular weight (in thousands). (c) The amount of proteins at 10 hrs post-infection in VR3- and VRTK mutant-infected cells were analyzed using western blotting. β actin was also visualized as an internal control. In all panels, IFN, samples were cultured in the absence of IFN-α; IFN+, cultured in the presence of 1 unit/mL IFN-α both before and after infection.

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2.4 Efficiency of viral DNA replication

US11 is a true late gene and its expression is dependent on viral genome replication [40]. Therefore, the efficiency of DNA replication in the wild-type and TK strains was investigated by real-time PCR (Table 1). Similar amounts of DNA synthesis were observed in IFN-treated VR3- and VRTK-infected cells; IFN treatment had no inhibitory effect on DNA replication in either the VR3 or VRTK strains. These results indicate that type I IFN-induced reduction in US11 expression in the TK mutant was not caused by inhibition of viral DNA replication.

Table 1. DNA replication of HSV-1 strains in infected cells
VirusMean copy no. (/cell)
IFN (hrs)IFN+ (hrs)
010010
  • , values are given as the means ± SD of three independent experiments;

  • , DNA replication in HSV-1-infected hTERT-BJ1 cells cultivated with and without 1 unit/mL α-IFN was evaluated by real-time PCR.

  • *

    , P-value 0.0246;

  • **

    , P-value > 0.05.

VR317.75 ± 1.10**2980 ± 469.51*18.31 ± 4.91**1860 ± 923.25**
VRTK25.08 ± 5.31**1990 ± 135.84*25.57 ± 10.83**2670 ± 1060**

2.5 Comparison of the amount of US11 in wild-type and thymidine kinase deficient mutant virions

US11 is a true late gene; however, the inhibitory effect of US11 on PKR phosphorylation occurred during the early stages of viral replication. To clarify whether prevention of PKR phosphorylation is induced by importation of US11 polypeptide into the cells from the virion or by its synthesis within the infected cells, the amount of US11 polypeptide in the virus particles was estimated by western blot analysis. When the virus titer had been normalized according to infectivity (pfu), the amount of US11 in the VRTK strain was found to be lower than that in the VR3 strain (Fig. 5). In contrast, the amount of VP5 in the VRTK strain was the same as that in the VR3 parental strain. These results suggest that, per virus particle, the VRTK strain possesses less US11 polypeptide than does the VR3 parent strain. Considering these results and the timing of the function of US11 in virus replication, inhibition of PKR phosphorylation for escape from the antiviral effects of IFN appears to be caused by US11 packaged within the tegument.

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Figure 5. Amounts of VP5 and US11 protein in TK-mutant and wild-type particles. Purified virus (2.7 × 106 pfu) was resolved on SDS–polyacrylamide gels and analyzed by western blot analysis.

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2.6 The amount of virion US11 and susceptibility to interferon

Acyclovir is reportedly an inhibitor of HSV DNA synthesis and IFN treatment shows synergistic anti-HSV activity [41-43]. It was speculated that this synergistic antiviral activity could be explained by the reduction in the level of US11 in the virion caused by ACV treatment-induced reduction in US11 expression via suppression of DNA synthesis. As expected, viral DNA replication and US11 expression were reduced by ACV in a dose-dependent manner (Fig. 6a, b). However, when the virus titers were normalized by infectivity (pfu), equal amounts of US11 and VP5 proteins were observed in the virus particles of ACV-treated and -untreated HSV-1 virus (Fig. 6c). As expected from the amount of US11 present, there was no difference in the susceptibilities of the two viruses, replicated with or without ACV-treatment, to IFN (Fig. 6d).

image

Figure 6. Analysis of ACV-treated HSV-1. (a) DNA replication in HSV-1-infected hTERT-BJ1 cells cultivated with 0 (●), 0.04 (▪), 0.1 (○) and 0.4 µg/mL (□) ACV was evaluated by real-time PCR. Error bars represent the SD of three independent experiments. (b) Expression of US11 in HSV-1-infected cells. In the presence of the indicated concentrations of ACV, the amount of US11 at 10 hrs post-infection in the VR3-infected cells was analyzed by western blotting. β actin was also visualized as an internal control. (c) Amounts of VP5 and US11 protein in VR3 virus particles in the presence of 0.1 µg/mL ACV. The purified virus (2.7 × 106 pfu) was resolved on SDS–polyacrylamide gels and analyzed by western blot analysis. (d) Yields of ACV-treated and -untreated HSV-1 strains in IFN-α-pretreated hTERT-BJ1 cells relative to that in the control culture. ○, ACV treated; ●, ACV not treated. Error bars represent the SD.

