Interferon-induced exonuclease ISG20 exhibits an antiviral activity against human immunodeficiency virus type 1

Lucile Espert1,{dagger}, Geneviève Degols1,{dagger}, Yea-Lih Lin2, Thierry Vincent3, Monsef Benkirane2 and Nadir Mechti1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Interferons (IFNs) encode a family of secreted proteins that provide the front-line defence against viral infections. It was recently shown that ISG20, a new 3'->5' exoribonuclease member of the DEDD superfamily of exonucleases, represents a novel antiviral pathway in the mechanism of IFN action. In this report, it was shown that ISG20 expression is rapidly and strongly induced during human immunodeficiency virus type 1 (HIV-1) infection. In addition, it was demonstrated that the replication kinetics of an HIV-1-derived virus expressing the ISG20 protein (HIV-1NL4-3ISG20) was delayed in both CEM cells and peripheral blood mononuclear cells. No antiviral effect was observed in cells overexpressing a mutated ISG20 protein defective in exonuclease activity, suggesting that the antiviral effect was due to the exonuclease activity of ISG20. Paradoxically, despite the antiviral activity of ISG20 protein, virus rescue observed in HIV-1NL4-3ISG20-infected cells was not due to mutation or partial deletion of the ISG20 transgene, suggesting that the virus was able to counteract the cellular defences. In addition, HIV-1-induced apoptosis was significantly reduced in HIV-1NL4-3ISG20-infected cells suggesting that emergence of HIV-1NL4-3ISG20 was associated with the inhibition of HIV-1-induced apoptosis. Altogether, these data reflect the ineffectiveness of virus replication in cells overexpressing ISG20 and demonstrate that ISG20 represents a new factor in the IFN-mediated antiviral barrier against HIV-1.

{dagger}These authors contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Interferons (IFNs) are a family of multifunctional secreted proteins characterized by their abilities to interfere with virus infection and replication (Espert et al., 2003b; Player & Torrence, 1998; Samuel, 2001). In particular, IFNs display a potent antiviral effect on primary and immortalized human immunodeficiency virus type 1 (HIV-1) permissive human T cells and monocytes/macrophages (Karpov, 2001; Pitha, 1991). IFNs act by interfering with the different steps of the virus life cycle, both in acute and chronic HIV-1 infections (Karpov, 2001; Pitha, 1994). For instance, during acute infection, IFNs inhibit virus cell penetration by downregulating both the CD4 receptor and the CXCR4 co-receptor of HIV-1 in peripheral blood mononuclear cells (PBMCs) and in monocytes (Dhawan et al., 1995; Shirazi & Pitha, 1998). In de novo HIV infection of monocytes, IFNs have been reported to interrupt early events in virus replication prior to provirus DNA integration (Gendelman et al., 1990b; Meylan et al., 1993; Shirazi & Pitha, 1992, 1993). In contrast, in chronically infected cells, IFN antiviral action seems to be mostly due to specific RNA and protein synthesis inhibition, as well as the inhibition of later stages of the HIV replication cycle leading to defective assembly, budding and release of virions (Coccia et al., 1994; Gendelman et al., 1990a; Hansen et al., 1992; Poli et al., 1989).

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-{kappa}B (I-{kappa}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{beta} isoform was reported to be involved in repression of HIV-1 replication by IFN-{beta} 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell cultures and antibodies.
Human T-lymphoblastoid CEM cells were cultured in RPMI 1640 supplemented with 10 % fetal bovine serum (FBS) (PAA Laboratories). Human embryonic kidney HEK293 and CD4+ HeLa P4 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10 % FBS. PBMCs were cultured in RPMI 1640 with 10 % FBS containing 10 U interleukin 2 (IL2) ml–1. Human {alpha}2bIFN (Hu-{alpha}2bIFN) was purchased from Schering-Plough. Polyclonal rabbit antiserum against ISG20 was obtained by immunization of rabbit with recombinant purified protein. The human antiserum against HIV-1 and the HIV-1 p24 rabbit antiserum were obtained from the NIH AIDS Research & Reference Reagent Program.

