Human Immunodeficiency Virus Protease Cleaves Poly(a) Binding Protein

The poly(A)-binding protein (PABP) is able to interact with the 3’ poly(A) tail of eukaryotic mRNA, promoting its translation. Cleavage of PABP by viral proteases encoded by several picornaviruses and caliciviruses plays a part in the abrogation of cellular protein synthesis. We report that infection of MT-2 cells with HIV-1 leads to an efficient proteolysis of PABP. Analysis of PABP integrity was carried out in BHK-21 and COS-7 cells upon individual expression of the protease from several members of the Retroviridae family, e.g. Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus (MMTV), human T-cell leukemia virus type I (HTLV-I), simian immunodeficiency virus (SIV), human immunodeficiency virus type 1 (HIV-1) and HIV2. Moreover, protease activity against PABP was tested in a HeLa cell free system. Only MMTV, HIV-1 and HIV-2 proteases were able to cleave PABP in absence of other viral proteins. Purified HIV-1 and HIV-2 proteases directly cleave PABP at positions 237, 410 and 477, separating the two first RNA recognition motifs from the C-terminal domain of PABP. These findings indicate that some retroviruses may share with picornaviruses and caliciviruses, the capacity to proteolyze PABP. INTRODUCTION The initiation of translation is a multistep process, being a major regulatory target for translational control in animal virus infected cells. In the early steps of translation the 5’ cap structure of the mRNA is recognized by eIF4F complex. eIF4F also binds to the small ribosome subunit by its interaction with eIF3 forming the 48S complex. Then, the small ribosomal subunit migrates along the 5’-untranslated region until an AUG initiation codon is encountered in a favorable context. eIF4F complex is composed of three polypeptides: the cap-binding protein eIF4E, the ATP-dependent RNA helicase eIF4A and the scaffolding protein eIF4G (Hentze, 1997). Another motif recognized by the translation machinery in the mRNA is the poly(A) tail, which is achieved by means of the poly(A) binding protein (PABP). In addition, PABP interacts with the N-terminal domain of eIF4G, promoting the circularization of the mRNA (Jacobson, 1996, Jacobson & Favreau, 1983). Moreover, eIF4G-PABP interaction may induce several changes in the translation initiation complex that increase the eIF4E affinity for the cap structure (Borman et al., 2000, Luo & Goss, 2001). In this regard, simultaneous interactions between the 5’ cap structure and the 3’ poly(A) tail of the mRNA synergistically estimulate translation, both in vitro and in vivo (Gallie, 1991, Munroe & Jacobson, 1990, Tarun et al., 1997). In human cells there are three lineages of PABP proteins, with several isoforms encoded by different genes and with different localizations and functions: cytoplasmic PABPs (PABPC group), nuclear PABP (PABPN) and X-linked PABP. Cytoplasmic PABP is a highly conserved protein that contains two domains linked by an unstructured region rich in proline and methionine residues: an N-terminal domain with four conserved RNA recognition motifs (RRMs) and a C-terminal helical domain that is not required for RNA recognition, but is essential for PABP oligomerization and for the interaction with several regulatory proteins implicated in deadenylation of the poly(A) tail and the initiation and termination of translation (Adam et al., 1986, Sachs et al., 1986). PABP interacts with translation initiation factors such as eIF4G and eIF4B (Imataka et al., 1998, Le et al., 1997, Tarun et al., 1997), with the eukaryotic release factor eRF3 (Cosson et al., 2002, Hoshino et al., 1999, Kozlov et al., 2001), and with two regulatory proteins termed as PABPinteracting proteins 1 and 2 (Paip1 and Paip2), which are implicated in regulation of translation (Craig et al., 1998, Khaleghpour et al., 2001a, Khaleghpour et al., 2001b, Roy et al., 2002). Animal viruses have evolved mechanisms to manipulate the host translational machinery in order to maximize efficient viral mRNA translation and to facilitate the selective translation of viral mRNA. For example, in picornavirus-infected cells, the proteolytic