Role of the protein kinase PKR in the inhibition of varicella-zoster virus replication by beta interferon and gamma interferon

Nathalie Desloges, Markus Rahaus and Manfred H. Wolff

University of Witten/Herdecke, Institute of Microbiology and Virology, Stockumer Str. 10, D-58448 Witten, Germany

Correspondence
Manfred H. Wolff
mhwolff{at}uni-wh.de


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Varicella-zoster virus (VZV) is sensitive to type I and type II interferons (IFNs), which mediate antiviral effects. In this study, it was demonstrated that IFN-{beta} and IFN-{gamma} inhibited the replication of VZV in vitro. Although IFN-{beta} was more effective than IFN-{gamma}, the level of inhibition of VZV replication achieved by the combination of both IFNs was more than additive and it was concluded that these two cytokines acted synergistically. Expression of the IFN-induced, double-stranded RNA-activated protein kinase PKR and its phosphorylation level were not modulated strongly during ongoing replication of VZV. However, in the presence of IFN-{beta}, but not IFN-{gamma}, PKR expression and its phosphorylation were increased, explaining in part the inhibition of virus replication by IFNs. The expression of herpes simplex virus Us11, a viral protein with several functions, including prevention of PKR activation, strongly increased the level of VZV replication.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
To establish infections, viruses must overcome the antiviral response that is provided by the cellular interferon (IFN) system. IFNs are secreted proteins that mediate antiviral effects, regulate cell growth and modulate the activation of immune responses (Goodbourn et al., 2000). IFNs can be classified into two distinct types: type I IFNs, which are produced in direct response to viral infection, consist of the products of the IFN-{alpha} and IFN-{beta} genes, whereas type II IFN, the product of the IFN-{gamma} gene, is synthesized in response to the recognition of infected cells by natural killer cells and T lymphocytes (Goodbourn et al., 2000).

It is known that varicella-zoster virus (VZV) replication is inhibited in vitro by IFN-{alpha}/{beta} and IFN-{gamma}, and that VZV is more sensitive than herpes simplex virus (HSV) to IFNs (Balachandra et al., 1994). To determine the sensitivity of VZV to IFN more precisely, MeWo cells grown in six-well plates were treated with 100 or 1000 U IFN-{beta} (Sigma) or IFN-{gamma} (PBL Biomedical Laboratories) ml–1 for 16 h and then infected with VZV. At 24 h post-infection (p.i.), cells were fixed and stained with haematoxylin and eosin to evaluate the number of visible plaques by light microscopy (Rahaus & Wolff, 2003). A minimum of 250 plaques was counted in an area of 2 cm2 for the control experiment. The number of plaques detected in the untreated, VZV-infected cells was set as 100 % (Fig. 1a and b). Dramatic reductions in the levels of VZV replication were observed at both concentrations of IFN-{beta}, with the number of plaques reduced to 24·3 and 13·6 % of the untreated-cell levels at 100 and 1000 U ml–1, respectively (Fig. 1a). Reductions were also observed at both concentrations of IFN-{gamma}, with plaque numbers reduced to 30·5 and 28·3 % at 100 and 1000 U ml–1, respectively (Fig. 1b). Due to similar levels of inhibition at both concentrations of IFN-{gamma}, it appeared that the antiviral activity of IFN-{gamma} against VZV was limited and maximal at low concentrations (<=100 U ml–1). However, the antiviral activity of IFN-{beta} was stronger at 1000 than at 100 U ml–1 and was, in general, stronger than that of IFN-{gamma}, suggesting that IFN-{beta} is more effective at inhibiting VZV replication than IFN-{gamma}.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. IFN-{beta} and IFN-{gamma} act synergistically to inhibit VZV replication. MeWo cells were treated with 0, 100 or 1000 U IFN-{beta} (a) or IFN-{gamma} (b) ml–1, or with a combination of IFN-{beta} and IFN-{gamma} at 50, 100 or 500 U of each cytokine ml–1 (c), prior to VZV infection. At 24 h p.i., the level of virus replication was determined by VZV plaque assay from triplicate samples. The number of viral plaques in untreated cells was set as 100 %. Results are shown as means±SD.