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 pNL4-3{Delta}nef 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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Fig. 2. Structure and analysis of the various recombinant viruses. (a) Schematic representation of the different HIV-1-derived proviral DNA constructs. The human ISG20 cDNA in sense (pNL4-3ISG20) or antisense (pNL4-3asISG20) orientation and cDNA encoding an inactive mutated ISG20 protein (pNL4-3mutISG20) were inserted in-frame for expression into the nef gene of the modified HIV-1 proviral pNL4-3{Delta}nef DNA. (b) Whole cellular extracts from HEK293 cells transfected with pNL4-3{Delta}nef, pNL4-3ISG20, pNL4-3asISG20 and pNL4-3mutISG20 DNAs were prepared and analysed by Western blotting with specific antibodies for ISG20 and HIV-1 proteins. The major HIV-1 proteins Pr55Gag, IN(32) and p24, as well as the ISG20 protein, are indicated.

 
HIV-1 infections.
For massive infections, 5x106 CEM cells were co-cultured with plated HEK293 cells previously transfected with 5 µg of each modified HIV-1 proviral DNA construct using the Lipofectamine 2000 reagent. For CEM cell infections, 5x105 cells were infected with the recombinant viruses in a final volume of 130 µl and incubated at 37 °C. After 1 h, the cells were collected by centrifugation to remove the viruses and then resuspended in 1 ml culture medium on six-well plates. For PBMC infections, cells were activated for 24 h in medium containing 10 % FBS, 10 U IL2 ml–1 and 5 µg phytohaemagglutinin (PHA) ml–1. Then, the cells were washed twice in fresh medium and resuspended to a concentration of 2x106 cells ml–1 in RPMI 1640 containing 10 % FBS and 8 µg polybrene ml–1 and incubated overnight at 37 °C in the presence of virus. The cells were then washed twice with PBS to remove the viruses and resuspended in RPMI 1640 with 10 % FBS and IL2.

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) ml–1, 0·16 µg oligo(dT) ml–1, [{alpha}-32P]dTTP (1 µCi ml–1; 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 V–Fluos 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).

{beta}-Galactosidase activity assay.
CD4+ HeLa P4 cells were infected with each recombinant virus and {beta}-galactosidase activity was measured using the {beta}-Galactosidase enzyme assay system with reporter lysis buffer (Promega) according the manufacturer's instructions.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
ISG20 protein expression is induced during HIV-1 infection of CEM cells
Host-cell factors are required for both virus replication and the establishment of an efficient antiviral response. In this way, HIV-1 infections activate the transcription of a large number of cellular genes (Corbeil et al., 2001; van 't Wout et al., 2003). Activation occurs either directly through activation of cellular transcription factors or indirectly through prior production of type I IFN (Bolt et al., 2002; Chang & Laimins, 2001; Cheung et al., 2002; Korth & Katze, 2002; Simmen et al., 2001; van 't Wout et al., 2003). To analyse the effect of HIV-1 infection on ISG20 gene expression, the human T-lymphoblastoid cell line CEM was subjected to a massive HIV-1 infection. To this aim, CEM cells were incubated in the presence of human embryonic kidney HEK293 cells transfected with the HIV-1 proviral {Delta}nef DNA (pNL4-3{Delta}nef) to generate HIV-1NL4-3{Delta}nef virus, as described in Methods. This HIV-1-derived recombinant virus has previously been shown to have similar in vitro infectivity to the HIV-1NL4-3 wild-type virus (Benkirane et al., 1997; Huang et al., 1994; Maitra & Silverman, 1998). At various times after infection, CEM cells were collected and protein extracts prepared and analysed by immunoblotting with a specific rabbit anti-ISG20 antibody. The results, presented in Fig. 1, showed a strong induction of ISG20 protein after the onset of HIV-1 infection. The level of ISG20 expression was maximal at 16 h post-infection and remained constant up to 24 h.



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Fig. 1. ISG20 expression is induced in CEM cells during HIV infection. CEM cells were co-cultured with HEK293 cells previously transfected with pNL4-3{Delta}nef proviral DNA to ensure a substantial infection. At various times after infection, CEM cells were collected and protein extracts prepared and analysed by immunoblotting with a specific rabbit polyclonal antibody directed against the ISG20 recombinant protein. The sizes (kDa) of the molecular mass markers and the position of the ISG20 protein are indicated. Expression of {alpha}-tubulin was used as an invariant control.

