1 CNRS, UMR-5160, EFS, 240 avenue Emile Jeanbrau, 34094 Montpellier Cedex 5, France
2 Institut de Genetique Humaine, CNRS, UPR-1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France
3 Laboratoire d'Immunologie, Hôpital St-Eloi, 80 Avenue A. Fliche, 34295 Montpellier Cedex 5, France
Correspondence
Nadir Mechti
nadir.mechti{at}ibph.pharma.univ-montp1.fr
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Two enzymes in the host-mediated antiviral response, the dsRNA-dependent protein kinase R (PKR) (Gale & Katze, 1998; Meurs et al., 1990
, 1992
) and the RNase L (Stark et al., 1998
; Zhou et al., 1993
), have been principally implicated in the IFN-induced antiviral response against HIV-1 (for reviews, see Espert et al., 2003b
; Katze et al., 2002
). After binding to dsRNA, PKR phosphorylates the protein synthesis initiation factor eIF2 and the inhibitor of NF-
B (I-
B) leading to a translational shut down and specific transcription regulation, both detrimental for virus development (Clemens & Elia, 1997
; D'Acquisto & Ghosh, 2001
; Williams, 2001
). The overexpression of PKR has been shown to prevent reactivation of HIV-1 replication in latently infected cells (Benkirane et al., 1997
; Muto et al., 1999
). RNase L is a dormant cytosolic endoribonuclease activated by short oligoadenylates produced, in the presence of dsRNA, by the 2'-5' oligoadenylate synthetase following viral infection or IFN exposure (Player & Torrence, 1998
; Stark et al., 1998
). Overexpression of RNase L has been reported to impair severely HIV replication associated with acceleration of death of infected cells (Maitra & Silverman, 1998
). In addition, the IFN-induced 16 kDa inhibitory C/EBP
isoform was reported to be involved in repression of HIV-1 replication by IFN-
in THP-1 cell-derived macrophages (Honda et al., 1998
).
There is now clear evidence that the effectiveness with which the host's antiviral response can clear virus infections requires multiple and complementary antiviral pathways (Gale & Katze, 1998; Kumar & Carmichael, 1998
; Player & Torrence, 1998
; Stark et al., 1998
; Williams, 2001
). We have isolated a human IFN-induced gene that we have termed ISG20 (Gongora et al., 1997
, 2000
; Mattei et al., 1997
), which encodes a 3'
5' exonuclease with specificity for ssRNA (Nguyen et al., 2001
). We showed that stable and constitutive expression of ISG20 conferred resistance to vesicular stomatitis virus (VSV), influenza virus and encephalomyocarditis virus (EMCV) infection in HeLa cells, providing an alternative antiviral pathway against RNA genomic viruses (Espert et al., 2003a
). In this report, to investigate the potential of ISG20 for controlling HIV infection, we generated HIV-1 recombinant viruses expressing the ISG20 protein. This approach has been successfully used to analyse the effect of PKR and RNase L overexpression on HIV-1 replication (Benkirane et al., 1997
; Maitra & Silverman, 1998
). We demonstrated that ISG20 overexpression delays HIV-1 replication in both CEM cells and PBMCs. Our findings clearly demonstrate that ISG20 represents a novel antiviral pathway in the mechanism of IFN action against HIV-1.
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METHODS |
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Construction of recombinant HIV-1 proviral DNAs and virus stocks.
The human ISG20 cDNA in sense or antisense orientation and the cDNA encoding an inactive mutated ISG20 protein were inserted into the modified HIV-1 proviral pNL4-3nef DNA (Benkirane et al., 1997
; Huang et al., 1994
) to generate the recombinant HIV-1 DNAs, pNL4-3ISG20, pNL4-3asISG20 and pNL4-3mutISG20 (Espert et al., 2003a
; Nguyen et al., 2001
), respectively (see Fig. 2
). The different recombinant constructs were verified by sequencing. The different virus stocks were prepared from the culture supernatant of HEK293 cells 48 h after transfection with the appropriate HIV-1 recombinant proviral DNAs using Lipofectamine 2000 reagent, according to the manufacturer's instructions (Invitrogen). Virus stocks were quantified by measuring their reverse transcriptase (RT) activities.
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RT assays.
RT assays were performed as previously described (Huang et al., 1994). Each reaction contained 5 µl viral supernatant in 25 µl RT cocktail [60 mM Tris/HCl pH 8, 75 mM KCl, 5 mM MgCl2, 0·1 % NP-40, 1 mM EDTA, 5 µg poly(rA) ml1, 0·16 µg oligo(dT) ml1, [
-32P]dTTP (1 µCi ml1; 37 kBq)]. The reaction was incubated for 3 h at 37 °C. Then 10 µl of each reaction was spotted on to DEAE paper, washed three times in 2x SSC, dried and quantified using an Instant Imager (Packard).
Detection of apoptotic cells and flow cytometry analysis.
Apoptotic cells were detected by using the fluorescein isothiocyanate-labelled annexin V method (Boehringer Mannheim). Cells were washed, labelled with annexin VFluos according to the manufacturer's recommendations and analysed by flow cytometry.