 
To determine whether the simultaneous activation of IFN-{beta} and IFN-{gamma} receptors could increase the inhibition of VZV replication, MeWo cells grown in six-well plates were treated with a combination of IFN-{beta} and IFN-{gamma} at 50, 100 or 500 U ml–1 for each cytokine for 16 h, followed by infection with VZV for 24 h. After staining, VZV plaque formation was examined. A dramatic decrease in the number of plaques to 14·3 % of the untreated-cell level was detected in infected cells that were treated with both IFNs at a concentration of 50 U ml–1 (Fig. 1c). By increasing the concentration of both cytokines (to 100 or 500 U ml–1), the number of plaques decreased to 10·8 and 3·5 %, respectively (Fig. 1c). The inhibition level of VZV replication that was achieved by the combination of both IFNs was thus more than additive. We concluded that the antiviral activities of these two molecules acted synergistically against VZV, as has been shown to occur for HSV-1 (Sainz & Halford, 2002).

HSV encodes at least two proteins, {gamma}134.5 and Us11, that modulate IFN-related pathways (He et al., 1997; Poppers et al., 2000). These two proteins are known to inhibit the action of PKR, an IFN-induced, double-stranded RNA (dsRNA)-activated serine/threonine protein kinase (Clemens & Elia, 1997; Clemens et al., 1993). This latent enzyme needs to be activated by autophosphorylation, which requires dimerization of two PKR molecules. Once activated, PKR phosphorylates the {alpha} subunit of translation initiation factor 2 (eIF-2), whose phosphorylation causes inhibition of translation and, therefore, inhibition of virus replication (Gale & Katze, 1998).

VZV does not possess homologues of the HSV {gamma}134.5 and Us11 proteins. In this study, we investigated the importance of PKR in IFN-induced antiviral defence against VZV by analysing the modulation of PKR expression levels during the infectious cycle. We designed a quantitative RT-PCR to clarify whether PKR transcription was affected during the viral life cycle. After reverse transcription of total RNA [isolated from VZV-infected MeWo cells at different time points as indicated in Fig. 2(b)], competitive PCR was done by using 0·7 µg cDNA and 5x102–5x107 molecules of the internal standard (initial denaturation for 4 min at 94 °C, followed by 28 cycles of 30 s at 94 °C, 30 s at 54·1 °C and 1 min at 72 °C). PCR amplification resulted in a 465 bp fragment for PKR and a 335 bp fragment for the internal standard, which represented a truncated form of the PKR target sequence (Fig. 2a). Evaluation of the amplified products was done by densitometry. The PKR transcript was found to remain at a steady-state level during the initial stages of infection (from 0 to 12 h p.i.) and had decreased slightly by 24 h p.i. (Fig. 2b).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2. Expression and phosphorylation of PKR in VZV-infected MeWo cells. (a) A PKR fragment of 465 bp was amplified and the number of molecules was compared with known amounts (5x102–5x107 molecules) of an internal standard, which was amplified as a shortened fragment of 335 bp. (b) Cells were infected with VZV and at the indicated times p.i.; PKR mRNA levels were determined by densitometrically comparing the intensities of the PKR fragments with the internal standard fragments after gel electrophoresis. All data are given as means of three independent experiments±SD. (c) MeWo cells were infected with VZV and at the indicated times p.i., infected cell protein lysates were fractionated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Blots were reacted with antiserum against PKR (PKR), PKR phosphorylated at Thr-446/451 (PKR-P) and the IE63 protein (IE63) to confirm infection of the cells. (d) Protein lysates from MeWo cells treated or not with IFN-{beta}, IFN-{gamma} or a combination of both cytokines at the indicated concentrations were fractionated by SDS-PAGE and electrotransferred onto nitrocellulose membranes. Blots were reacted with antiserum directed against PKR (PKR) or PKR phosphorylated at Thr-446/451 (PKR-P).

