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Journal of Virology, December 2003, p. 12392-12400, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12392-12400.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Eukaryotic Translation Initiation Factor 4GI Is Cleaved by Different Retroviral Proteases

Enrique Álvarez,* Luis Menéndez-Arias, and Luis Carrasco

Centro de Biología Molecular (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain

Received 8 May 2003/ Accepted 29 August 2003


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ABSTRACT
 
The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.


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INTRODUCTION
 
For most eukaryotic mRNAs, the initiation of protein synthesis commences with the recognition of the cap structure by several initiation factors, followed by the binding of the 40S ribosomal subunit. Then, the 40S subunit migrates along the 5' untranslated region until an AUG initiation codon is encountered in a favorable context. Binding of the ternary complex eIF2/Met-tRNA/GTP to the small ribosomal subunit leads to the formation of the initiation complex, which triggers interaction with the 60S subunit to form the 80S ribosome, proceeding to translation of the mRNA coding region.

A number of eukaryotic initiation factors participate in both mRNA binding and scanning by the small ribosomal subunit of the 5' untranslated sequence of the mRNA. The cap-binding protein eIF4E, together with the ATP-dependent RNA helicase eIF4A and the translation initiation factor eIF4G, forms the protein complex eIF4F, which binds to the 5' cap structure. This interaction is required for recruiting the 40S subunit onto the mRNA molecule. eIF4G, the larger polypeptide of eIF4F, functions as an adaptor molecule that bridges the mRNAs to ribosomes via interactions with factors eIF4E and eIF3 (18, 34). In addition, eIF4G interacts with other polypeptides involved in translation, such as eIF4A, which is required to unwind the secondary structure within the 5' leader sequence that would otherwise inhibit ribosome scanning; the poly(A)-binding protein, which promotes circularization of the mRNA and facilitates the initiation of new rounds of translation (19, 54); and Mnk-1, a mitogen-activated protein kinase responsible for the phosphorylation of eIF4E (15, 19, 21, 38, 53). Two forms of eIF4G have been identified in mammalian cells. Both proteins, called eIF4GI and eIF4GII, respectively, show 46% amino acid sequence identity and are functionally interchangeable (17). Five isoforms of eIF4GI (designated a to e) have recently been described (9). The largest is the eIF4GI-a isoform, which contains 1,600 residues. Other isoforms, such as eIF4GI-b, -c, -d, and -e, are shorter variants of 1,560, 1,513, 1,435, and 1,404 amino acids, respectively, that result from an alternate usage of multiple initiation codons.

Viral replication has evolved mechanisms to manipulate the host translational machinery to maximize efficiency and to facilitate the selective translation of viral mRNA over the endogenous host transcripts. For example, in picornaviruses, the proteolytic cleavage of eIF4G by rhinovirus or poliovirus 2A protease (2Apro) or aphtovirus protease L (Lpro) inhibits translation of capped cellular mRNAs. In contrast, translation of the uncapped picornavirus RNA, which occurs through a cap-independent mechanism that involves the internal ribosome entry site (IRES), is not affected by eIF4G hydrolysis. Cleavage of eIF4GI/II by picornaviral proteases produces two polypeptides: the N-terminal fragment contains binding sites for the poly(A)-binding protein and eIF4E, while the C-terminal moiety of eIF4G retains the binding sites for eIF3 and eIF4A.

Retroviral genomic RNAs are capped at their 5' ends and contain a 3' poly(A) tail, resembling cellular transcripts. However, retroviral RNAs have a relatively long 5' untranslated region, and the presence of stable secondary structures between the cap and the initiation codon has been proposed to strongly interfere with the scanning mechanism that operates during the initiation of translation (4). Interestingly, IRES motifs have been described in gammaretroviruses (e.g., Moloney murine leukemia virus [MoMLV] [48], Friend murine leukemia virus [5, 10], and Harvey murine sarcoma virus [6]), alpharetroviruses (e.g., avian reticuloendotheliosis virus REV-A [27] and Rous sarcoma virus [11]), deltaretroviruses (e.g., human T-cell leukemia virus type 1 [HTLV-1] [3]), and lentiviruses (e.g., simian immunodeficiency virus [SIV] [35] and human immunodeficiency virus type 1 [HIV-1]). The HIV-1 IRES has recently been identified as a nucleotide segment which overlaps the primer binding site, the dimer initiation site, the major splice donors, and major determinants of the encapsidation signal in the viral genomic RNA (7). In all cases, IRES motifs have been shown to promote cap-independent translation of retroviral genomic RNAs.

HIV-1 protease (PR) cleaves eIF4GI into three smaller fragments of 140 (N-terminal domain), 102 (central domain), and 57 (C-terminal domain) kDa (51). This hydrolysis leads to the inhibition of cap-dependent translation (36, 51). These findings, together with the presence of IRES elements in HIV-1 (8), suggested similar mechanisms of translational control of viral gene expression in HIV-1 and some picornavirus species. In the present work, we extend this concept to other members of the family Retroviridae. Thus, MoMLV, mouse mammary tumor virus (MMTV), SIV, HIV-2, and HTLV-1 PRs hydrolyze eIF4GI. Furthermore, eIF4G cleavage by PRs from MoMLV and HIV-2 inhibits cap-dependent translation without affecting IRES-driven protein synthesis.