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2.7 Ectopic US11 and viral thymidine kinase expression do not reverse the susceptibility of the VRTK strain to interferon

VRTK strain produced less US11 and its virion contained a smaller amount of US11 than did the wild-type strain. Whether ectopic expression of viral TK and US11 reverses the reduction of VRTK in IFN-pretreated cells was investigated. Even though ectopic viral TK expression was found in the VRTK infected cells, US11 expression was not restored. Ectopic US11 expression also had no effect (Fig. 7a). Furthermore, VRTK derived from ectopic viral TK-expressing cells and US11-expressing cells was also sensitive to IFN at the same level as VRTK derived from cells without viral TK or US11 expression (Fig. 7b).

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Figure 7. Ectopic expression of viral TK and US11 in HSV-1-infected cells, and the susceptibility of the VRTK strain derived from ectopic viral TK- and US11-expressing FL cells to IFN-α in hTERT-BJ1 cells. (a) The amount of proteins at 10 hrs post-infection in ectopic viral TK- and US11-expressing cells was analyzed by western blotting. MOCK, virus-uninfected cells; VR3, i VR3 strain-infected cells; VRTK, VRTK strain-infected cells. (b) Yield of VRTK strains in IFN-α-pretreated hTERT-BJ1 cells harboring viral TK- or US11-expressing plasmids relative to that in the control culture. β actin was also visualized as an internal control. ○, VR3/pcDNA3.1; □, VRTK/pcDNA3.1; ●, VRTK/pcDNA3.1(+)-HSV-1-TK; ▪,VRTK/pcDNA US11. Error bars represent the SD.

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3 DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

In this paper, we have shown that a HSV-1 TK-deficient mutant strain, VRTK, is more sensitive to IFN-α when the host cells are pre-treated with IFN-α prior to infection, but is as sensitive as the VR3 parental strain without pretreatment (Fig. 1). IFN(s) plays important roles in the antiviral immune response and escape from the antiviral activity of IFN is essential for pathogenic viruses. In particular, HSV-1 possesses various strategies for counteracting IFN-induced antiviral responses, namely: (i) HSV-1 suppresses the signaling pathway of IFN through up-regulation of SOCS3 by an unknown pathway [44] and degradation of nuclear interferon inducible protein 16 (IFI16) by ICP0 inhibits IRF3 signaling [4-7, 15, 38]; and (ii) the PKR pathway plays a major role in the antiviral function of the IFN system. This pathway functions through the sequential phosphorylation of PKR and eIF2α. PKR phosphorylation is induced by binding with virus-induced dsRNA and regulates conversion of PKR from an inactive to an active form. Activated PKR then phosphorylates eIF2α, causing repression of protein synthesis. HSV-1 US11 inhibits the first step, PKR activation, by competitive binding of dsRNA and γ34.5 dephosphorylates phospho-eIF2α, releasing host cells from the antiviral state [11, 12, 19, 21, 45-47]. Based on this knowledge, we focused on US11 and ICP0 to clarify the mechanism for the hyper-sensitivity of the TK-deficient HSV mutant to IFN.

Compared with the wild-type strain, the VRTK, TK mutant virus, expresses less US11 and has less US11 protein packaged into the tegument of the virus particles, preventing sufficient inhibition of PKR phosphorylation at the early stage of virus infection (Figs. 2, 4 and 5). When the VR3 parent strain is replicated in ACV-treated cells, although the virus expresses less US11, the virion contains a normal amount of US11 protein and these viruses have the same susceptibility to IFN as do viruses replicated in the absence of ACV (Fig. 6). Proteins packaged into the tegument, many of which are late gene products, are thought to be important for control of host cells during the early stage of the virus replication cycle. Therefore, the functions of the tegument proteins should be divided into two distinct phases: the functions that occur in the cells expressing them and those that occur in cells into which they are introduced by infected virions. In this study, we showed US11 derived from the tegument acts as the initial factor in the evasion of IFN in IFN-pretreated cells. γ34.5 mutants with intact US11 are reportedly unable to overcome PKR-mediated shut-off of protein synthesis in cells infected at an MOI of 100 pfu/cell [9]; however, that study was performed without IFN pretreatment. On the other hand, a γ34.5 recombinant mutant expressing US11 as an immediate-early protein has been found to reverse inhibition of protein synthesis due to IFN pretreatment [48]. This indicates that US11 is required at the early stage of viral replication in IFN-pretreated cells and supports the findings of our study. Virion US11 appears to play an important role in escape from antiviral functions in IFN-pretreated cells. However, it is not important in IFN-untreated cells because HSV up-regulates a cellular factor, SOCS3, which blocks the IFN signal pathway at the early stage of infection. The results of this study, together with those of previous reports, suggest that US11 plays an important role in secondary HSV-1-infected cells that have been exposed to type 1 IFN secreted from primary infected cells or macrophages activated by the HSV-1 antigen (Fig. 8).