 
Characterization of HIV-1-derived virus expressing ISG20 protein
We have recently shown that ISG20 is an active player in the antiviral response of IFNs against various RNA viruses (Espert et al., 2003a). As it was induced during HIV-1 infection, we decided to determine the effect of its overexpression on HIV-1 replication. To this end, we subcloned human ISG20 cDNA into the modified HIV-1 proviral pNL4-3{Delta}nef DNA (Fig. 2a). To synthesize ISG20 coincidently with peak periods of viral gene expression, ISG20 cDNA was inserted in-frame for expression into the nef gene (pNL4-3ISG20). As negative controls, we generated HIV-1-derived proviral DNAs expressing either the ISG20 cDNA in an antisense orientation (pNL4-3asISG20) or an ISG20-inactive RNase mutant (pNL4-3mutISG20) as previously described (Espert et al., 2003a; Nguyen et al., 2001). The different proviral DNAs were then transfected into the HEK293 cells and the recombinant viruses produced were collected in the culture supernatant 48 h after transfection. To ascertain that the pNL4-3ISG20 and pNL4-3mutISG20 HIV-1-derived proviral constructs were able to express wild-type or mutated ISG20 protein, respectively, whole cellular extracts from transfected HEK293 cells were prepared and analysed by Western blotting with specific antibodies for ISG20 or HIV-1 antigens. The data presented in Fig. 2(b) indicated that both pNL4-3ISG20 (lane 2) and pNL4-3mutISG20 (lane 4) accurately expressed the transgene. No detectable differences in the HIV-1 protein pattern expressed by the four different proviral DNAs were observed, suggesting that the insertion of DNA into the HIV-1 nef gene did not influence viral protein expression. In addition, purified virions exhibited similar protein patterns, indicating that viral integrity was preserved for all the recombinant viruses produced (data not shown). The amount of virus obtained from the proviral DNAs was determined by measuring the level of RT activity, as described in Methods. The different virus stocks were normalized for RT activity and then used to infect CEM cells.

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{Delta}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{Delta}nef and HIV-1NL4-3asISG20. In addition, Nef–ISG20 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 Nef–ISG20 expression by the virus. The fact that strong expression of Nef–mutISG20 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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Fig. 3. ISG20 overexpression strongly delays HIV replication in CEM cells. (a) HIV-1NL4-3{Delta}nef, HIV-1NL4-3ISG20, HIV-1NL4-3asISG20 and HIV-1NL4-3mutISG20 produced from transfected HEK293 cells were normalized for RT activity and used to infect CEM cells. Virus replication was monitored at regular intervals after infection by measuring the accumulation of RT activity in the culture supernatant. (b) Protein extracts prepared from CEM cells infected with the indicated recombinant viruses were analysed for ISG20 expression at the RT peak by Western blotting with specific antibodies against ISG20. Expression of {alpha}-tubulin was used as an invariant control. The Nef-fused ISG20 recombinants and endogenous ISG20 proteins are indicated. (c) The experiments described in (a) were reproduced using 100-fold more virus.

 
Expression of ISG20 protein from a recombinant HIV-1 does not alter transcription of the long terminal repeat (LTR) promoter of HIV-1
To determine whether the integration of ISG20 affected the first steps of viral infection before viral LTR promoter expression, the HIV-1-derived viruses were used in a single-round virus infection to challenge CD4+ HeLa P4 cells, which contain an integrated {beta}-galactosidase reporter gene under the control of the LTR promoter of HIV-1 (Charneau et al., 1994). Because activation of the LTR {beta}-galactosidase gene requires HIV-1 gene expression, this method allows global analysis of the early steps of HIV-1 replication, from virus penetration to DNA integration and early gene expression. Twenty-four hours after infection, cellular extracts from infected cells were prepared and the {beta}-galactosidase activities determined, as described in Methods. As shown in Fig. 4, a similar {beta}-galactosidase activity was obtained with each of the different recombinant HIV-1 viruses tested, indicating that ISG20 did not affect LTR promoter expression.



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Fig. 4. Integration of ISG20 cDNA does not affect the early steps of HIV-1 replication. CD4+ HeLa P4 cells containing a {beta}-galactosidase reporter gene under the control of the LTR promoter of HIV-1 were infected with the indicated recombinants. At 24 h post-infection, {beta}-galactosidase activity was measured as described in Methods. Standard deviation was determined for at least three independent experiments.