 
To clarify whether PKR protein accumulation was affected during the VZV replication cycle, Western blotting of VZV-infected MeWo cell protein lysates harvested at different time points was performed. At these time points, cells were treated with 0·1 µM calyculin A, an inhibitor of protein phosphatases, for 15 min and collected in 10 µM PMSF in PBS. After determination of protein concentrations by the method of Bradford (1976), samples were lysed in SDS sample buffer and fractionated by SDS-PAGE. The blot was reacted with an antibody directed against PKR (K-17, diluted 1 : 500 in 5 % Blotto; Santa Cruz), which detected the corresponding 68 kDa protein (Fig. 2c, row PKR). The protein level remained constant throughout the VZV replicative cycle, demonstrating that PKR protein expression was not modulated during infection. In contrast, total protein levels of PKR have been shown to decrease during late times of pseudorabies virus (PRV) infection (Wong & Yen, 1998).

It is known that PKR is activated by phosphorylation at Thr-446 and Thr-451 in the activation loop (Romano et al., 1998). Western blotting of VZV-infected MeWo cell protein lysates obtained at different time points was performed by using an anti-phospho-PKR (Thr-446/451) antibody (diluted 1 : 1000 in 5 % BSA; Cell Signaling Technology), which detects PKR when phosphorylated at Thr-446/451. A 74 kDa protein was detected in the mock-infected sample, indicating that a basal level of phosphorylated PKR was present in MeWo cells, as is also the case in PRV mock-infected HeLa cells (Wong & Yen, 1998). During the ongoing replicative cycle of VZV, the phosphorylation level of PKR remained constant (Fig. 2c, row PKR-P). This is in contrast to HSV infection, where Chou et al. (1995) showed that PKR phosphorylation increases in HSV-infected cells, whereas PRV infection leads to a decrease in PKR phosphorylation (Wong & Yen, 1998). Our results demonstrated that VZV develops a useful strategy to avoid the effect of active PKR in infected cells, either by repressing the activation of PKR following the production of a signal or by producing no signal, such as dsRNA, that activates PKR.

Knowing that VZV replication was sensitive to the presence of IFN and that PKR was induced by IFN, we investigated PKR expression and its phosphorylation in IFN-treated cells. Immunoblotting of MeWo cell protein lysates obtained after treating cells for 16 h with 100 or 1000 U IFN-{beta} or IFN-{gamma} ml–1, or with a combination of IFN-{beta} and IFN-{gamma} each at a concentration of 50 or 500 U ml–1, was performed. Blots were reacted with antibodies directed against PKR or phospho-PKR (Fig. 2d). Densitometric analyses of these Western blots showed that the presence of IFN-{beta} resulted in a 2·2-fold increase in PKR expression and a 2·3-fold increase in PKR phosphorylation. IFN-{gamma} had no effect on PKR expression or on its phosphorylation level. The combination of both IFNs increased the PKR expression 2·0-fold and its phosphorylation 2·3-fold as a logical consequence of the presence of IFN-{beta}. Identical results were also obtained in IFN-treated MeWo cells infected with VZV. These results demonstrated that induction of the PKR pathway by IFN-{beta} can explain in part the inhibition of VZV replication by IFNs. However, IFN-{gamma} inhibits VZV replication without the capacity to induce the PKR pathway, indicating that different defence mechanisms are involved in inhibition of virus replication.

HSV-1 Us11 is a multifunctional protein with several functions, such as the control of mRNA transport and translation, as well as regulation of PKR activation (Cassady & Gross, 2002; Duc Dodon et al., 2000; Giraud et al., 2004; Khoo et al., 2002). This protein compensates for the {gamma}134.5 gene if present before activation of PKR by precluding its phosphorylation and that of eIF-2{alpha} (Cassady et al., 1998). Two cell lines were developed by stably transfecting MeWo cells with either pcDNA3 (empty vector) or pcDNA/Us11, which was constructed by PCR-amplifying Us11 from HSV-1 strain F and inserting this fragment into pcDNA3 (Invitrogen). By performing RT-PCR analyses, we demonstrated that both selected clones were positive for the presence of the neomycin gene and that only the cell line MeWo/Us11 was positive for the presence of Us11 (Fig. 3a). These two cell lines were infected with VZV for 24 h and cells were then stained and the number of plaques evaluated. VZV replicated to similar levels in MeWo/pcDNA3-transfected cells and non-transfected MeWo cells (data not shown). The number of plaques in VZV-infected MeWo/Us11 cells increased to 565 % when compared with VZV-infected MeWo/pcDNA3 cells (Fig. 3b), indicating that Us11 seems to facilitate VZV replication. In order to determine whether the action of Us11 was due to inhibition of the PKR pathway, the presence of PKR and its phosphorylation state were analysed by Western blotting using PKR and phospho-PKR antibodies. As shown in Fig. 3(c), the levels of PKR and its phosphorylation were similar in both cell lines.