cleavage of eIF4G by rhinovirus or poliovirus 2A proteases (2A) or aphtovirus protease L inhibits translation of capped cellular mRNAs (Prevot et al., 2003). In contrast, translation of the uncapped picornavirus RNA, that occurs by a capindependent mechanism involving the internal ribosome entry site (IRES) is not affected by eIF4G hydrolysis. This mechanism is not exclusive of some picornaviruses. Thus, recent reports have illustrated that eIF4G is cleaved by the protease (PR) of several members of the family Retroviridae, including mouse mammary tumor virus (MMTV), Moloney murine leukemia virus (MoMLV), human T-cell leukemia virus type 1 (HTLV-1), simian immunodeficiency virus (SIV), human immunodeficiency virus type 1(HIV-1) and HIV-2 (Alvarez et al., 2003, Ventoso et al., 2001). The hydrolysis of eIF4G leads to the inhibition of cap-dependent translation without affecting IRES-driven protein synthesis (Alvarez et al., 2003, Ohlmann et al., 2002, Ventoso et al., 2001). PABP plays an important role in translation and it is not surprising that certain viruses have developed mechanisms to target this protein to abrogate cellular translation. Rotavirus nonstructural protein 3 (NSP3) interacts with the N-terminus region of eIF4G, replacing PABP. This interaction may lead to the shut-off of cellular protein synthesis, whereas viral mRNA translation is maintained unaffected by pseudocircularization via NSP3 interaction with the 3’UTR of viral transcripts (Piron et al., 1998). Several reports have shown that PABP is cleaved by 2A and 3C during enterovirus infection (Joachims et al., 1999, Kerekatte et al., 1999, Kuyumcu-Martinez et al., 2002). Poliovirus 2A bisects PABP, while poliovirus 3C cleaves this factor at three sites separating its C-terminal and N-terminal domains. It has been proposed that the removal of the C-terminal domain inhibits cellular translation without affecting binding of the N-terminal domain of PABP to the poly(A) tail and eIF4G (KuyumcuMartinez et al., 2004b). A similar strategy has recently been described in caliciviruses, in which the 3C-like protease (3CL), like poliovirus 3C, cleaves PABP during infection (Kuyumcu-Martinez et al., 2004a). In the present work, we present evidence that the protease from some retroviruses such as MMTV, HIV-1 and HIV-2 PRs are capable to hydrolyze PABP. MATERIALS AND METHODS Plasmids and in vitro Transcription. pT7SVHIV-1PR and pT7SV-2A were generated by inserting a PCR product digested with XbaI/BamHI in the same sites of pH3 ́2J. The subgenomic promoter cassette of pH3,2J1-HIV-1PR and pH3,2J-2A was inserted into the SV cDNA clone pT7SVwt using the ApaI and XhoI restriction sites. pT7SVwt was previously described in detail (Sanz & Carrasco, 2001). The pTM1-derived plasmids containing the PR-coding regions of several retroviruses were described in detail earlier (Alvarez et al., 2003, Ventoso et al., 2001). The pTM1-Luc plasmid, which contains the luciferase gene, and the plasmid pTM12A have also been described (Aldabe et al., 1995, Ventoso et al., 1998). The plasmid pGEX-2T-PABP1 containing the sequence encoding the human PABP1, lacking the first nine amino acids and fused to the glutathione S-transferase (GST) gene, was described earlier (a gift from Dr. Amelia Nieto (Centro Nacional de Biotecnología, CSIC, Madrid, Spain)(Burgui et al., 2003). Capped genomic SV mRNAs were synthesized in vitro using the T7 RNA polymerase kit (Promega). Plasmids used as DNA templates in these assays were linearized with XhoI. Cell Culture, virus infection and Transfections. MT-2 cells were grown in RPMI medium 1640 containing 10 % fetal calf serum. MT-2 cells were infected with HIV-1 virus (NL3.4 strain) by using a multiplicity of infection of ≈ 1 plaque-forming units per cell (Dr. Balbino Alarcón provided HIV-1 infected cells), and at three days post-infection (dpi) cells were lysed in sample buffer as described previously (Ventoso et al., 1998). BHK-21 cells were electroporated with 30 μg of recombinant SV genomic RNAs in final volume of 50 μl. Electroporated-cells were grown in DMEM (Dulbecco’s modified Eagle’s minimal essential medium) containing 10 % fetal calf