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MATERIALS AND METHODS
 
Plasmids. The PR-coding regions of several retroviruses were cloned in the plasmid pTM1 (14). For this purpose, they were amplified by PCR using primers containing NcoI and BamHI sites. The plasmids pGEX-2T/MMTV PR, pGEX-2T/MoMLV PR, and pGEX-2T/HTLV-1 PR (24, 29, 32) were used as templates, and the PCR products obtained were digested with NcoI and BamHI and cloned in the corresponding sites of the pTM1 vector. The pTM1-SIV PR plasmid was constructed by cloning the digested AflIII/BamHI PCR product, obtained using the construct pBK28-SIV as a template, in the NcoI and BamHI sites of pTM1. The plasmid pBK28-SIV (25) was obtained from J. Mullins through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH. The construct pTM1-HIV-2 PR was obtained by inserting the PCR product obtained using a pROD10 (modified) plasmid as a template, after digestion with NcoI and BamHI, in the corresponding sites of pTM1.

The pKS-HIV-2GagPol plasmid was constructed by inserting the digested SacI/SalI PCR product, obtained using the pROD10 (modified) plasmid as a template, in the corresponding SacI and SalI sites of the pKS vector (Stratagene). The pROD10 (modified) plasmid was supplied by the Centralised Facility for AIDS Reagents, supported by the European Union program EVA/MRC and the United Kingdom Medical Research Council, and by J.-M. Bechet (Institute Pasteur, Paris, France) and A. M. L. Lever (University of Cambridge, Cambridge, United Kingdom).

The pTM1-Luc plasmid, which contains the luciferase gene, was constructed by cloning the PCR product obtained after amplification of the luciferase gene in the SpeI and XhoI sites of pTM1, using the pKS-Luc plasmid (50) as a template. The plasmids pTM1-HIV-1 PR, pTM1-2Apro, and pTM1-2C were described previously (2, 50, 51).

The construct pKS-5'NCMo-Luc contains the luciferase gene under the control of the entire 5' noncoding region of MoMLV derived from pRR88 (56). The 5' noncoding region of MoMLV was PCR amplified using pRR88 as a template, digested with NotI and SpeI, and cloned in the corresponding sites of the pKS-Luc vector. The plasmid pRR88 was a generous gift of Alan Rein (HIV Drug Resistance Program, National Cancer Institute, Frederick, Md.).

Cell culture and transfections. COS-7 cells were grown in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal calf serum and nonessential amino acids. Coupled infection-DNA transfection of COS-7 cells with recombinant vT7 virus and pTM1-derived plasmids has been described in detail (2). The cells were labeled with 50 µCi of [35S]Met-[35S]Cys (Promix; Amersham Pharmacia)/ml for 1 h and lysed in sample buffer as described previously (50). Cell extracts were loaded onto polyacrylamide gels containing sodium dodecyl sulfate (SDS). The gels were analyzed by fluorography. For this purpose, the gels were soaked in a 1 M sodium salicylate solution, dried, and exposed to X-ray film (Agfa). Alternatively, proteins were transferred to a 0.45-µm-pore-size nitrocellulose membrane (Bio-Rad) for Western blot analysis. The Western blots were developed with anti-eIF4GI antisera raised against peptides derived from the N- and C-terminal regions of human eIF4GI (2) at 1:1,000 dilution or with rabbit antisera against the N- and C-terminal regions of eIF4GII (a gift from N. Sonenberg) at 1:500 dilution. Goat anti-rabbit immunoglobulin G antibody coupled to peroxidase (Pierce) was used at 1:5,000 dilution.

Determination of transfection efficiencies. Transfection efficiencies were determined by immunofluorescence microscopy using an anti-luciferase antiserum (Promega) after the cells were transfected with the plasmid pTM1-Luc. At 8 h posttransfection, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 30 min. Then, samples were washed twice with PBS, permeabilized with 0.1% (vol/vol) Triton X-100 for 20 min, blocked with 5% bovine serum albumin in 0.1% (vol/vol) Triton X-100 for 30 min, and treated with rabbit anti-luciferase polyclonal antiserum for 1 h at room temperature. After the cells were washed twice with PBS, they were treated with goat anti-rabbit immunoglobulin G antiserum conjugated to rhodamine (Pierce). Then, samples were incubated for 1 h at room temperature, washed twice with PBS, and treated with 1 µg of DAPI (4',6'-diamidino-2-phenylindole dihydrochloride) (Sigma)/ml. Finally, the cells were washed and the coverslips were mounted on a slide with a drop of mowiol (Calbiochem) and were then observed under a Zeiss Axioskop2 plus model fluorescence microscope.

Protein purification. HIV-1 PR was purchased from I. Pichova (Centralised Facility for AIDS Reagents). Purified HIV-2 PR (39) was obtained from B. Shirley and M. Cappola (Boehringer Ingelheim Pharmaceuticals) through the NIH AIDS Research and Reference Reagent Program. MoMLV PR was expressed and purified as previously described (29). The chimeric maltose binding protein (MBP)-2Apro was purified as described previously (49). The pGEX-4GI plasmid (12) (a gift from M. Hentze) containing the sequence encoding the C-terminal half of the human eIF4GI factor (residues 682 to 1600) fused to the glutathione S-transferase (GST) gene was used to purify GST-{delta}eIF4GI(682-1600) by affinity chromatography, using a glutathione-agarose column (Sigma) as described previously (12).

