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1 Department of Virology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany
2 Department of Immunology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225 Langen, Germany
Correspondence
Gerd Sutter
sutge{at}pei.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E3L) results in high-level synthesis of intermediate RNA, but lacks viral late transcription. The viral E3 protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase)/RNase L and protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2'-5'OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate proteins, as demonstrated for vaccinia late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2
(eIF2) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and late-protein production were restored in MVA-E3L-infected mouse embryonic fibroblasts from Pkr0/0 mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes. Supplementary figures are available in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sutter & Staib, 2003; Drexler et al., 2004). The virus was found to be replication-deficient in most cells of mammalian origin (Sutter & Moss, 1992; Carroll & Moss, 1997; Blanchard et al., 1998; Drexler et al., 1998), probably because MVA lacks many of the viral gene products exploited by other orthopoxviruses to regulate virus–host interactions (Antoine et al., 1998). However, several important regulatory-gene sequences of Vaccinia virus are still conserved within the MVA genome, including vaccinia virus genes K3L and E3L (Antoine et al., 1998; Moss & Shisler, 2001; Staib et al., 2005).
The vaccinia virus E3L gene encodes the 25 kDa polypeptide E3, which is synthesized early during the viral-infection cycle (Chang et al., 1992) and harbours an amino-terminal Z-DNA-binding domain (Patterson & Samuel, 1995; Herbert et al., 1997; Kim et al., 2003, 2004; Kahmann et al., 2004), as well as a carboxyl-terminal domain with a typical double-stranded RNA (dsRNA)-binding motif (Chang & Jacobs, 1993; Chang et al., 1995; Ho & Shuman, 1996; Shors et al., 1997). The amino-terminal domain of E3 is dispensable for infection of cells in culture, but both amino- and carboxy-terminal domains of E3 are required for pathogenesis in mice (Shors et al., 1998; Brandt & Jacobs, 2001; Brandt et al., 2005). A vaccinia virus E3L-deletion mutant has been shown to be highly sensitive to the antiviral activity of alpha/beta interferon (IFN-/
) and replication-deficient in Vero and HeLa cells, but retained full replicative capacity in CEF, hamster BHK and rabbit RK13 cells (Beattie et al., 1991, 1995, 1996; Chang et al., 1995; Langland & Jacobs, 2002). By binding dsRNA, the E3 protein is thought to inhibit stimulation of protein kinase (PKR) and activation of 2'-5'-oligoadenylate synthetase (2'-5'OA synthetase), two enzymes that are activated by dsRNA (Chang et al., 1992; Chang & Jacobs, 1993; Rivas et al., 1998; Langland & Jacobs, 2004). Upon stimulation with dsRNA, 2'-5'OA synthetase polymerizes oligoadenylates with 2'–5' linkages (Kerr & Brown, 1978). These synthesized 2'-5'-oligoadenylates activate an endoribonuclease (RNase L), which is suggested to affect virus replication by cleaving cellular and viral RNA, thereby leading to a general inhibition of protein synthesis (Floyd-Smith et al., 1981; Wreschner et al., 1981; Silverman et al., 1982, 1983; Kumar et al., 1988; Díaz-Guerra et al., 1997; Shors et al., 1997; Stark et al., 1998). The IFN-inducible PKR can bind to dsRNA, resulting in enzyme activation and phosphorylation of the subunit of the eukaryotic translation initiation factor eIF2 (eIF2), with consequent global inhibition of translation (Thomis & Samuel, 1993; Clemens & Elia, 1997; Goodbourn et al., 2000).
An MVA E3L-deletion mutant (MVA-E3L) was unable to replicate in CEFs; however, it possesses full replicative capacity in mammalian BHK-21 cells (Hornemann et al., 2003). This vaccinia virus host-range phenotype in CEFs was associated with induction of apoptosis, enhanced production of chicken IFN-/ and reduced viral DNA replication and protein biosynthesis (Hornemann et al., 2003).