 
Expression of ISG20 protein from a recombinant HIV-1 severely delays virus replication in PBMCs
To assess the physiological relevance of our observations, we next examined the efficiency of HIV-1NL4-3{Delta}nef and HIV-1NL4-3ISG20 replication in PBMCs. To this aim, PBMCs obtained from healthy donors were cultured and activated in the presence of 4 µg PHA ml–1and 10 U IL2 ml–1 for 24 h. Infections were performed as described above and virus replication was monitored by measuring RT activity in the culture supernatant. Similar to the results observed for CEM cells, the replication kinetics for HIV-1NL4-3ISG20 were strongly delayed compared with HIV-1NL4-3{Delta}nef; the RT peak occurred at day 8 for HIV-1NL4-3{Delta}nef and at day 21 for HIV-1NL4-3ISG20 (Fig. 5). These data strengthened our findings on the antiviral activity of ISG20 against HIV-1.



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Fig. 5. ISG20 protein expression delays HIV-1 infection of PBMCs. PBMCs from healthy donors were infected with HIV-1NL4-3{Delta}nef or HIV-1NL4-3ISG20 recombinant virus, as described in Methods. RT activity from the supernatant was measured at regular intervals after infection.

 
Expression of ISG20 protein from a recombinant HIV-1 results in inhibition of HIV-induced apoptosis of CEM cells
Strategies used by HIV-1 to escape cellular antiviral processes include rapid mutations and gene deletions. In particular, the negative selective pressure imposed by a transgene present in the viral genome frequently results in mutation or partial deletion of the transgene. To determine whether the viral rescue resulted from alteration of the nef–ISG20 transgene by the virus, viral recombinant ISG20 gene was PCR amplified and sequenced. Interestingly, neither deletion nor mutation was observed (data not shown), indicating that ISG20 protein was not altered, contrary to what has been described for RNase L (Maitra & Silverman, 1998) and PKR (M. Benkirane, unpublished data). In accordance with the data presented in Fig. 3(b), these findings demonstrated that the emergence of virus at late times following infection with HIV-1NL4-3ISG20 was not due to mutation or partial deletion of the ISG20 transgene. Despite the antiviral activity of the ISG20 protein, its cDNA appeared to be stable in HIV-1NL4-3ISG20, suggesting that the virus is able to bypass the mechanism of ISG20 action.

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{Delta}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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Fig. 6. HIV-1-induced apoptosis is inhibited in HIV-1NL4-3ISG20-infected cells. CEM cells were infected with HIV-1NL4-3{Delta}nef ({bullet}), HIV-1NL4-3ISG20 ({blacktriangleup}), HIV-1NL4-3asISG20 ({blacksquare}) or HIV-1NL4-3mutISG20 ({circ}) recombinant virus. At regular time intervals after infection, the number of living cells was determined by the trypan blue staining method (a). Virus replication was monitored by measuring the accumulation of RT activity in the culture supernatant of infected cells (b). Concurrently, apoptotic cells were detected by flow cytometry using the annexin V staining method (c). The graph represents the percentage of apoptotic cells for each type of infected cell.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mammalian cells have developed several antiviral pathways to interfere efficiently with viral multiplication (Espert et al., 2003b; Gale & Katze, 1998; Kumar & Carmichael, 1998; Player & Torrence, 1998; Samuel, 2001; Stark et al., 1998; Williams, 2001). Among the gene products known to contribute to the antiviral activity of IFN, the 2'-5' adenylate/RNase L system and PKR are reported to be involved in cellular protection against HIV-1 virus (for review, see Karpov, 2001). Indeed, a recombinant HIV-1 provirus encoding PKR has been shown to suppress virus replication of wild-type HIV-1 provirus in co-transfection experiments (Benkirane et al., 1997; Maitra & Silverman, 1998). In the same way, a recombinant HIV-1 provirus expressing RNase L replicated less efficiently than its wild-type counterpart in Jurkat cells and peripheral blood lymphocytes (Maitra & Silverman, 1998). We have recently shown that ISG20, an IFN-induced 3'->5' exonuclease specific for ssRNA, confers cell resistance to various RNA viruses, including VSV, EMCV and influenza virus, suggesting that ISG20 represents a novel antiviral pathway in the mechanism of IFN action (Espert et al., 2003a; Gongora et al., 1997; Nguyen et al., 2001). In the present report, we analysed the potential antiviral activity of ISG20 against HIV-1.

