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
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ABSTRACT |
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MAIN TEXT |
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It is known that varicella-zoster virus (VZV) replication is inhibited in vitro by IFN-/
and IFN-
, 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-
(Sigma) or IFN-
(PBL Biomedical Laboratories) ml1 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. 1
a and b). Dramatic reductions in the levels of VZV replication were observed at both concentrations of IFN-
, with the number of plaques reduced to 24·3 and 13·6 % of the untreated-cell levels at 100 and 1000 U ml1, respectively (Fig. 1a
). Reductions were also observed at both concentrations of IFN-
, with plaque numbers reduced to 30·5 and 28·3 % at 100 and 1000 U ml1, respectively (Fig. 1b
). Due to similar levels of inhibition at both concentrations of IFN-
, it appeared that the antiviral activity of IFN-
against VZV was limited and maximal at low concentrations (
100 U ml1). However, the antiviral activity of IFN-
was stronger at 1000 than at 100 U ml1 and was, in general, stronger than that of IFN-
, suggesting that IFN-
is more effective at inhibiting VZV replication than IFN-
.
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HSV encodes at least two proteins, 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
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 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 5x1025x107 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
).
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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- or IFN-
ml1, or with a combination of IFN-
and IFN-
each at a concentration of 50 or 500 U ml1, 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-
resulted in a 2·2-fold increase in PKR expression and a 2·3-fold increase in PKR phosphorylation. IFN-
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-
. Identical results were also obtained in IFN-treated MeWo cells infected with VZV. These results demonstrated that induction of the PKR pathway by IFN-
can explain in part the inhibition of VZV replication by IFNs. However, IFN-
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
134.5 gene if present before activation of PKR by precluding its phosphorylation and that of eIF-2
(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. 3
a). 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.
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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-, but not IFN-
, 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.
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ACKNOWLEDGEMENTS |
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Received 21 July 2004;
accepted 17 September 2004.