serum and non-essential amino acids, and at indicated times cells were labeled with 50 μCi/ml of [S]Met/[S]Cys (Promix, Amersham Pharmacia) for 1 h and lysed in sample buffer. Cell extracts were loaded onto polyacrylamide gels containing SDS. Gels were analyzed by fluorography. For this purpose, gels were soaked in a 1 M sodium salicylate solution, dried and exposed to an X-ray film (Agfa). COS-7 cells were grown in DMEM 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 have been described in detail (Aldabe et al., 1995). Transfection efficiencies were determined by immunofluorescence using an anti-luciferase antiserum (Promega) after transfecting the cells with the plasmid pTM1-Luc as described previously (Alvarez et al., 2003). Western blotting. Proteins were transferred to a 0.45 μm nitrocellulose membrane (Bio-Rad) for Western blot analysis. Western blots were developed with the following antibodies: mouse monoclonal anti-PABP antibody (Abcam) at a 1:250 dilution; anti-eIF4GI antisera raised against peptides derived from the Nand C-terminal regions of the human eIF4GI (Aldabe et al., 1995) at a 1:1000 dilution; or rabbit antisera against the C-terminal region of eIF4GII (a gift from N. Sonenberg) at 1:500 dilution; hybridoma supernatant of a monoclonal antibody against eIF4A factor at a 1:50 dilution (a gift from H. Trachsel) and mouse ascites of a monoclonal antibody against HIV-1 p24 antigen (Centralised Facility for AIDS reagent) was used at a 1:100 dilution. Goat anti-rabbit IgG antibody coupled to peroxidase and goat anti-mouse IgG antibody coupled to peroxidase (Pierce) were used at a 1:10000 dilution. Protein Purification. HIV-1 PR was purchased from the Centralised Facility for AIDS reagents, from Dr. I. Pichova. Purified HIV-2 PR (Rittenhouse et al., 1990) was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH, from B. Shirley and M. Cappola (Boehringer Ingelheim Pharmaceuticals). MoMLV PR was expressed and purified as previously described (Menendez-Arias et al., 1993). The chimeric maltose binding protein (MBP)-2A was purified by affinity chromatography, as described previously (Ventoso et al., 1999). The pGEX-2T-PABP1 plasmid was used to purify the GST-PABP1 fusion protein by affinity chromatography, using a glutathione-agarose 4B resin (Pharmacia Biotechnology) as described previously (Burgui et al., 2003). Protease cleavage assays. To detect PABP processing by HIV-1 PR and HIV-2 PR in cell-free systems, crude HeLa S10 extracts were incubated with 2.5 ng/μl of recombinant HIV-1 PR, HIV-2 PR, and MBP-2A,and at indicated times, reactions were stopped by adding sample buffer. Lysates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. Degradation kinetics of translation factors was determined by densitometric scanning of the protein band corresponding to each factor analyzed using a GS-710 calibrated Imagin Densitometer (Bio-Rad). In order to map the cleavage sites of HIV-1 PR and HIV-2 PR on PABP, 50 μg of recombinant GST-PABP1 protein were incubated with 1 μg of recombinant HIV-1 PR or HIV-2 PR in a total volume of 200 l for 3 h at 30oC in a buffer containing 50 mM Na2HPO4, pH 6.0, 25 mM NaCl, 5 mM EDTA, and 1 mM DTT. Cleavage products were separated by SDS-polyacrylamide gel electrophoresis and transferred to an Inmobilon polivinylidene difluoride membrane (Bio-Rad) and then subjected to automated Edman degradation with an Applied Biosystems Procise Sequencer in the proteomic service of Centro de Investigaciones Biológicas (CSIC). RESULTS Cleavage of PABP in HIV-1-infected cells. The hydrolysis of both PABP and eIF4G may contribute to the drastic shut-off of host translation in poliovirus-infected cells. In order to investigate the susceptibility of PABP to proteolysis after HIV-1 infection, MT-2 cells were infected with HIV-1 at a multiplicity of ≈1 plaque-forming units per cell. The integrity of PABP was assessed by Western blotting at 3 dpi using anti-PABP antiserum (Figure 1A, upper panel). Proteohydrolysis of PABP was clearly apparent in MT-2 cells infected with HIV-1. Thus, intact PABP decreased considerably, while a smaller immunoreactive protein band with molecular mass