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{Delta}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 stem–loop 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.


   ACKNOWLEDGEMENTS
 
The human antiserum against HIV-1 and the HIV-1 p24 rabbit antiserum were obtained through the NIH AIDS Research & Reference Reagent Program, Division AIDS, NIAID, NIH. This work was supported by grants from the Association pour la Recherche contre le Cancer, the Centre National de la Recherche Scientifique. L. E. was supported by fellowships from the Association pour la Recherche contre le Cancer and Sidaction-Ensemble Contre le Sida.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baca, L. M., Genis, P., Kalvakolanu, D., Sen, G., Meltzer, M. S., Zhou, A., Silverman, R. & Gendelman, H. E. (1994). Regulation of interferon-{alpha}-inducible cellular genes in human immunodeficiency virus-infected monocytes. J Leukoc Biol 55, 299–309.[Abstract/Free Full Text]

Barber, G. N. (2001). Host defence, viruses and apoptosis. Cell Death Differ 8, 113–126.[CrossRef][Medline]

Benkirane, M., Neuveut, C., Chun, R. F., Smith, S. M., Samuel, C. E., Gatignol, A. & Jeang, K. T. (1997). Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR. EMBO J 16, 611–624.[Abstract/Free Full Text]

Bolt, G., Berg, K. & Blixenkrone-Moller, M. (2002). Measles virus-induced modulation of host-cell gene expression. J Gen Virol 83, 1157–1165.[Abstract/Free Full Text]

Brand, S. R., Kobayashi, R. & Mathews, M. B. (1997). The Tat protein of human immunodeficiency virus type 1 is a substrate and inhibitor of the interferon-induced, virally activated protein kinase, PKR. J Biol Chem 272, 8388–8395.[Abstract/Free Full Text]

Cai, R., Carpick, B., Chun, R. F., Jeang, K. T. & Williams, B. R. (2000). HIV-I TAT inhibits PKR activity by both RNA-dependent and RNA-independent mechanisms. Arch Biochem Biophys 373, 361–367.[CrossRef][Medline]

Chang, Y. E. & Laimins, L. A. (2001). Interferon-inducible genes are major targets of human papillomavirus type 31: insights from microarray analysis. Dis Markers 17, 139–142.[Medline]

Charneau, P., Mirambeau, G., Roux, P., Paulous, S., Buc, H. & Clavel, F. (1994). HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol 241, 651–662.[CrossRef][Medline]

Cheung, C. Y., Poon, L. L., Lau, A. S., Luk, W., Lau, Y. L., Shortridge, K. F., Gordon, S., Guan, Y. & Peiris, J. S. (2002). Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 360, 1831–1837.[CrossRef][Medline]

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]

Coccia, E. M., Krust, B. & Hovanessian, A. G. (1994). Specific inhibition of viral protein synthesis in HIV-infected cells in response to interferon treatment. J Biol Chem 269, 23087–23094.[Abstract/Free Full Text]

Corbeil, J., Sheeter, D., Genini, D. & 12 other authors (2001). Temporal gene regulation during HIV-1 infection of human CD4+ T cells. Genome Res 11, 1198–1204.[Abstract/Free Full Text]

D'Acquisto, F. & Ghosh, S. (2001). PACT and PKR: turning on NF-{kappa}B in the absence of virus. Sci STKE 2001, RE1.

Daher, A., Longuet, M., Dorin, D. & 9 other authors (2001). Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression. J Biol Chem 276, 33899–33905.[Abstract/Free Full Text]

Demarchi, F., Gutierrez, M. I. & Giacca, M. (1999). Human immunodeficiency virus type 1 tat protein activates transcription factor NF-{kappa}B through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR. J Virol 73, 7080–7086.[Abstract/Free Full Text]

de Veer, M. J., Holko, M., Frevel, M., Walker, E., Der, S., Paranjape, J. M., Silverman, R. H. & Williams, B. R. (2001). Functional classification of interferon-stimulated genes identified using microarrays. J Leukoc Biol 69, 912–920.[Abstract/Free Full Text]