Western blotting analysis.
Cells (3x106) were lysed in 50 mM Tris/HCl pH 7·5, 150 mM NaCl, 1·5 mM MgCl2, 1 mM EDTA, 0·1 % NP-40, 10 % glycerol, 1 mM PMSF, 5 mM NaF and complete mini protease inhibitor cocktail (Roche). Lysate was cleared by centrifugation at 10 000 g for 10 min. Proteins were fractionated by 12 % SDS-PAGE and transferred on to PVDF membrane. After a blocking step, the membrane was hybridized with the appropriate antibody and developed using a chemiluminescent detection system (ECL Plus; Amersham Pharmacia Biotech).
-Galactosidase activity assay.
CD4+ HeLa P4 cells were infected with each recombinant virus and -galactosidase activity was measured using the
-Galactosidase enzyme assay system with reporter lysis buffer (Promega) according the manufacturer's instructions.
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RESULTS |
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Expression of ISG20 protein from a recombinant HIV-1 severely delays virus replication in CEM cells
CEM cells were infected with the four different recombinant viruses, as described in Methods. At regular intervals after infection, virus replication was monitored by measuring the RT activity in the culture supernatant. A typical experiment, presented in Fig. 3(a), showed that the RT peaks for HIV-1NL4-3
nef, HIV-1NL4-3asISG20 and HIV-1NL4-3mutISG20 were observed at day 7 post-infection. In contrast, the RT peak for HIV-1NL4-3ISG20 was strongly delayed and occurred at day 14, demonstrating that ISG20 expression was detrimental for HIV-1 replication. Similar data were obtained in various independent experiments performed with different preparations of virus stocks (data not shown). Concurrently, ISG20 expression was monitored for each virus, at the RT peak, by Western blot analysis (Fig. 3b
). As expected on the basis of the data presented in Fig. 1
, induction of endogenous ISG20 was observed in CEM cells infected with HIV-1NL4-3
nef and HIV-1NL4-3asISG20. In addition, NefISG20 fusion protein was strongly expressed at the RT peak (day 16) in CEM cells infected with HIV-1NL4-3ISG20, suggesting that the delayed emergence of virus was not viral rescue due to inactivation of NefISG20 expression by the virus. The fact that strong expression of NefmutISG20 fusion protein was also observed in CEM cells infected with HIV-1NL4-3mutISG20 (day 7) demonstrated that the antiviral effect of ISG20 did not result from overexpression of a foreign protein by the virus and suggested that this effect was dependent on its exonuclease activity. To determine whether the viral resistance observed was dependent on the number of viral particles used for infection, an additional experiment was performed using 100-fold more virus. As expected, the replication kinetics of all viruses were faster when the cells were infected with higher virus concentrations. However, a significant delay between the RT peaks of HIV-1NL4-3ISG20 and the three other viruses was still observed (Fig. 3c
).
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It has been suggested that HIV-1 has evolved mechanisms of blocking or delaying the cellular suicide programme at least until high levels of progeny virus are produced (Selliah & Finkel, 2001; Gougeon, 2003
). We observed that HIV-1NL4-3ISG20-infected cells died less frequently at the RT peak compared with cells infected with HIV-1NL4-3
nef, HIV-1NL4-3asISG20 or HIV-1NL4-3mutISG20 (Fig. 6a
), although, they produced similar amounts of infectious particles, monitored by measuring the accumulation of RT activity in the culture supernatant of the infected cells (Fig. 6b
). The percentage of apoptotic cells was then evaluated in massively infected cells by flow cytometry analysis with the annexin V staining method. A typical experiment presented in Fig. 6(c)
showed that apoptosis was significantly reduced in HIV-1NL4-3ISG20-infected cells compared with cells infected with the other recombinant viruses, suggesting that the delayed emergence of HIV-1NL4-3ISG20 was associated with inhibition of HIV-1-induced apoptosis.
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DISCUSSION |
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We showed that ISG20 expression was rapidly and strongly induced during HIV-1 infection. These data are not surprising because it is now clearly established that HIV can induce expression of cellular genes involved in the host-mediated antiviral response, independently of IFN secretion (Baca et al., 1994; Corbeil et al., 2001
; de Veer et al., 2001
). More recently, it has been shown that HIV-1 infection or expression of Tat alone, using an adenovirus-mediated gene transfer system (adeno-Tat), induces IFN-responsive gene expression in immature human dendritic cells (Izmailova et al., 2003
). Interestingly, ISG20 induction was observed both in HIV-1 and adeno-Tat infections, in the absence of detectable IFNs in the culture supernatants, suggesting that ISG20 overexpression is mediated by Tat. As, the transcriptional regulators of IFN-inducible genes, interferon regulatory factor-7 (IRF-7) and signal transducer and activator of transcription 1 (STAT1), were also induced by adeno-Tat, the authors speculated that they could be responsible for the induction of the other set of IFN-induced genes (Izmailova et al., 2003
). Accordingly, we have previously shown that the enhancer sequence element involved in the response to these transcription factors is present in the ISG20 promoter region (Gongora et al., 2000
).