For regulation of vaccinia virus gene expression, a cascade-like model was suggested (Vos & Stunnenberg, 1988; Keck et al., 1990). Early transcription starts immediately upon entry of the virus, because all necessary components are present in the infectious particle. The early proteins that are newly synthesized are enzymes for DNA replication, RNA polymerase subunits, intermediate transcription factors (VITF-1 and 3 and capping enzyme) and viral host-range or immunomodulatory proteins. Activation of intermediate and late transcription requires the onset of viral DNA replication. Intermediate genes encode, for example, the vaccinia late transcription factors VLTF 1, VLTF 2 and VLTF 3, encoded by G8R, A1L and A2L, respectively (Keck et al., 1990). Late-gene products include RNA polymerase, early-gene transcription factors, poly(A) polymerase and proteins required for virion morphogenesis (reviewed by Moss, 1990; Condit & Niles, 2002; Broyles, 2003).
Previously, we have demonstrated unimpaired late-protein synthesis and complete cascade-like transcription of early, intermediate and late temporal-gene classes following MVA infection of human HeLa cells (Sutter & Moss, 1992; Ludwig et al., 2005). In contrast, we found that only early and intermediate transcripts were made abundantly in MVA-E3L-infected HeLa cells, suggesting an essential role of E3L for completion of the viral molecular life cycle in human cells. Consistent with the proposed function of the vaccinia virus E3L-encoded protein to bind and sequester dsRNA, the dsRNA-activated antiviral 2'-5'OA synthetase/RNase L system was found to be induced in MVA-E3L mutant virus-infected human HeLa cells (Ludwig et al., 2005). The cellular factor(s) responsible for the dramatic block of viral late-gene expression and the interrupted MVA-E3L molecular life cycle has not been investigated.
In this work, we addressed the question of whether, in the context of an MVA-E3L infection, enough dsRNA is formed to induce the 2'-5'OA synthetase/RNase L pathway and/or PKR. Additionally, we determined that the interruption of the MVA-E3L molecular life cycle in human cells is associated with a host cell-dependent differential activation of 2'-5'OA synthetase/RNase L and/or PKR activity. Consequently, we identified a failure in intermediate-protein synthesis as a major impediment to MVA-E3L late-gene transcription. Finally, in mouse embryonic fibroblasts (MEFs), we demonstrate that activation of the PKR pathway is sufficient for the arrest of viral infection at the level of intermediate-gene expression.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and human embryonic kidney 293T (ATCC CRL-11268) cells were grown in RPMI 1640 medium supplemented with 10 % fetal calf serum (FCS). MEFs were prepared from C57BL/6 mice (Pkr+/+) or mice devoid of functional PKR (Pkr0/0) (Yang et al., 1995) and cultured in Dulbecco's modified Eagle's medium with 10 % FCS. Vaccinia virus MVA (cloned isolate F6 at 582nd CEF passage) (Meyer et al., 1991), MVA-E3L and MVA-E3Lrev (Hornemann et al., 2003) were routinely propagated and titrated on BHK-21 cells.
Northern blot analysis.
Cells were mock-infected or infected with MVA or MVA-E3L at an m.o.i. of 5. Total RNA was isolated with TRIzol reagent (Invitrogen), following the manufacturer's instructions. Total RNA was separated by electrophoresis in 1 % agarose formaldehyde gels. Subsequently, RNA was transferred onto positively charged nylon membranes (Roche Diagnostics). Riboprobes for detection of MVA-encoded mRNAs 005R (C11R), 078R (G8R) and 047R (F17R) were synthesized by in vitro transcription using PCR products generated from viral DNA templates via primer pairs HLG5 (5'-TTATCTGATGTTGTTGTTGTTCGC-3')/HLG6 (5'-CTAATACGACTCACTATAGGGAGAGTTTAGTTCGTCGAGTGAAC-3'), HLG3 (5'-CGATAAACTGCGCCAAATTG-3')/HLG4 (5'-CTAATACGACTCACTATAGGGAGACATAATAGCCAAATGCTGATG-3') and HLG1 (5'-ATTCTCATTTTGCATCTGCTC-3')/HLG2 (5'-CTAATACGACTCACTATAGGGAGACTAGAAGCTACATTATCGCG-3'), respectively. A riboprobe specific for human 18S rRNA was synthesized by in vitro transcription using a PCR product amplified from HeLa cell DNA via primer pair HLPEI92 (5'-GCGAATGGCTCATTAAATCAG-3')/HLPEI91 (5'-CTAATACGACTCACTATAGGGAGACGCTGAGCCAGTCAGTGTAG-3'). Reverse primers contained a T7 RNA polymerase promoter-recognition sequence (underlined). Digoxigenin (DIG)-labelled riboprobes were obtained by in vitro transcription with T7 RNA polymerase (Roche Diagnostics), using PCR-generated DNA fragments as templates. In vitro RNA labelling, hybridization and signal detection were carried out according to the manufacturer's instructions (DIG RNA labelling kit and Anti-DIG detection chemicals; Roche Diagnostics), applying 68 °C for hybridization and high-stringency wash in 0·1x SSC containing 0·1 % SDS buffer.