Dhawan, S., Heredia, A., Wahl, L. M., Epstein, J. S., Meltzer, M. S. & Hewlett, I. K. (1995). Interferon-{gamma}-induced downregulation of CD4 inhibits the entry of human immunodeficiency virus type-1 in primary monocytes. Pathobiology 63, 93–99.[Medline]

Espert, L., Degols, G., Gongora, C., Blondel, D., Williams, B. R., Silverman, R. H. & Mechti, N. (2003a). ISG20, a new interferon-induced RNase specific for single-stranded RNA, defines an alternative antiviral pathway against RNA genomic viruses. J Biol Chem 278, 16151–16158.[Abstract/Free Full Text]

Espert, L., Gongora, C. & Mechti, N. (2003b). Interferon: antiviral mechanisms and viral escape (in French). Bull Cancer 90, 131–141.[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]

Gatignol, A., Buckler-White, A., Berkhout, B. & Jeang, K. T. (1991). Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science 251, 1597–1600.[Medline]

Gendelman, H. E., Baca, L., Turpin, J. A., Kalter, D. C., Hansen, B. D., Orenstein, J. M., Friedman, R. M. & Meltzer, M. S. (1990a). Restriction of HIV replication in infected T cells and monocytes by interferon-{alpha}. AIDS Res Hum Retroviruses 6, 1045–1049.[Medline]

Gendelman, H. E., Baca, L. M., Turpin, J., Kalter, D. C., Hansen, B., Orenstein, J. M., Dieffenbach, C. W., Friedman, R. M. & Meltzer, M. S. (1990b). Regulation of HIV replication in infected monocytes by IFN-{alpha}. Mechanisms for viral restriction. J Immunol 145, 2669–2676.[Abstract/Free Full Text]

Gil, J. & Esteban, M. (2000). Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5, 107–114.[CrossRef][Medline]

Gongora, C., David, G., Pintard, L., Tissot, C., Hua, T. D., Dejean, A. & Mechti, N. (1997). Molecular cloning of a new interferon-induced PML nuclear body-associated protein. J Biol Chem 272, 19457–19463.[Abstract/Free Full Text]

Gongora, C., Degols, G., Espert, L., Hua, T. D. & Mechti, N. (2000). A unique ISRE, in the TATA-less human Isg20 promoter, confers IRF-1-mediated responsiveness to both interferon type I and type II. Nucleic Acids Res 28, 2333–2341.[Abstract/Free Full Text]

Gougeon, M. L. (2003). Apoptosis as an HIV strategy to escape immune attack. Nat Rev Immunol 3, 392–404.[CrossRef][Medline]

Gurer, C., Cimarelli, A. & Luban, J. (2002). Specific incorporation of heat shock protein 70 family members into primate lentiviral virions. J Virol 76, 4666–4670.[Abstract/Free Full Text]

Hansen, B. D., Nara, P. L., Maheshwari, R. K., Sidhu, G. S., Bernbaum, J. G., Hoekzema, D., Meltzer, M. S. & Gendelman, H. E. (1992). Loss of infectivity by progeny virus from alpha interferon-treated human immunodeficiency virus type 1-infected T cells is associated with defective assembly of envelope gp120. J Virol 66, 7543–7548.[Abstract]

Honda, Y., Rogers, L., Nakata, K., Zhao, B. Y., Pine, R., Nakai, Y., Kurosu, K., Rom, W. N. & Weiden, M. (1998). Type I interferon induces inhibitory 16-kD CCAAT/enhancer binding protein (C/EBP){beta}, repressing the HIV-1 long terminal repeat in macrophages: pulmonary tuberculosis alters C/EBP expression, enhancing HIV-1 replication. J Exp Med 188, 1255–1265.[Abstract/Free Full Text]

Huang, L. M., Joshi, A., Willey, R., Orenstein, J. & Jeang, K. T. (1994). Human immunodeficiency viruses regulated by alternative trans-activators: genetic evidence for a novel non-transcriptional function of Tat in virion infectivity. EMBO J 13, 2886–2896.[Abstract]

Izmailova, E., Bertley, F. M., Huang, Q., Makori, N., Miller, C. J., Young, R. A. & Aldovini, A. (2003). HIV-1 Tat reprograms immature dendritic cells to express chemoattractants for activated T cells and macrophages. Nat Med 9, 191–197.[CrossRef][Medline]