PR cleavage assays. To detect eIF4G processing without the use of specific antibodies, rabbit reticulocyte lysates (Promega) were programmed with eIF4GI or eIF4GII mRNA synthesized in vitro, using as DNA templates the pcDNA3 HAeIF4G-I and pcDNA3 HAeIF4G-II plasmids (17, 20) (a gift from N. Sonenberg) linearized with XhoI and AvrII, respectively. The synthesized proteins were labeled with [35S]Met-[35S]Cys (Promix) for 90 min and then treated for another 90 min with 50 ng of recombinant MBP-2Apro, HIV-1 PR, HIV-2 PR, or MoMLV PR in a total volume of 50 µl. The lysates were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. In order to map the cleavage sites of HIV-2 PR and MoMLV PR on eIF4GI, 50 µg of recombinant GST-{delta}eIF4GI(682-1600) protein was incubated with 1 µg of recombinant HIV-2 PR or MoMLV PR in a total volume of 200 µl for 3 h at 30°C in a buffer containing 50 mM Na2HPO4, pH 6.0, 25 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol. The cleavage products were separated by SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon polyvinylidene difluoride membrane (Bio-Rad), and subjected to automated Edman degradation with an Applied Biosystems (model ABI493A) sequenator in an in-house proteomic service.

In vitro transcription and translation stimulation assays. Capped and uncapped mRNAs were synthesized in vitro using the T7 RNA polymerase kit (Promega). Plasmids used as DNA templates in these assays were linearized with an appropriate restriction enzyme: pTM1-2A was digested with SalI, pTM1 HIV-2 PR was digested with PstI, and pTM1 MoMLV PR and pKS-derived plasmids were digested with XhoI.

The effects of viral PRs on translation in intact cells were determined upon RNA cotransfection of COS-7 cells with 2 µg of mRNAs derived from the corresponding construct carrying the luciferase gene and 2 µg of capped mRNAs containing the analyzed PR-coding region under conditions described previously (52). Eight hours posttransfection, the cells were lysed in buffer containing 0.5% Triton X-100, 25 mM glycylglycine (pH 7.8), and 1 mM dithiothreitol. Luciferase activity was determined using a Monolight 2010 apparatus (Analytical Luminescence Laboratory) as described previously (52).

RNA isolation and real-time RT-PCR. Luciferase RNA levels in transfected cells were determined by real-time quantitative reverse transcription (RT)-PCR. For this purpose, total RNA was extracted from 107 cells at 8 h posttransfection using Trizol reagent (Gibco BRL) following the manufacturer's recommendations. The isolated RNA was resuspended in 5 µl of nuclease-free water, and 1 µl was subjected to analysis.

Real-time quantitative RT-PCRs were performed with the LightCycler thermal cycler system (Roche Diagnostics) using the RNA Master SYBR Green I kit (Roche Diagnostics) as described by the manufacturer. The primers LUC-forward (5'-GGC GTT AAT CAG AGA GGC GA-3') and LUC-reverse (5'-CCG CAA TAT TTG GAC TTT CCG CCC-3') were used to amplify a sequence of 500 bp to maximize the efficiency of the reaction. RT-PCRs were carried out in 20 µl of LightCycler RNA Master SYBR Green I solution containing 3 mM manganese acetate and a 1 µM concentration of each primer. RT was carried out at 61°C for 20 min. Then, PCR amplification was initiated with incubation at 95°C for 2 min, followed by 45 cycles of 95°C for 5 s, 50°C for 5 s, and 72°C for 20 s. Data analysis was done using the Roche Molecular Biochemicals LightCycler software (version 3.3). The specificity of the amplification reactions was confirmed by analyzing their corresponding melting curves.


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RESULTS
 
Retroviral PRs cleave eIF4GI and eIF4GII in transfected cells. The susceptibility of eIF4G to proteolysis mediated by retroviral PRs encoded by MoMLV, MMTV, HTLV-I, SIV, HIV-1, and HIV-2 was first tested in cell culture. For this purpose, the corresponding retroviral PR-coding regions were cloned in the pTM1 vector and expressed in COS-7 cells upon transfection using the vaccinia virus vT7 system. Protein synthesis was monitored by [35S]Met-[35S]Cys labeling for 1 h, followed by the analysis of cell lysates by polyacrylamide gel electrophoresis (Fig. 1A). In most cases, significant expression of the corresponding viral PRs was evident by gel analysis, revealing products of sizes similar to those expected for the viral PRs. In agreement with previous observations using poliovirus 2Apro and HIV-1 PR (51), we detected a significant reduction in host cell translation upon expression of all tested retroviral PRs. Inhibition of cap-dependent translation was estimated to range between 50 and 75%, depending on the analyzed PR (Fig. 1A). The efficiency of transfection in these experiments was ~70% (data not shown), as determined by the immunofluorescence of cells transfected with the pTM1-Luc plasmid, using an anti-luciferase antiserum.