of about 50 kDa appeared. To analyze the integrity of other initiation factors these samples were blotted using antibodies against eIF4GI, eIF4GII (Figure 1B) and eIF4A (Figure 1A, lower panel). The amount of eIF4A remained constant after infection, indicating that this protein was not modified by HIV-1 and that similar amounts of proteins were loaded in all cases (Figure 1A, lower panel). Cleavage of eIF4GI was detected in MT-2 infected-cells, using antibodies that recognize epitopes at the N-terminal (Nt) or C-terminal (Ct) regions of the initiation factor. Hydrolysis of eIF4GI was estimated to be around 60%. In agreement with previous reports, eIF4GII was much less affected by HIV-1 (Ohlmann et al., 2002, Ventoso et al., 2001). Finally, the activity of HIV-1 PR in infected cells was also tested by western blotting using antip24 antiserum (Figure 1C). The Gag (p55) precursor was processed to intermediate CA-MA product and to CA (p24) by HIV-1 PR. HIV-1 PR cleaves PABP Next, the ability of HIV-1 PR to cleave PABP was tested when this protease was expressed alone in culture cells. For this purpose, the HIV-1 pr gene was placed under the control of a duplicated late promoter in a Sindbis virus (SV) construct in order to generate a recombinant SV (SV-PR) that express the HIV-1 PR. In addition, the poliovirus 2A gene was cloned using a similar strategy to generate a recombinant SV that express poliovirus 2A (SV-2A). The transcribed RNAs corresponding to wt SV, SV-PR and SV-2A were electroporated in BHK cells, and the integrity of PABP was examined at 8 h post-electroporation (hpe) (Figure 2A). The expression of HIV-1 PR led to over 85% cleavage of PABP, a result similar to that found after expression of poliovirus 2A. A polypeptide of around 50 kDa, which could be a proteolytic product of PABP, was detected in cells electroporated with SV-PR RNA. The decrease of PABP induced by expression of HIV-1 PR was abolished when 12 μM saquinavir, a specific inhibitor of the HIV PR, was present (Figure 2A). Cell extracts were further analyzed to determine the cleavage of eIF4GI and eIF4GII (Figures 2B and 2C). Previous findings revealed the presence of two forms of eIF4G of 220 and 150 KDa in BHK cells. These different eIF4G forms may be the result of post-translational modifications. The 150 KDa product may be a breakdown product of eIF4GI. Both polypeptides of 220 and 150 KDa disappeared in 2A and in HIV-1PR expressing cells. According to previous data, the expression of HIV-1 PR induces an effective cleavage of eIF4GI, while eIF4GII remained intact (Figures 2B and 2C). The C-terminal proteolytic fragment of eIF4GI and eIF4GII was only detected in BHK cells. The kinetics of the hydrolysis of PABP, eIF4GI and eIF4GII by HIV-1 PR were analyzed after electroporation of BHK cells (Figure 2D). The cleavage of PABP by HIV-1 PR was similar to that observed with eIF4GI proteolysis. In addition, cleavage of eIF4GII by HIV-1 PR was very inefficient in this assay. Other retroviral PRs cleave PABP in transfected cells The susceptibility of PABP to proteolysis mediated by different retroviral PRs encoded by MoMLV, MMTV, HTLV-I, SIV, HIV-1 and HIV-2 was tested in transfected cells. For this purpose, the pTM1-derived plasmids containing the corresponding retroviral PR gene were transfected in COS-7 cells utilizing the vaccinia virus vT7 system. Poliovirus 2A was expressed in parallel to serve as a control of PABP cleavage. The integrity of PABP was assessed by western blot at 16 h posttransfection (hpt) (Figure 3A). Expression of HIV-1, HIV-2 and MMTV PR induced a substantial decrease in intact PABP comparable to that obtained after transfection of poliovirus 2A. The addition of 2 μM saquinavir prevented a loss of PABP caused by HIV-1 and HIV-2 PRs, suggesting that the lower level of intact PABP in HIV PRtransfected cells was due to their proleolytic activity. On the other hand, a decrease in PABP was not detectable in cells transfected with plasmids containing the retroviral PR-coding regions of MoMLV, HTLV-I and SIV. The integrity of eIF4GI and eIF4GII were also tested in PR-transfected cells (Figures 3B and 3C). In agreement with