Karpov, A. V. (2001). Endogenous and exogenous interferons in HIV-infection. Eur J Med Res 6, 507–524.[Medline]

Katze, M. G., He, Y. & Gale, M., Jr (2002). Viruses and interferon: a fight for supremacy. Nat Rev Immunol 2, 675–687.[CrossRef][Medline]

Korth, M. J. & Katze, M. G. (2002). Unlocking the mysteries of virus–host interactions: does functional genomics hold the key? Ann N Y Acad Sci 975, 160–168.[Abstract/Free Full Text]

Kumar, M. & Carmichael, G. G. (1998). Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes. Microbiol Mol Biol Rev 62, 1415–1434.[Abstract/Free Full Text]

Maitra, R. K. & Silverman, R. H. (1998). Regulation of human immunodeficiency virus replication by 2',5'-oligoadenylate-dependent RNase L. J Virol 72, 1146–1152.[Abstract/Free Full Text]

Marin, M., Rose, K. M., Kozak, S. L. & Kabat, D. (2003). HIV-1 Vif protein binds the editing enzyme APOBEC3G and induces its degradation. Nat Med 9, 1398–1403.[CrossRef][Medline]

Martinand, C., Montavon, C., Salehzada, T., Silhol, M., Lebleu, B. & Bisbal, C. (1999). RNase L inhibitor is induced during human immunodeficiency virus type 1 infection and down regulates the 2-5A/RNase L pathway in human T cells. J Virol 73, 290–296.[Abstract/Free Full Text]

Mattei, M. G., Tissot, C., Gongora, C. & Mechti, N. (1997). Assignment of ISG20 encoding a new interferon-induced PML nuclear body-associated protein, to chromosome 15q26 by in situ hybridization. Cytogenet Cell Genet 79, 286–287.[Medline]

McMillan, N. A., Chun, R. F., Siderovski, D. P. & 7 other authors (1995). HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR. Virology 213, 413–424.[CrossRef][Medline]

Mehle, A., Strack, B., Ancuta, P., Zhang, C., McPike, M. & Gabuzda, D. (2004). Vif overcomes the innate antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome pathway. J Biol Chem 279, 7792–7798.[Abstract/Free Full Text]

Meurs, E., Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R. & Hovanessian, A. G. (1990). Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon. Cell 62, 379–390.[CrossRef][Medline]

Meurs, E. F., Watanabe, Y., Kadereit, S., Barber, G. N., Katze, M. G., Chong, K., Williams, B. R. & Hovanessian, A. G. (1992). Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth. J Virol 66, 5805–5814.[Medline]

Meylan, P. R., Guatelli, J. C., Munis, J. R., Richman, D. D. & Kornbluth, R. S. (1993). Mechanisms for the inhibition of HIV replication by interferons-{alpha}, -{beta}, and -{gamma} in primary human macrophages. Virology 193, 138–148.[CrossRef][Medline]

Muto, N. F., Martinand-Mari, C., Adelson, M. E. & Suhadolnik, R. J. (1999). Inhibition of replication of reactivated human immunodeficiency virus type 1 (HIV-1) in latently infected U1 cells transduced with an HIV-1 long terminal repeat-driven PKR cDNA construct. J Virol 73, 9021–9028.[Abstract/Free Full Text]

Nguyen, L. H., Espert, L., Mechti, N. & Wilson, D. M., III (2001). The human interferon and estrogen-regulated ISG20/HEM45 gene product degrades single-stranded RNA and DNA in vitro. Biochemistry 40, 7174–7179.[Medline]

Ott, D. E., Coren, L. V., Kane, B. P., Busch, L. K., Johnson, D. G., Sowder, R. C., II, Chertova, E. N., Arthur, L. O. & Henderson, L. E. (1996). Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 70, 7734–7743.[Abstract]

Ott, D. E., Coren, L. V., Johnson, D. G. & 7 other authors (2000). Actin-binding cellular proteins inside human immunodeficiency virus type 1. Virology 266, 42–51.[CrossRef][Medline]

Pantaleo, G. & Fauci, A. S. (1995). Apoptosis in HIV infection. Nat Med 1, 118–120.[CrossRef][Medline]