Western blot analysis.
Cell monolayers were washed with PBS, scraped off and incubated with lysis buffer [50 mM Tris (pH 7·0), 150 mM NaCl, 0·5 % NP-40, 1 mM PMSF, 1 mM sodium vanadate, 20 mM sodium fluoride and 1 mM sodium molybdate] for 10 min on ice. The cell debris was removed by centrifugation. Lysates were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking, membranes were incubated with antibodies specific for eIF2 (Cell Signaling Technology) or eIF2 phosphorylated at Ser51 (eIF2-P) (Sigma-Aldrich) at a 1 : 1000 dilution in 5 % skimmed milk powder including 50 µM sodium vanadate overnight at 4 °C. Polyclonal antisera from rabbits, specific for vaccinia virus E3 and A1 proteins and ectromelia virus interleukin 18-binding protein (IL-18bp), were applied at 1 : 1000, 1 : 500 and 1 : 2000 dilutions, respectively. mAbs directed against the A27 envelope protein (Czerny et al., 1994) and -actin (Sigma-Aldrich) were used at 1 : 2000 and 1 : 10 000 dilutions, respectively.
Detection of dsRNA.
HeLa cells were mock-infected or infected with MVA or MVA-E3L at an m.o.i. of 5 in the absence or presence of 40 µg cytosine arabinoside (AraC) ml–1. Total RNA was isolated at indicated hours post-infection (h p.i.) with TRIzol reagent (Invitrogen), following the manufacturer's instructions. For denaturation (90 °C), reannealing (56 °C) and RNase A/T1 treatment (30 °C), 5 µg total RNA was applied per reaction, using an RPA kit (BD RiboQuant) according to the manufacturer's instructions. Following phenol/chloroform extraction and ammonium acetate precipitation, samples were separated electrophoretically and blotted as described for Northern blot analysis. To detect RNase-resistant RNA species, an MVA-specific DIG-labelled probe was synthesized by random-primed labelling with 2 µg EcoRV-digested MVA DNA as template, using random hexameric primers and a DIG-High Prime kit (Roche Diagnostics). Prehybridization and hybridization were performed at 50 °C by using EasyHyb (Roche Diagnostics). For low- and high-stringency washes, 2x SSC containing 0·1 % SDS (at room temperature) and 0·1x SSC containing 0·1 % SDS (at 50 °C) were used, respectively. Signal detection was carried out as described for Northern blot analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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following MVA infection of HeLa cells). Here, we used a previously generated revertant MVA-E3L virus containing a reinserted E3L gene copy under transcriptional control of its authentic promoter (MVA-E3rev) (Hornemann et al., 2003) to ascertain that the above-described phenotypes are solely due to the lack of E3L gene function. Upon infection of HeLa cells, we analysed the integrity of rRNA species and monitored by Northern blot for transcription of the MVA 047R late gene (encoding the vaccinia virus 11 kDa DNA-binding protein F17). In MVA-E3rev-infected cells, we were able to detect abundant late viral transcripts and fully preserved rRNA, very similar to infections with wild-type MVA, yet in sharp contrast to MVA-E3L infection (see Supplementary Fig. S1, available in JGV Online). Therefore, we concluded that the E3L gene product is sufficient to allow for late transcription and to inhibit the 2'-5'OA synthetase/RNase L pathway. In addition, we monitored for dsRNA-activated PKR, which is responsible for phosphorylation of eIF2 and represents another prime-candidate host protein described to be regulated by E3 activity (García et al., 2002; Langland & Jacobs, 2002). We determined PKR activation in association with E3L gene function in cell lysates prepared from MVA- and MVA-E3L-infected HeLa cells by Western blot using an antibody directed against the phosphorylated form of the PKR substrate eIF2. Indeed, we detected phosphorylation of eIF2 exclusively in MVA-E3L-infected HeLa cells, starting at 3 h p.i. (Fig. 1). These data show clearly that both dsRNA-stimulated host responses, i.e. the 2'-5'OA synthetase/RNase L system and PKR-dependent eIF2 phosphorylation, are activated in MVA-E3L-infected HeLa cells.