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 3. HSV-1 Us11 protein increases the level of VZV replication. MeWo cell lines stably transfected with pcDNA3 or pcDNA/Us11 were established. After selection, cell lines were tested by RT-PCR for the presence of Us11 and neomycin genes (a). Cells were infected with VZV for 24 h and then stained and the number of VZV plaques was determined (b). Cell protein lysates were also recovered and analysed by Western blotting to detect PKR and its level of phosphorylation in both cell lines (c). MeWo/Us11 cells were treated with IFN-{beta}, IFN-{gamma} or a combination of both cytokines at the indicated concentrations and protein lysates were tested by Western blotting for the presence of PKR and its level of phosphorylation (c) or viral plaques were counted at 24 h p.i. (d–f). The number of plaques in MeWo/pcDNA3 cells (b) and in untreated cells (d–f) was set as 100 %. Results are shown as means of three experiments±SD.

 
To determine whether the presence of Us11 led to protection against the antiviral effects of IFNs, MeWo/Us11 cells were treated with 100 or 1000 U IFN-{beta} or IFN-{gamma} ml–1, or with a combination of both cytokines, for 16 h and cell protein lysates were analysed by Western blotting with antibodies directed against PKR and phospho-PKR (Fig. 3c). Densitometric analyses of the Western blots demonstrated that only the presence of IFN-{beta} resulted in a 2·3-fold increase in PKR expression in MeWo/Us11 cells, similar to the data obtained with MeWo cells (Fig. 2d). Moreover, PKR phosphorylation was elevated by the presence of IFN-{beta} in MeWo/Us11 cells (1·6-fold), but clearly to a lesser extent than in MeWo cells (2·3-fold). MeWo/Us11 cells were also treated with IFNs, as indicated in Fig. 3(d–f), prior to infection with VZV. Plaques were counted after staining the cells. Dramatic reductions were observed at both concentrations of IFN-{beta}, resulting in plaque numbers decreasing to 34·3 and 20·7 % of untreated-cell levels, respectively (Fig. 3d), as well as at both concentrations of IFN-{gamma}, resulting in plaque numbers decreasing to 34·2 and 29·4 % at 100 and 1000 U ml–1, respectively (Fig. 3e). Concentrations of 50 or 500 U ml–1 of both cytokines resulted in a decrease to 13·3 and 6·5 % of the number of untreated-cell plaques, respectively (Fig. 3f). Comparing these results with the IFN-treated, VZV-infected MeWo cells (Fig. 1) indicated that the presence of HSV Us11 had no consequence on the inhibition of virus replication caused by IFN-{gamma}, as similar inhibition levels were achieved in both cell lines. However, the presence of Us11 seemed to decrease the efficiency of IFN-{beta}, as demonstrated by a lower capacity to phosphorylate PKR and a higher level of VZV replication in MeWo/Us11 cells compared with MeWo cells in the presence of IFN-{beta} or a combination of both IFNs.

In summary, this study showed that VZV is sensitive to the action of both types of IFN and that a combination of both cytokines inhibits its replication synergistically. The PKR pathway, which is induced by the presence of IFN-{beta}, but not IFN-{gamma}, can explain in part the inhibition of virus replication by IFNs. However, other antiviral mechanisms are implicated and further studies are in progress to clarify this question further.