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  		FIG. 1. Cleavage of eIF4GI in transfected cells. (A) COS-7 cells were transfected with the unmodified pTM1 vector or with pTM1 carrying inserts containing the PR-coding region of several retroviruses or encoding poliovirus 2Apro. The cells were labeled with [35S]Met-[35S]Cys from 15 to 16 h posttransfection. Equal amounts of protein extract were loaded in a 17% polyacrylamide gel and analyzed by autoradiography. Estimates of protein synthesis inhibition in cell extracts obtained from transfections with different constructs are indicated. The data refer to control experiments carried out with the unmodified pTM1 vector, and determinations were obtained by densitometric scanning of the protein band of ~45 kDa. (B) Detection of eIF4GI cleavage products by Western blotting using a mixture of antisera against its N- and C-terminal regions (top) or individual antisera against the N-terminal (middle) or C-terminal region of eIF4GI (bottom). c.p., cleavage fragments; Nt, N-terminal fragments of eIF4GI; Ct, C-terminal fragments of eIF4GI. The position of the intact eIF4GI is also indicated. The amount of hydrolyzed eIF4GI for each transfection experiment is indicated below the corresponding lane.

The integrity of eIF4GI and eIF4GII was assessed by Western blotting at 16 h posttransfection. Cleavage of eIF4GI was detected in COS-7 cells transfected with all retroviral PR constructs, using antibodies that recognize epitopes in the N-terminal or C-terminal region of the initiation factor (Fig. 1B). However, the cleavage pattern of eIF4GI depended on the viral PR assayed. In most cases, antibodies directed against the N-terminal region revealed the presence of three bands of ~140 kDa, similar in size to those that appeared in cells transfected with poliovirus 2Apro. An additional band of ~100 kDa was also observed with HTLV-1, which was barely detectable with MMTV, SIV, HIV-1, and HIV-2 PRs. Cleavage of eIF4GI produced a 102-kDa product with all PRs tested, as revealed by using antibodies recognizing the C-terminal region of the initiation factor. Although the eIF4GI cleavage patterns were very similar for all retroviral PRs and poliovirus 2Apro, we found additional polypeptides of 53 and 49 kDa in MMTV PR-transfected cells. These polypeptides were also observed upon transfection with plasmids containing the MoMLV PR-coding region. In this case, another band of 72 kDa was also detected. Both HIV-1 and HIV-2 PRs generated similar C-terminal proteolytic eIF4GI products. Thus, bands of ~102 and 57 kDa were revealed by specific antisera against the C-terminal region of the protein. In HIV-2 PR-transfected cells, we also found polypeptides of 53 and 49 kDa, which were similar in size to those observed in MMTV and MoMLV PR-transfected cells. The fragments described above and obtained with viral PRs were different from those observed in cells treated with the apoptotic inducer actinomycin D (data not shown), suggesting that they did not result from activation of cell caspases during apoptosis.

Unlike in the case of eIF4GI, Western blot analysis carried out with a mixture of antisera recognizing the N- and C-terminal regions of eIF4GII showed that this factor was a poor substrate for HIV PRs. On the other hand, cleavage of eIF4GII was barely detectable in cells transfected with plasmids containing the retroviral PR-coding regions of MoMLV, HTLV-1, SIV, and MMTV (data not shown).

Protein synthesis was also studied in cells transfected with a pKS-derived plasmid containing the HIV-2GagPol region. In these experiments, we observed 20% inhibition of cellular protein synthesis (Fig. 1A), while cleavage of eIF4GI was estimated to be ~5% in the same transfected cells (Fig. 1B). The cleavage pattern induced by HIV-2GagPol was very similar to that observed in HIV-2 PR-transfected cells.

Effects of HIV-2 and MoMLV PRs on translation. To examine the effects of retroviral PRs on translation in intact cells, COS cell cultures were transfected with different mRNAs. Cap-dependent translation was analyzed using a capped mRNA of the luciferase gene (cap-Luc mRNA). IRES-driven translation was assayed using two different luciferase-encoding mRNAs containing either the encephalomyocarditis virus IRES or the MoMLV leader region (5'NCMo) ahead of the luciferase-coding sequence (Fig. 2A). The 5'NCMo sequence has been reported to promote the internal initiation of translation in murine leukemia virus (MLV). The three mRNAs were cotransfected either with a capped mRNA encoding the poliovirus 2C protein whose expression does not affect protein synthesis or with capped mRNAs encoding the retroviral PR of HIV-2 or MoMLV or poliovirus 2Apro. After transfection, cell extracts were collected at 8 h posttransfection to estimate luciferase activity (Fig. 2B). A remarkable inhibition of luciferase activity was observed with all three viral PRs (>70% in all cases) in cells transfected with the cap-Luc mRNA. This effect could be a consequence of an impaired luciferase mRNA translation rate. However, luciferase mRNA levels could change from cell to cell and affect the activity measurements. Real-time RT-PCR quantitations showed that the amounts of luciferase mRNA in transfected cells were within a threefold range relative to the corresponding control experiments. For example, the number of RNA molecules per 106 cells obtained from cultures cotransfected with capped luciferase mRNAs and RNAs derived from pTM1-2A, pTM1 HIV-2 PR, and pTM1 MoMLV PR were estimated at 1.8 x 108, 5.4 x 108, and 0.9 x 108, respectively, while in control experiments this value was 2.3 x 108. To avoid the effects of these fluctuations on our estimates of translation stimulation, the luciferase activity relative to the number of RNA molecules was determined for each case and normalized to the values obtained in the corresponding control experiments (Fig. 2C). Our data showed that inhibition of cap-dependent translation in cells transfected with RNAs encoding viral PRs ranged from 27% ± 10% (MoMLV PR) to 89% ± 7% (HIV-2 PR).