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E3L infectionE3L infection of HeLa cells allows for unimpaired initiation and prolonged activity of viral intermediate transcription (Ludwig et al., 2005). Thus, it was desirable to address the question of whether viral intermediate transcripts would be necessary or sufficient to form enough dsRNA for activation of the 2'-5'OA synthetase/RNase L and PKR pathways in the absence of the dsRNA-binding E3 protein. We therefore analysed intermediate transcripts synthesized during MVA-E3L infection for their capacity to form dsRNA and to stimulate rRNA degradation. HeLa cells were infected with MVA or MVA-E3L in the absence or presence of AraC to block viral DNA replication and, thereby, intermediate and late transcription. In the presence of AraC, DNA synthesis of MVA and residual genome replication of MVA-E3L were reduced to background levels (see Supplementary Fig. S2, available in JGV Online). As expected, transcription of the early gene 005R (encoding MVA VGF/C11R homologue) was not affected by the AraC block of DNA replication, but was rather prolonged and enhanced, as has been shown previously (Sanz & Moss, 1999) (Fig. 2a). Transcription of the intermediate gene 078R (MVA G8R homologue encoding VLTF 1) was blocked following infection with both viruses in the presence of AraC, as shown by Northern blot (Fig. 2a). Remarkably, upon abrogation of DNA synthesis and, consequently, intermediate transcription, rRNA also remained intact during MVA-E3L infection of HeLa cells. Additionally, phosphorylation of eIF2 was also inhibited in the presence of AraC (Fig. 2a). These results also suggest that intermediate transcripts can form dsRNA and have the capacity to activate the 2'-5'OA synthetase/RNase L and PKR pathways in the absence of E3L expression.
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E3L infection with the transfection of synthetic dsRNA poly(I : C) as an approach for 2'-5'OA synthetase/RNase L activation, we also tested rRNA degradation in a highly sensitive Northern blot, using a probe specific for human 18S rRNA. This assay markedly demonstrated the need for E3L gene function to prevent massive RNA degradation during MVA infection of HeLa cells. Again, we detected a substantial repression of rRNA degradation in MVA-E3L-infected cells in the presence of AraC (Fig. 2b
, lanes 3 and 9). However, the degradation of rRNA induced by transfection of synthetic dsRNA poly(I : C) was not affected by the addition of AraC to the medium, excluding a direct inhibitory effect of the AraC nucleoside on the 2'-5'OA synthetase/RNase L pathway (Fig. 2b, lanes 5 and 11).
Next, we wished to ascertain the capacity of MVA intermediate transcripts to form dsRNA species. To address this question, we quantified RNase A/T1-resistant RNA species as an indicator for intracellular dsRNA synthesized during vaccinia virus infection (Colby & Duesberg, 1969; Langland & Jacobs, 2002). In MVA-infected HeLa cells, strong signals corresponding to MVA-specific RNase-resistant RNAs were detectable at late times of infection (Fig. 3a, b). This finding is in agreement with the suggestion that dsRNA species originate predominantly from abundant late viral transcripts produced after DNA replication. The latter was confirmed by the lack of dsRNA-specific signals in samples prepared from infections in the presence of AraC as an inhibitor of DNA replication (Fig. 3a, b). Total RNA isolated from MVA-E3L-infected cells also included RNase-resistant RNA, although to a much lower amount than for MVA infection (Fig. 3a, b). These dsRNAs should be formed by intermediate transcripts, as no late transcription is initiated in the absence of E3L expression (Ludwig et al., 2005). These results suggest that intermediate transcripts, synthesized during infection of HeLa cells with MVA-E3L, form dsRNA with the potential to stimulate rRNA degradation by RNase L and phosphorylation of eIF2 by PKR.
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E3L-infected HeLa cells in other human cells, we infected HaCaT and 293T cells with MVA or MVA-E3L and monitored for degradation of rRNA. Starting at 6 h p.i. of HaCaT cells with MVA-E3L, almost-complete degradation of 28S and 18S rRNA was detectable (Fig. 4). In contrast, after infection of 293T cells with MVA-E3L, we noticed only weak RNase L activity (Fig. 4). Thus, compared with the infections of HaCaT or 293T cells, we observed an intermediate level of rRNA degradation in MVA-E3L-infected HeLa cells (Figs 2a and 4).