   ACKNOWLEDGEMENTS
 
This work was supported by the Alfried Krupp von Bohlen und Halbach Stiftung, Germany. N. D. is supported by the Fonds québécois de la Recherche sur la Nature et les Technologies and the Natural Sciences and Engineering Research Council of Canada.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Balachandra, K., Thawaranantha, D., Ayuthaya, P. I., Bhumisawasdi, J., Shiraki, K. & Yamanishi, K. (1994). Effects of human alpha, beta and gamma interferons on varicella zoster virus in vitro. Southeast Asian J Trop Med Public Health 25, 252–257.[Medline]

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]

Cassady, K. A. & Gross, M. (2002). The herpes simplex virus type 1 US11 protein interacts with protein kinase R in infected cells and requires a 30-amino-acid sequence adjacent to a kinase substrate domain. J Virol 76, 2029–2035.[Abstract/Free Full Text]

Cassady, K. A., Gross, M. & Roizman, B. (1998). The herpes simplex virus US11 protein effectively compensates for the {gamma}134.5 gene if present before activation of protein kinase R by precluding its phosphorylation and that of the {alpha} subunit of eukaryotic translation initiation factor 2. J Virol 72, 8620–8626.[Abstract/Free Full Text]

Chou, J., Chen, J.-J., Gross, M. & Roizman, B. (1995). Association of a Mr 90,000 phosphoprotein with protein kinase PKR in cells exhibiting enhanced phosphorylation of translation initiation factor eIF-2{alpha} and premature shutoff of protein synthesis after infection with {gamma}134.5 mutants of herpes simplex virus 1. Proc Natl Acad Sci U S A 92, 10516–10520.[Abstract]

Clemens, M. J. & Elia, A. (1997). The double-stranded RNA-dependent protein kinase PKR: structure and function. J Interferon Cytokine Res 17, 503–524.[Medline]

Clemens, M. J., Hershey, J. W. B., Hovanessian, A. C. & 7 other authors (1993). PKR: proposed nomenclature for the RNA-dependent protein kinase induced by interferon. J Interferon Res 13, 241.[Medline]

Duc Dodon, M., Mikaélian, I., Sergeant, A. & Gazzolo, L. (2000). The herpes simplex virus 1 Us11 protein cooperates with suboptimal amounts of human immunodeficiency virus type 1 (HIV-1) Rev protein to rescue HIV-1 production. Virology 270, 43–53.[CrossRef][Medline]

Gale, M., Jr & Katze, M. G. (1998). Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase. Pharmacol Ther 78, 29–46.[CrossRef][Medline]

Giraud, S., Diaz-Latoud, C., Hacot, S., Textoris, J., Bourette, R. P. & Diaz, J.-J. (2004). US11 of herpes simplex virus type 1 interacts with HIPK2 and antagonizes HIPK2-induced cell growth arrest. J Virol 78, 2984–2993.[Abstract/Free Full Text]

Goodbourn, S., Didcock, L. & Randall, R. E. (2000). Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J Gen Virol 81, 2341–2364.[Free Full Text]

He, B., Gross, M. & Roizman, B. (1997). The {gamma}134.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1{alpha} to dephosphorylate the {alpha} subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase. Proc Natl Acad Sci U S A 94, 843–848.[Abstract/Free Full Text]

Khoo, D., Perez, C. & Mohr, I. (2002). Characterization of RNA determinants recognized by the arginine- and proline-rich region of Us11, a herpes simplex virus type 1-encoded double-stranded RNA binding protein that prevents PKR activation. J Virol 76, 11971–11981.[Abstract/Free Full Text]

Poppers, J., Mulvey, M., Khoo, D. & Mohr, I. (2000). Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein. J Virol 74, 11215–11221.[Abstract/Free Full Text]

Rahaus, M. & Wolff, M. H. (2003). Reciprocal effects of Varicella-zoster virus (VZV) and AP1: activation of jun, fos and ATF-2 after VZV infection and their importance for the regulation of viral genes. Virus Res 92, 9–21.[CrossRef][Medline]

Romano, P. R., Garcia-Barrio, M. T., Zhang, X. & 7 other authors (1998). Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2{alpha} kinases PKR and GCN2. Mol Cell Biol 18, 2282–2297.[Abstract/Free Full Text]

Sainz, B., Jr & Halford, W. P. (2002). Alpha/beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type 1. J Virol 76, 11541–11550.[Abstract/Free Full Text]

Wong, M.-L. & Yen, Y.-R. (1998). Protein synthesis in pseudorabies virus-infected cells: decreased expression of protein kinase PKR, and effects of 2-aminopurine and adenine. Virus Res 56, 199–206.[CrossRef][Medline]

Received 21 July 2004; accepted 17 September 2004.