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  		FIG. 2. Effects of eIF4G cleavage by HIV-2 PR and MoMLV PR on cap-dependent and IRES-driven translation. (A) Schematic diagram of the three synthetic luciferase mRNAs used to transfect COS-7 cells. (B) Luciferase activity shown by COS-7 cells cotransfected with 2 µg of capped luciferase mRNAs (Cap-Luc) or mRNAs encoding the luciferase protein under the control of the IRES element from encephalomyocarditis virus (EMC-Luc) or capped luciferase mRNAs with the 5' noncoding region from MoMLV (5'NCMo-Luc) and 2 µg of capped mRNAs encoding HIV-2 PR, MoMLV PR, poliovirus 2Apro, or poliovirus 2C protein (used here as a control). Determinations were done 8 h posttransfection. Control experiments representing 100% of the luciferase activity in each experiment were performed by cotransfecting cells with the poliovirus 2C protein capped mRNA. The effects of the PRs on luciferase activity were calculated based on those values. Standard deviations determined from three independent experiments are indicated by the error bars. (C) Normalized translation stimulation in cotransfected cells. RNA was isolated from transfected cells at 8 h posttransfection, and the amount of luciferase mRNA was determined by real-time quantitative RT-PCR as described in Materials and Methods. The translation stimulation values were normalized to the relative amount of luciferase mRNA in each experiment and calculated based on values obtained for the corresponding controls: 2.3 x 108 RNA molecules/106 cells for Cap-Luc, 1.8 x 109 RNA molecules/106 cells for EMC-Luc, and 4.2 x 108 RNA molecules/106 cells for Cap-5'NCMo-Luc.

In contrast, IRES-driven translation promoted by the encephalomyocarditis virus IRES was not inhibited when the retroviral PRs were synthesized. In fact, in experiments carried out with both retroviral PRs, the normalized translation levels were two- to threefold higher than in controls without PR, an effect similar to that with poliovirus 2Apro. In the presence of poliovirus 2Apro or retroviral PRs, luciferase synthesis directed by the 5'NCMo increased, although the effects were more pronounced with poliovirus 2Apro and MoMLV PR. These results reveal that MoMLV PR inhibits cap-dependent translation without impairing protein synthesis directed by mRNA bearing the IRES-like sequence in its genome.

In vitro cleavage of eIF4GI and eIF4GII by MoMLV and HIV-2 PRs. The proteolysis of eIF4G was further studied in cell-free systems, using purified recombinant PRs from MoMLV and HIV-2 to analyze the resulting products. The HIV-1 PR was included as a control. HeLa cell extracts were incubated for 90 min at 30°C with 50 ng of each purified PR. The cleavage patterns of eIF4GI observed using a mixture of antibodies that react with the N-terminal or the C-terminal region of the initiation factor (Fig. 3A) were similar to those found in transfection experiments (Fig. 1B). In agreement with the results obtained with other viral PRs (e.g., poliovirus 2Apro), a heterogeneous mixture of N-terminal polypeptides was observed in reactions carried out with retroviral PRs. This heterogeneity is likely a consequence of the different sizes of the eIF4GI isoforms found in eukaryotic cells (57). Several N-terminal products in the range of 110 to 140 kDa were detected with HIV-1, HIV-2, and MoMLV PRs, and these bands were equivalent to those observed in transfected cells. Smaller products that appeared in reactions carried out with HIV-1 PR could originate from further degradation of the larger ones. Using an antibody that specifically recognizes the C-terminal region of eIF4GI (Fig. 3A, right), three products (designated Ct1, Ct2, and Ct4) were detected in samples treated with HIV-2 PR. The polypeptides of 102 (Ct2) and 57 (Ct4) kDa were identical to those generated by HIV-1 PR. However, an additional product of 120 kDa (Ct1) appeared only in samples treated with HIV-2 PR. The 120-kDa band originating in HIV-2 PR-treated samples appears to be a larger product which is further hydrolyzed to the smaller polypeptides of 102 (Ct2) and 57 (Ct4) kDa. Processing of eIF4GI by the MoMLV PR produces four C-terminal fragments of ~102 (Ct2), 72 (Ct3), 53 (Ct5), and 49 (Ct6) kDa.