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E3L, respectively. However, in both cell lines, viral intermediate transcription was prolonged and no late transcripts were detectable in the absence of E3L, confirming the pattern of viral transcription observed for MVA-E3L-infected HeLa cells [Fig. 4; compare with Fig. 2a and Ludwig et al. (2005)].
Thus, in the context of obvious differences in the levels of MVA-E3L-induced RNase L activities in human HeLa, HaCaT and 293T cells, we observed a clear-cut block of the mutant virus life cycle, being arrested precisely at the level of intermediate transcription in all of these cells. Therefore, we speculated that another antiviral pathway might be involved in triggering this phenotype of infection, at least in 293T cells. As we had noticed strong phosphorylation of eIF2 in infected HeLa cells (Fig. 1), we also determined PKR activity in HaCaT and 293T cells. Indeed, following infection of HaCaT and 293T cells with MVA-E3L, we observed either moderate (HaCaT) or stronger (293T) phosphorylation of eIF2 (Fig. 5). These data suggested that infection of different human cells with MVA-E3L can cause differential activation of the 2'-5'OA synthetase/RNase L pathway or PKR, resulting in different levels of either rRNA degradation or eIF2 phosphorylation.
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E3L virusE3L-infected human cells (Figs 2a and 4), first we wished to analyse early-protein synthesis and monitored lysates prepared from MVA- and MVA-E3L-infected HeLa cells by Western blot analysis. When analysing the synthesis of the early vaccinia virus E3 protein in an initial control experiment, we detected two previously described immunoreactive species, p25 and p20 (Watson et al., 1991; Chang et al., 1992), being present at peak levels in MVA-infected cells at 6 and 9 h p.i. (Fig. 6a). As expected, no E3 protein was visualized in lysates from MVA-E3L-infected cells (Fig. 6a). To compare synthesis of a viral early protein in the presence or absence of E3L gene expression, we monitored protein levels of the MVA-encoded IL-18bp (Born et al., 2000; Smith et al., 2000). At 3 h p.i. with MVA or MVA-E3L, similar levels of IL-18bp were detectable, showing clearly that translation of viral early transcripts is independent of E3 protein synthesis. However, at later time points of infection, we noticed reduced levels of IL-18bp in MVA-E3L-infected HeLa cells (Fig. 6a).
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). MVA-E3L is still capable of transcribing intermediate genes, as shown for the mRNA encoding the MVA 078R gene (G8R homologue) (Fig. 2a), but no late mRNA is made and no late-protein biosynthesis was detectable (Ludwig et al., 2005). Therefore, it was tempting to look for the presence of late viral transcription factors in MVA-E3L-infected HeLa cells. Western blot analysis using an antiserum against the A1 protein (VLTF 2) (Keck et al., 1993) verified its production in MVA-infected HeLa cells at later times of infection (Fig. 6b). In clear contrast, the late transcription factor A1 was not detectable in the absence of E3L gene expression. Similarly, as expected for intermediate gene products, A1 synthesis was inhibited by the addition of AraC (Fig. 6b). Moreover, comparable to the infection of HeLa cells, A1 protein synthesis was also absent upon MVA-E3L infection of human HaCaT and 293T cells (Fig. 6b). These results show clearly that a block of translation of intermediate mRNA causes the failure of viral late-gene transcription in MVA-E3L-infected human cells. Interestingly, this inhibition of intermediate protein synthesis in the absence of the E3 protein was observed in all human cell lines tested, irrespective of either the RNase L or the PKR antiviral pathway being dominant (see Figs 1, 5 and 6b).