Pitha, P. M. (1991). Multiple effects of interferon on HIV-1 replication. J Interferon Res 11, 313–318.[Medline]

Pitha, P. M. (1994). Multiple effects of interferon on the replication of human immunodeficiency virus type 1. Antiviral Res 24, 205–219.[CrossRef][Medline]

Player, M. R. & Torrence, P. F. (1998). The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation. Pharmacol Ther 78, 55–113.[CrossRef][Medline]

Poli, G., Orenstein, J. M., Kinter, A., Folks, T. M. & Fauci, A. S. (1989). Interferon-{alpha} but not AZT suppresses HIV expression in chronically infected cell lines. Science 244, 575–577.[Medline]

Samuel, C. E. (2001). Antiviral actions of interferons. Clin Microbiol Rev 14, 778–809.[Abstract/Free Full Text]

Selliah, N. & Finkel, T. H. (2001). Biochemical mechanisms of HIV induced T cell apoptosis. Cell Death Differ 8, 127–136.[CrossRef][Medline]

Sheehy, A. M., Gaddis, N. C., Choi, J. D. & Malim, M. H. (2002). Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418, 646–650.[CrossRef][Medline]

Sheehy, A. M., Gaddis, N. C. & Malim, M. H. (2003). The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif. Nat Med 9, 1404–1407.[CrossRef][Medline]

Shirazi, Y. & Pitha, P. M. (1992). Alpha interferon inhibits early stages of the human immunodeficiency virus type 1 replication cycle. J Virol 66, 1321–1328.[Abstract]

Shirazi, Y. & Pitha, P. M. (1993). Interferon {alpha}-mediated inhibition of human immunodeficiency virus type 1 provirus synthesis in T-cells. Virology 193, 303–312.[CrossRef][Medline]

Shirazi, Y. & Pitha, P. M. (1998). Interferon downregulates CXCR4 (fusin) gene expression in peripheral blood mononuclear cells. J Hum Virol 1, 69–76.[Medline]

Simmen, K. A., Singh, J., Luukkonen, B. G., Lopper, M., Bittner, A., Miller, N. E., Jackson, M. R., Compton, T. & Fruh, K. (2001). Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B. Proc Natl Acad Sci U S A 98, 7140–7145.[Abstract/Free Full Text]

Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. & Schreiber, R. D. (1998). How cells respond to interferons. Annu Rev Biochem 67, 227–264.[CrossRef][Medline]

Tanaka, N., Sato, M., Lamphier, M. S., Nozawa, H., Oda, E., Noguchi, S., Schreiber, R. D., Tsujimoto, Y. & Taniguchi, T. (1998). Type I interferons are essential mediators of apoptotic death in virally infected cells. Genes Cells 3, 29–37.[Abstract/Free Full Text]

van 't Wout, A. B., Lehrman, G. K., Mikheeva, S. A., O'Keeffe, G. C., Katze, M. G., Bumgarner, R. E., Geiss, G. K. & Mullins, J. I. (2003). Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4+-T-cell lines. J Virol 77, 1392–1402.[CrossRef][Medline]

Varela, N., Munoz-Pinedo, C., Ruiz-Ruiz, C., Robledo, G., Pedroso, M. & Lopez-Rivas, A. (2001). Interferon-{gamma} sensitizes human myeloid leukemia cells to death receptor-mediated apoptosis by a pleiotropic mechanism. J Biol Chem 276, 17779–17787.[Abstract/Free Full Text]

Williams, B. R. (2001). Signal integration via PKR. Sci STKE 2001, RE2.[Medline]

Yu, X., Yu, Y., Liu, B., Luo, K., Kong, W., Mao, P. & Yu, X. F. (2003). Induction of APOBEC3G ubiquitination and degradation by an HIV-1 Vif-Cul5-SCF complex. Science 302, 1056–1060.[Abstract/Free Full Text]

Zhou, A., Hassel, B. A. & Silverman, R. H. (1993). Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action. Cell 72, 753–765.[CrossRef][Medline]

Zimmerman, C., Klein, K. C., Kiser, P. K., Singh, A. R., Firestein, B. L., Riba, S. C. & Lingappa, J. R. (2002). Identification of a host protein essential for assembly of immature HIV-1 capsids. Nature 415, 88–92.[CrossRef][Medline]

Received 31 March 2005; accepted 5 May 2005.