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  		FIG. 3. Cleavage of eIF4GI and eIF4GII by purified recombinant HIV-2 and MoMLV PRs. (A) Fifty micrograms of crude HeLa S10 extracts was incubated with 50 ng of each recombinant PR in a total volume of 20 µl for 90 min, and eIF4GI degradation products were detected by Western blotting using a mixture of antisera raised using eIF4GI peptides contained within the N-terminal and the C-terminal regions of the protein (left) or individual antisera recognizing the N-terminal (middle) or the C-terminal (right) region of eIF4GI. Nt1, Nt2, Ct1, Ct2, Ct3, Ct4, Ct5, and Ct6 are products generated after cleavage with retroviral PRs. SQ, saquinavir at 2 µM concentration; RTV, ritonavir at 20 µM concentration; -, without. (B) Detection of eIF4GII degradation products by Western blotting in crude HeLa S10 extracts, upon incubation with recombinant PRs, under the conditions described above. Western blots were obtained using a mixture of antisera against N-terminal and C-terminal regions of eIF4GII (left) or using the C-terminal antisera alone (right). Nt1, Nt2, Ct1, Ct2 and Ct3 are products generated after cleavage of eIF4GII with retroviral PRs.

Under the experimental conditions used, there is complete proteolytic processing of the intact eIF4GI by the HIV-1 and MoMLV PRs, while there is still some intact eIF4GI upon incubation with HIV-2 PR. This result indicates that the HIV-2 PR hydrolyzes the initiation factor with less efficiency than the MoMLV and the HIV-1 PRs. The specificity of these cleavages was evidenced by using HIV-1 PR inhibitors, such as saquinavir or ritonavir, which blocked cleavage of the initiation factor by both HIV-1 and MoMLV PRs (Fig. 3A).

Proteolysis of eIF4GII by retroviral PRs has been tested in vitro by using antibodies generated against polypeptides derived from the N-terminal or C-terminal regions of this factor. Two high-molecular-mass products of ~180 (N-terminal fragment 1 [Nt1]) and 170 (Ct1) kDa were found in HeLa cell extracts incubated with both HIV PRs (Fig. 3B). Cleavage products of 140 (Nt2) and 110 (Ct2) kDa appeared with HIV-1 PR. The pattern of eIF4GII cleavage found with MoMLV PR was more complex: two N-terminal products of 180 (Nt1) and 140 (Nt2) kDa were observed. In addition, two C-terminal products of 110 (Ct2) and 60 (Ct3) kDa were generated upon incubation with the MoMLV PR. More than 90% of the initial eIF4GII was cleaved after incubation with the MoMLV PR. In contrast, eIF4GII was very inefficiently proteolyzed by both HIV-1 and HIV-2 PRs. As with eIF4GI, cleavage of eIF4GII was totally inhibited in the presence of saquinavir or ritonavir.

Activity of viral PRs on radioactively labeled eIF4G. Detection of eIF4G and its cleavage products has been carried out using antibodies directed to epitopes located at positions 231 to 251 or 1194 to 1219 on eIF4GI (2) or 445 to 604 on eIF4GII (17). Therefore, eIF4G degradation products lacking those amino acid sequences may not be detectable by Western blot analysis. To overcome this limitation, eIF4G was radioactively synthesized in cell-free systems using mRNAs encoding either eIF4GI or eIF4GII. These factors, labeled with [35S]Met-[35S]Cys, were used as substrates to measure their hydrolysis by the viral PRs. Radioactive eIF4Gs were incubated with 50 ng of the corresponding PR in the absence or presence of inhibitors (e.g., 2 µM saquinavir in the case of HIV-1 PR or HIV-2 PR and 20 µM ritonavir in MoMLV PR assays). Several of the retroviral PR cleavage products of eIF4GI, shown in Fig. 4, correspond to bands which were detected by Western blotting (Fig. 3A). For all PRs analyzed, major cleavage products included two or three polypeptides of 100 to 140 kDa. An additional major product of 57 kDa was detected with HIV-1 PR.



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  		FIG. 4. Cleavage of radioactive eIF4GI and eIF4GII synthesized in vitro after incubation with recombinant HIV-2 and MoMLV PRs. mRNAs encoding eIF4GI or eIF4GII were translated in rabbit reticulocyte lysates (Promega) and labeled with [35S]Met-[35S]Cys. Then, the extracts were incubated for 90 min with 50 ng of each recombinant retroviral PR or MBP-2Apro and analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Saquinavir (SQ) was used at a final concentration of 2 µM, and ritonavir (RTV) was used at 20 µM. -, without; +, with.

In agreement with previous reports, hydrolysis of eIF4GII by poliovirus 2Apro and HIV-1 and HIV-2 PRs was much less efficient than eIF4GI degradation under the conditions used (Fig. 4, bottom). In contrast, several cleavage products of eIF4GII were detected with MoMLV PR. The generation of polypeptides of 102, 72, 53, and 49 kDa was consistent with the degradation pattern found in cell-free systems using purified recombinant PR. Cleavage of eIF4GII was inhibited by ritonavir. Taken together, our findings indicate that all retroviral PRs have the ability to cleave eIF4G, both in intact cells and in cell-free systems.