PKR activity is sufficient to block intermediate- and late-protein expression in MVA-E3L-infected MEFs
To further explore the role of PKR-mediated phosphorylation of eIF2 for interruption of the vaccinia viral life cycle, we analysed late-gene expression upon MVA-E3L infection in the presence and absence of a functional PKR. For this purpose, we infected MEFs derived from wild-type mice (Pkr+/+) or from mice devoid of functional PKR (Pkr0/0) (Yang et al., 1995) with MVA or MVA-E3L. Interestingly, we observed no signs of RNase L-mediated rRNA degradation in MVA-E3L-infected Pkr+/+ or Pkr0/0 MEFs (data not shown). To analyse the impact of PKR on protein synthesis of intermediate genes, we monitored for A1 (VLTF 2) expression by Western blot analysis. In Pkr+/+ MEFs, the late transcription factor A1 was detectable following MVA, but not MVA-E3L, infection (Fig. 7a), a finding comparable to the results obtained with different human cells (see also Fig. 6b). Interestingly, in Pkr0/0 MEFs, both MVA and MVA-E3L were able to synthesize the late transcription factor A1 (Fig. 7a). This result further supported the hypothesis that activated PKR in MVA-E3L-infected cells blocks translation of viral intermediate genes, thus limiting late-gene expression. Finally, we tested the capacity of MVA and MVA-E3L to synthesize the late vaccinia virus 14 kDa envelope protein A27 (Rodriguez et al., 1987) in the presence or absence of a functional PKR. As expected, A27 protein was found easily by Western blot analysis of lysates from MVA-infected cells, but it was not detectable in those from MVA-E3L-infected Pkr+/+ MEFs (Fig. 7b). We revealed that A27 expression levels were fully restored in MVA-E3L-infected MEFs derived from mice deficient for functional PKR (Fig. 7b). These data clearly demonstrate prevention of PKR activation as a crucial role of the MVA-encoded E3 protein to ensure expression of intermediate and, consequently, late viral proteins.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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E3L was shown to initiate intermediate transcription, but completely failed to activate late transcription and late-protein biosynthesis in infected HeLa cells (Ludwig et al., 2005). Such a distinct interruption of the virus life cycle seemed surprising for a mutation targeting a viral immune-evasion factor. In this work, we first confirmed that this particular infection phenotype is linked to the inactivated E3L gene locus, as an MVA-E3L-revertant virus regained full capacity to activate late transcription in human cells (see Supplementary Fig. S1, available in JGV Online). We assumed that the failure of MVA-E3L to initiate late transcription in human cells should be a consequence of activated cellular antiviral activities, rather than being due to a direct function of the E3 protein in the MVA molecular life cycle. Indeed, upon MVA-E3L infection of HeLa cells, we had observed RNase L-mediated degradation of rRNA (Ludwig et al., 2005), and here we show clear evidence for activation of PKR, the other dsRNA-stimulated pathway, following infection of human cells (Figs 1 and 5).
Post-translational activation of the IFN-response effectors 2'-5'OA synthetase/RNase L and PKR requires the presence of dsRNA. Yet, the source of dsRNA during vaccinia virus MVA infection still remained to be investigated. In particular, viral late mRNA species are believed to provoke formation of dsRNA molecules, due to read-through transcription by the viral RNA polymerase in the late phase of infection generating heterogeneous 3' termini (Colby et al., 1971; Boone et al., 1979; Varich et al., 1979; Cooper et al., 1981; Mahr & Roberts, 1984). Interestingly, RNA 3'-end heterogeneity was also demonstrated for intermediate transcripts encoding the vaccinia virus G8 protein (Baldick & Moss, 1993). In MVA-E3L-infected human cells, we exclusively detected early and intermediate transcripts, including RNase A/T1-resistant RNA species. These latter transcripts were recognized as being derived from viral intermediate-gene products because their identification strictly required the unimpaired onset of viral DNA replication (Fig. 3). Thus, synthesis of intermediate transcripts during MVA-E3L infection seemed sufficient for dsRNA formation and critical activation of RNase L and/or PKR.
In addition, the absence of protective E3L function offered an excellent opportunity to define more precisely the step at which activation of RNase L and/or PKR can block the virus life cycle. Both host-response pathways associated with the non-permissive MVA-E3L infection trigger consequences – degradation of 28S and 18S rRNA and the phosphorylation of eIF2 – that are linked to inhibition of the cellular translation machinery. Whilst one might expect that cessation of translation could result in a more insidious inhibition of the virus life cycle, we hypothesized that a failure of viral protein synthesis could explain the peculiarity of MVA-E3L to transcribe intermediate, but not late, viral genes. Activation of viral intermediate transcription requires viral DNA replication and can be prevented by addition of the inhibitor AraC, whilst post-replicative de novo protein biosynthesis, in contrast to the requirements for late transcription, is dispensable (Vos & Stunnenberg, 1988) (Fig. 2a). Indeed, upon MVA-E3L infection of different human cells, we found robust levels of intermediate transcription, but failed to detect intermediate proteins, as shown for the A1 protein (VLTF 2) (Figs 2a, 4 and 6b). These results suggest that a block of translation of intermediate mRNA can abruptly prevent the production of essential viral late transcription factors and, thus, cause the complete failure of viral late-gene expression in MVA-E3L-infected human cells.