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Figure 8. Model for inhibition of PKR phosphorylation by US11 incorporated into the virion. In primary infected cells, virus infection and replication lead to activation of IRF3, resulting in IFN-α/β synthesis and release from primary infected cells. Released IFN binds to type I IFN receptors in both an autocrine and paracrine manner and induces PKR as an interferon-stimulating gene via the JACK/STAT pathway. The induced PKR binds to double-stranded RNA and changes to an active state, as shown by the “secondary infected cell” in the figure. US11, transported into the infected cell by the virion, binds to dsRNA and PKR, thus inhibiting PKR phosphorylation. dsRNA, double-stranded RNA; IFNAR, type I IFN receptor.

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Compared to that of VR3, ICP0 protein expression by the VRTK strain is slightly reduced or delayed without IFN pretreatment; however, pretreatment with IFN leads to a marked reduction in ICP0 expression in the VRTK strain (Fig. 3a). ICP0 reportedly inhibits expression of IFN-β by degradation of IFI16 and inhibition of IRF3-activation [15]. The antiviral effects of exogenously added IFN are more pronounced in PML+/+ than in PML−/− [49]. PML and Sp100 are both recruited to sites associated with their parental viral genomes and then repress HSV-1 gene expression [50, 51]. ICP0 interacts with PML and its E3 ubiquitin ligase activity induces PML degradation [16]. In IFN-pretreated HSV-1-infected cells, ICP0 may mediate escape from PML-mediated antiviral activity. In this study, the reduction of ICP0 expression in IFN-pretreated VRTK-infected cells may have been caused by insufficient inhibition of PKR phosphorylation by the reduced amount of virion-derived US11. Furthermore, there were differences between the VR3 and VRTK strains in the copy numbers of ICP0 mRNA (Fig. 3b). Recently, Sanfilippo and Blaho reported that ICP0 mRNA can trigger the cell death cascade and induce apoptosis [52]. The mechanism of this function is unclear, but the observation suggests a novel field of herpesvirus virology. The question of whether ICP0 mRNA contributes to the escape functions of HSV from the antiviral activity of interferon remains.

Thymidine kinase activities are not essential for virus replication in actively dividing cells in vitro, but are important for the expression of virulence [53, 54]. The low virulence of the TK mutant has been explained simply by the lower yield caused by the lack of TTP necessary for DNA replication. Leib et al. reported that attenuation of TK mutants in a mouse model depends on the IFN system [31]. Our observations indicate that the VRTK strain has a lower tolerance to IFN than does the VR3 parent virus, whereas ACV-treated VR3 virus has the same tolerance to IFN as the untreated virus. VRTK and ACV-treated VR3 both express less US11, but only the former packages less US11 in virus particles and is hyper-sensitive to IFN. These results indicate that the lower tolerance of the VRTK strain to IFN is caused by the reduced amount of US11 packaged in the virions. These results also raise questions as to why TK activity influences degree of protein expression by a mechanism other than reduction of DNA replication, and why the VRTK strain shows abortive packaging of US11 into virus particles. It is probable that the VRTK strain possesses mutations other than that in the TK gene. Indeed, ectopic viral TK expression does not restore US11 expression and ectopic TK and US11 expression does not improve the susceptibility of the VRTK strain to IFN (Fig. 7). For example, the ICP22 mutant has a reduced level of US11 expression, a smaller amount of US11 in the virion and hyper-sensitivity to IFN [7, 55]. Further studies on the expression of other true late genes, particularly gC and gE, which are involved in escape from the host defense response and in the mechanisms for regulation of tegument protein packaging, are required in TK mutant-infected cells.

In this study, we showed that the amount of structural protein US11 in the virion controls virus susceptibility to type 1 IFN. Similarly, ICP22-deletion changes the ratio of structural proteins and confers complement-labile characteristics on the virions by lowering the copy number of glycoprotein C therein [55]. Copy numbers of capsid proteins in the virion have been critically defined as 960 copies of VP5 incorporated into one virion [56, 57]. However, the amount of tegument proteins and (glyco)proteins inserted into the envelope per virion is uncertain. Differences in the degree of expression, localization and post-translational modification, such as phosphorylation, of viral proteins in infected cells may influence the balance between virion components, resulting in different virion phenotypes. To better understand the pathogenesis of HSV-1 in various diseases, the relationships between the characteristics of the progeny virus and their components replicated in various tissues should be studied in the future.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES

The authors thank the members of the Department of Microbiology for their valuable advice and comments in relation to this study.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. 1 MATERIALS AND METHODS
  4. 2 RESULTS
  5. 3 DISCUSSION
  6. ACKNOWLEDGMENTS
  7. 4 DISCLOSURE
  8. REFERENCES
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