Identification of the cleavage sites of retroviral PRs on the translation factor eIF4GI. Western blot analysis using antibodies recognizing the C-terminal region of eIF4GI revealed similar degradation patterns for HIV-1 and HIV-2 PRs with major bands at 102 and 57 kDa (see the results described above). Although a 120-kDa band is also found with HIV-2 PR, it is clear that there are at least two cleavage sites in eIF4GI which are sensitive to proteolysis by both HIV PRs. In order to precisely identify the PR cleavage sites, a chimeric protein containing GST fused to residues 682 to 1600 of eIF4GI was expressed in Escherichia coli and purified. Digestion of this fusion protein [designated GST-{delta}eIF4GI(682-1600)] with HIV-2 PR gives two fragments of 102 and 57 kDa (data not shown). N-terminal sequencing of the 102-kDa polypeptide produced TVLMTE, which corresponds to a cleavage site located between positions 718 and 719 of eIF4GI (Fig. 5). In addition, LQQAVP was identified as the N-terminal sequence of the 57-kDa fragment, which indicates a processing site located between positions 1126 and 1127 of eIF4GI. These data revealed that both HIV-1 and HIV-2 PRs cleave eIF4GI around the same locations (Fig. 5B). The 120-kDa product (Ct1) was not detected, suggesting that the additional N-terminal cleavage site found with HIV-2 PR could be located within the initial 681 residues absent in the GST-{delta}eIF4GI(682-1600) construct. The presence of additional cleavage sites in the N-terminal region of eIF4G could also explain, in part, the variability observed among the degradation products rendered by HIV-1, HIV-2, and MoMLV PRs and revealed by using specific antibodies recognizing residues 231 to 251 of eIF4G (Fig. 1B and 3A).



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  		FIG. 5. Identification of HIV-2 PR and MoMLV PR cleavage sites on eIF4GI. (A) Amino acid sequences of the eIF4GI cleavage sites identified using HIV-2 and MoMLV PRs. HIV-1 PR cleavage sites (51) are also shown. (B) Diagram showing the functional domains found in eIF4GI based on the available data, including the positions of mapped cleavage sites for HIV-2 and MoMLV PRs. The putative double-stranded-RNA binding domain (RRM) and the regions involved in binding to other translation factors are shown. The positions of peptides used to obtain antibodies are also indicated. PABP, poly(A)-binding protein. The sequence numbering refers to the eIF4GI-a isoform (9).

The GST-{delta}eIF4GI(682-1600) cleavage pattern found with MoMLV PR is more complex than the one observed with HIV-1 or HIV-2 PR (data not shown). N-terminal sequencing of the 102-kDa fragment produced an ~1:1 mixture of V/M, L/T, M/E, T/D, E/I, D/K, and I/L in sequencing cycles 1 to 7. This result indicates that there are two processing sites on eIF4GI (at positions 719 to 720 and 721 to 722) which are recognized by MoMLV PR. This heterogeneous processing was similar to that previously reported for HIV-1 PR (Fig. 4) (51). The other cleavage sites inferred from the Western blot patterns were not identified due to the small amounts of recovered products. The relative conservation of the eIF4GI cleavage sites of viral PRs supports the view that this initiation factor is also a genuine substrate for the HIV-2 and MoMLV PRs.


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DISCUSSION
 
Cell damage induced by virus infection is provoked by a variety of viral products. PRs encoded by animal viruses act on viral polypeptide precursors to produce the mature proteins. In addition, viral PRs recognize cellular proteins that, upon hydrolysis, affect a number of cellular functions. This is the case for eIF4G, which is cleaved by some picornavirus-encoded PRs, leading to the inhibition of translation directed by capped cellular mRNAs. Moreover, bisection of eIF4G by picornavirus PRs enhances the translation of the virus mRNA genome that contains an IRES motif. The finding that many retroviral genomic mRNAs also contain IRES-like structures suggested the possibility that their translation does not require an intact eIF4G. Previous work showing that HIV-1 PR cleaves eIF4GI at positions 718 to 719 and 1126 to 1127 (51) and a recent report demonstrating the cleavage of eIF4GI by HIV-2 PR at positions 1126 to 1127 (37) support this hypothesis.

The results reported in the present work suggest that eIF4G cleavage occurs not only in HIV-infected cells but also in cells infected by other retroviruses. In fact, all of the tested retroviral PRs were able to cleave eIF4GI efficiently, producing N-terminal and C-terminal polypeptides of different sizes. The identification of two cleavage sites in eIF4GI, recognized by different viral PRs, has shown that the region located around residues 674 and 675 (the cleavage site for aphtovirus Lpro) to 721 and 722 (the cleavage site for HIV-1 PR) (23, 51) is highly susceptible to proteolysis. This is the only cleavage site recognized by picornaviral PRs and leads to the release of an N-terminal fragment that interacts with eIF4E, implicated in cap recognition, while the resulting C-terminal moiety is necessary to translate picornaviral mRNAs containing IRESs (26). The C-terminal domain interacts with several polypeptides that participate in mRNA binding and unwinding of secondary structures (33). Apart from processing at positions 718 and 719, HIV PRs also hydrolyze eIF4GI at positions 1126 and 1127, generating a polypeptide extending from residues 722 to 1126 that may retain some of these activities. In addition, eIF4GI residues 682 to 721 exhibit RNA-binding properties and play a critical role in ribosomal scanning (37).

It is interesting that eIF4GII exhibits differential susceptibility to cleavage by the different retroviral PRs. While eIF4GII is a poor substrate for HIV-1 (36, 51) and HIV-2 PRs, this form of the initiation factor is hydrolyzed by the PRs of MoMLV, MMTV, HTLV-1, and SIV PRs. MoMLV PR generated eIF4GII cleavage products of sizes similar to those obtained using eIF4GI as a substrate. The differential effect on eIF4GI versus eIF4GII observed with HIV-1 (36) and HIV-2 PRs raises the possibility of testing the effect on translation of each form of eIF4G, both in intact cells and in cell-free systems.