Interestingly, the inhibition of intermediate protein synthesis in the absence of E3 protein was observed in all human cell lines tested, irrespective of either the RNase L or the PKR antiviral pathway being activated dominantly (Figs 1, 4 and 5). Despite barely detectable degradation of rRNA in MVA-E3L-infected 293T cells, we found no evidence for synthesis of proteins encoded by intermediate genes or late transcription. The strong phosphorylation of eIF2 in 293T cells implies that, in these host cells, PKR activity prevents translation of intermediate messages, rather than stimulation of the 2'-5'OA synthetase/RNase L pathway. In HaCaT cells, however, we observed nearly complete degradation of rRNA, indicating high levels of 2'-5'-oligoadenylates, but only weak eIF2 phosphorylation upon MVA-E3L infection (Fig. 4). Interestingly, activated RNase L can possibly degrade the intermediate viral mRNA encoding the vaccinia virus G8 protein (VLTF 1) directly, as suggested by our detection of faster-migrating G8R-specific transcripts (Fig. 4). This degradation of viral mRNA would be a first example of specific cleavage of a vaccinia virus mRNA by activated RNase L. Until now, specific cleavage of viral mRNA has rarely been demonstrated, e.g. during infection with Encephalomyocarditis virus in intact cells (Li et al., 1998) and for reovirus mRNA in a cell-free system (Baglioni et al., 1984). It seems most likely that this mRNA destruction effectively stops G8 protein synthesis, in addition to rRNA degradation. Interestingly, in MEFs derived from mice deficient for functional PKR, we found E3 to be dispensable for unimpaired expression of intermediate A1 (VLTF 2) and late envelope protein A27 (Fig. 7). These data demonstrate a pivotal role of activated PKR to inhibit MVA intermediate-, and thereby late-, protein synthesis in the absence of the dsRNA-binding protein E3. In contrast to the infection of human cells, we detected no signs of rRNA degradation in MEFs infected with MVA-E3L. Thus, inhibition of PKR alone is sufficient to restore viral protein synthesis completely, at least in MEFs. Interestingly, MEFs deficient for PKR, RNase L and Mx1 were shown to not allow for complete rescue of the replicative capacity of a vaccinia virus strain Copenhagen E3L-deletion mutant, possibly indicating the involvement of other cellular activities and/or additional viral regulatory factors (Xiang et al., 2002). In addition to E3, MVA encodes another PKR inhibitor, the viral regulatory protein K3, being suggested to act as a viral eIF2 decoy to competitively block phosphorylation of eIF2 (Beattie et al., 1991; Davies et al., 1992; Carroll et al., 1993; Jagus & Gray, 1994). Despite conservation of functional K3 protein-encoding sequences in the MVA-E3L genome, we observed eIF2 phosphorylation upon infection of different human cells. These data imply that, at least in human cells, the MVA-E3L-induced phosphorylation of eIF2 by PKR cannot be prevented fully by the virus-encoded K3 protein. The cell line-dependent impact of this viral decoy has been shown by the specific need for K3 function for permissive vaccinia virus infection of hamster BHK-21 cells (Langland & Jacobs, 2002).
Given the described function of a recently described RNA helicase (retinoic acid-inducible gene-I, RIG-I) (Yoneyama et al., 2004; Kato et al., 2005), it is tempting to speculate about activation of its downstream signalling modulators depending on the presence of the dsRNA-binding protein E3 during vaccinia virus infection, as was suggested for IRF-3 activity (Smith et al., 2001; Xiang et al., 2002). Moreover, stimulation of such innate responses has also been shown to enhance antigen-specific immune responses (Leitner et al., 2003; Schulz et al., 2005). Thus, in future experiments, the mutant virus MVA-E3L should represent a promising tool to investigate the potential impact of E3L on adaptive immune responses induced after MVA vaccination.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 October 2005;
accepted 14 January 2006.
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