To investigate whether ISG20 could interfere with HIV-1 infection, we developed the same approach successfully used to analyse the effect of PKR and RNase L (Benkirane et al., 1997; Maitra & Silverman, 1998
). We showed that the replication kinetics of an HIV-1-derived virus expressing the ISG20 protein was strongly delayed in both T cells and PBMCs. These data demonstrated that ISG20 can function as a potent suppressor of HIV-1 replication when it is overexpressed in infected cells and represents a new factor in the IFN-mediated antiviral barrier against HIV. Interestingly, the exonuclease activity of ISG20 seems to be required for its antiviral effect, since the HIV-1NL4-3mutISG20 and control viruses replicated with similar kinetics. Unfortunately, the ISG20 antisense HIV-1-derived virus was unable to downregulate endogenous ISG20 expression and exhibited the same phenotype as HIV-1NL4-3
nef and HIV-1NL4-3mutISG20.
Viruses have developed diverse non-immune strategies to counteract host-mediated antiviral mechanisms. For example, the HIV-1 Tat protein is able to bind directly to PKR and inhibit its function (Brand et al., 1997; Cai et al., 2000
; Demarchi et al., 1999
; McMillan et al., 1995
). Similarly, the cellular RNA-binding protein, identified by its ability to cooperate with HIV-1 Tat protein for binding to the 5'-termini TAR sequence of HIV-1 RNA, blocks the anti-HIV effect of PKR (Benkirane et al., 1997
; Daher et al., 2001
; Gatignol et al., 1991
). In the same way, HIV-1 inhibits the RNase L pathway by upregulating expression of the RNase L inhibitor (Martinand et al., 1999
), also known as the multifunctional cellular protein (HP68) (Zimmerman et al., 2002
). Finally, the virally encoded Vif protein turns away antiviral defences during the late stages of virus production through proteasome-mediated degradation of the antiviral protein CEM15 (Marin et al., 2003
; Mehle et al., 2004
; Sheehy et al., 2002
, 2003
; Yu et al., 2003
). Thus, we cannot exclude the possibility that the emergence of HIV-1NL4-3ISG20 virus was due to inactivation of ISG20 antiviral function. However, we observed that HIV-1NL4-3ISG20 virus reactivation did not result in mutation or deletion in the virally integrated ISG20 cDNA contrary to what has been reported for the reactivation of HIV-1NL4-3RNaseL virus (Maitra & Silverman, 1998
). Accordingly, HIV-1NL4-3ISG20 produced in the first-round infection was able to reinfect cells with a similar delayed replication (data not shown). These data suggested that the virus is able to bypass the antiviral activity of ISG20.
On the basis of its RNase activity, it is tempting to imagine that ISG20 acts directly by specifically degrading viral RNA. However, we previously showed that ISG20 specifically degrades ssRNA but not RNA sharing a stemloop structure at the 3' end. Thus, it is rational to speculate that the genomic HIV-1 RNA with the TAR structure is not a favourable substrate for ISG20, suggesting that ISG20 probably affects the expression of viral proteins or the late steps of virus replication such as budding and release. However, we cannot exclude the possibility that ISG20 acts indirectly on virus by global or specific degradation of cellular RNAs. Indeed, widespread activation of ISG20 leading to global degradation of RNA and resulting in cell death would be detrimental for cell survival but also for virus replication. In this way, IFN has been shown to be an essential mediator of the apoptosis induced during viral infection (Tanaka et al., 1998). Thus, it is conceivable to imagine that early ISG20-mediated destruction of infected cells might greatly reduce the ability of virus to replicate and might represent a major component of the IFN-induced host antiviral response (Barber, 2001
; Gil & Esteban, 2000
; Pantaleo & Fauci, 1995
; Samuel, 2001
; Varela et al., 2001
). The fact that HIV-1 rescue is associated with the reduction of HIV-1-induced apoptosis in HIV-1NL4-3ISG20-infected cells is in accordance with this hypothesis. Because the establishment of a functional HIV-1 life cycle requires a dynamic interplay between viral and host factors, we can also postulate that ISG20 specifically affects the stability of cellular RNAs encoding cellular factors required for virus replication or transcription (Gurer et al., 2002
; Ott et al., 1996
, 2000
). In accordance with this, we have previously demonstrated that ISG20 strongly inhibited VSV replication without any apparent global alteration in the cellular RNA profile (Espert et al., 2003a
). The exact mechanism by which ISG20 expression resulted in inhibition of virus replication remains unclear. More generally, the control of RNA turnover is involved in the regulation of critical functions such as cell cycling, apoptosis and stress response, suggesting that ISG20 might be involved in these processes. The identification of cellular targets of ISG20 remains a main challenge for the comprehension of the molecular mechanism of ISG20 and IFN action.
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ACKNOWLEDGEMENTS |
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Received 31 March 2005;
accepted 5 May 2005.