HIV-1 and HIV-2 PRs are structurally similar (for a recent review, see reference 55). Differences between the substrate specificities of the two enzymes are relatively small (45, 46), and HIV-2 PR retains susceptibility to all HIV-1 PR-specific inhibitors used in the clinical setting, including saquinavir (40), indinavir (47), and ritonavir (22). This evidence is consistent with our results indicating that eIF4GII is a poor substrate for both HIV-1 and HIV-2 PRs, while eIF4GI is cleaved at positions 718 to 719 and 1126 to 1127 by both enzymes. In both cases, saquinavir (2 µM) was an effective inhibitor of the proteolysis of eIF4GI.

Substrate specificity in retroviral PRs is governed by residues at positions P4 to P3' (where positions P1 and P1' correspond to residues flanking the cleavage site) which bind to the PR in an extended conformation (43, 55). The amino acid sequences of eIF4GI and eIF4GII around the cleavage sites recognized by both HIV PRs show significant differences (17) that could explain its resilience to hydrolysis. The differences include the deletion of one amino acid (Ala) at the P1 position in the cleavage site at residues 718 to 719, as well as the replacement of Gln by Pro at position P3' at the C-terminal cleavage site. Despite the structural similarities found between the PRs of MoMLV and HIVs (29, 31), there are significant differences between the substrate specificities of these enzymes (30, 31). Studies with oligopeptide substrates have shown that peptides with bulky hydrophobic residues at positions P4 and P2 are cleaved more efficiently by the MoMLV PR than by the HIV-1 and HIV-2 PRs, as suggested by the predicted larger size of the corresponding subsites (S4 and S2) in a model structure of MoMLV PR (30, 31). Interestingly, the N-terminal cleavage site in eIF4GI was shifted one amino acid position in reactions catalyzed by MoMLV PR compared with those carried out with HIV PRs. As a result, the P4 position in the sequence recognized by the MoMLV enzyme is occupied by isoleucine instead of lysine, which is the amino acid found at this position in the eIF4GI cleavage site recognized by HIV PRs.

Apart from eIF4G, a number of host proteins have been identified as substrates of HIV PRs (41, 44). The potential role of the cleavage of cellular proteins in the cytotoxic effects of HIV has been described by a number of authors, who observed degradation by the PR of microtubule-associated proteins, cytoskeletal proteins, and NF-{kappa}B, among others (references 13 and 55 and references therein). Although it is unclear if these cleavages contribute to the cytopathogenicity observed during HIV infection, a direct effect of the MLV PR on actin-containing stress fibers during the infection of mouse fibroblasts by MLV has been reported (28).

Several retroviruses, including HIV-1 and MoMLV, have been shown to promote translation of their gag gene products by internal ribosome entry. Cleavage of eIF4GI by HIV PRs (36, 37, 51) and subsequent inhibition of cap-dependent translation suggest that lentiviruses manipulate the host translational machinery through mechanisms which are similar to those described for picornaviruses. In this study, our proposal is further extended to other retroviruses, such as MoMLV, which contains an IRES sequence in the 5' untranslated region of the viral genome (5). As occurs with picornaviruses, the viral PR is able to inhibit cap-dependent translation through cleavage of the translation initiation factor eIF4G without affecting IRES-driven translation with mRNAs having the 5' leader of MLV.

As in the cases of other cellular proteins, the role of eIF4G cleavage in the retroviral life cycle or its implications for pathogenesis remain to be elucidated. It is generally accepted that most retroviruses do not affect cellular protein synthesis after infection. This is not the case for HIV, which blocks cellular protein synthesis more or less efficiently depending on the viral strain and cell line analyzed (1, 42). In HIV-1, the Vpr protein blocks proliferation of CD4+ T cells at the G2 cell cycle checkpoint (7, 16). At this stage, cap-dependent translation initiation is suppressed and IRES-mediated translation initiation ensures the synthesis of Gag and Gag-Pol. In this context, cleavage of eIF4GI could facilitate viral-gene expression while contributing to host protein synthesis shutoff. However, MoMLV lacks the G2/M cell cycle arrest function of Vpr and is able to replicate in proliferating cells. The presence of an IRES motif in its genomic RNA and the higher susceptibility of eIF4GI and eIF4GII to cleavage by MoMLV PR suggest that cleavage of eIF4G could have a role in the viral life cycle. Although inhibition of translation initiation during MoMLV replication has not been demonstrated and host protein synthesis shutoff has not been observed in cells persistently infected with MoMLV, this mechanism could improve the efficiency of translation of viral mRNAs over the endogenous host transcripts.


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ACKNOWLEDGMENTS
 
The financial support of grants DGICYT number PM99-0002 and CAM number 08.2/0024.2/2000 and the Institutional Grant to Centro de Biología Molecular "Severo Ochoa" from Fundación Ramón Areces are acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Centro de Biología Molecular (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain. Phone: 34-913978451. Fax: 34-913974799. E-mail: ealvarez{at}cbm.uam.es. Back


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Journal of Virology, December 2003, p. 12392-12400, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12392-12400.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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