previous data, all the retroviral PRs analyzed were able to cleave eIF4GI (Figure 3B), while the PRs of MMTV, MoMLV, HTLV-I and SIV cleave eIF4GII in transfected cells (Figure 3C). The efficiency of transfection in these experiments was around 70%, as determined by immunofluorescence of cells transfected with the pTM1-Luc plasmid, using an anti-luciferase antiserum (data not shown). In vitro cleavage of PABP by HIV-1 and HIV-2 PRs. The proteolysis of PABP was further studied in cell-free systems using purified recombinant PRs from HIV-1, HIV-2 and MoMLV. The recombinant MBP-2A was included as a control. HeLa cell extracts were incubated for 90 min at 30oC with 3 ng/μl of each purified PR. Under the experimental conditions used, complete proteolytic processing of intact PABP by HIV-1 and HIV-2 PRs occurs, while some intact PABP still remains upon incubation with MBP-2A (Figure 4A). This result indicates that the purified HIV-1 and HIV-2 PRs hydrolyze PABP with a better efficiency than MBP-2A. The MoMLV PR was unable to cleave PABP in this assay. The specificity of these cleavages was evidenced by using an HIV-1 PR inhibitor such as SQ, which blocked cleavage of PABP1 by both HIV-1 and HIV-2 PRs (Figure 4A). Consistent with the above results, the purified HIV-1 and HIV-2 PRs proteolyze eIF4GI in vitro, while eIF4GII was very inefficiently degraded by both PRs (data not shown). In addition, the integrity of other initiation factors such as eIF4E, eIF4A and the 100 kDa subunit of eIF3 was analyzed (Figure 4B upper, middle and lower panels, respectively). The amount of these translation factors remained constant after incubation with recombinant HIV-1 and HIV-2 PRs. To analyze the kinetics of proteolysis of PABP, eIF4GI and eIF4GII, HeLa cell extracts were incubated with recombinant HIV-1 PR and HIV-2 PR, and at the indicated times the reactions were stopped (Figures 5A and 5B). As observed with SV-PR in culture cells, the kinetics of cleavage of PABP by HIV-1 and HIV-2 PRs occurred in a similar fashion to that observed for eIF4GI proteolysis (Figures 5C and 5D). Consistent with previous observations using HIV-1 and HIV-2 PRs (Ohlmann et al., 2002, Ventoso et al., 2001), hydrolysis of eIF4GII does not occur with the retroviral proteases throughout the assay. Identification of the cleavage sites of HIV-1 and HIV-2 PRs on PABP1. To determine if the cleavage of HIV-1 and HIV-2 PRs on PABP was produced directly or indirectly by activation of cellular proteases, the recombinant GST-PABP1 (9-636) protein was incubated with the recombinant HIV-1, HIV-2 and MoMLV PRs. In some assays, 2 μM saquinavir (in the case of HIV-1 PR or HIV-2 PR) and 20 μM ritonavir (in the case of MoMLV PR) were added. The staining of SDS-polyacrylamide gels with Coomassie brilliant blue revealed the generation of several cleavage products (Figure 6A). Incubation of GST-PABP1 (9-636) with both HIV-1 PR and HIV-2 PR revealed a similar degradation pattern but with some slight differences (Figure, 6A left and middle panels). Cleavage of GST-PABP1 by HIV-1 and HIV-2 PRs was inhibited by addition of 2 μM saquinavir. In addition, the purified MoMLV PR was unable to cleave the recombinant GST-PABP1 under the conditions used in this assay (Figure 6A, right). In order to precisely identify the PR proteolysis sites, the cleavage products were sequenced by the Edman degradation method. N-terminal sequencing of the polypeptides ~44 and 27 kDa rendered FERHED, which corresponds to a cleavage site located between positions 237 and 238 of PABP1 (Figure 6B). Sequencing analysis of the 20 kDa polypeptide rendered a mixture of S:F, T:E, Q:R, R:H, V:E and A:D in sequencing cycles 1 to 6, which correspond to cleavage sites located at positions 477/478 and 237/238 of PABP1. In addition, AAIPQT was identified as the N-terminal sequence of the ~10 kDa fragment, which reveals a processing site located between positions 410 and 411 of PABP1 (Figure 6B). The other cleavage sites inferred by the Coomassie gel patterns were not identified due to the low amounts of recovered products. These findings reflect that both HIV-1 and HIV-2 PRs cleave PABP1 around the same sites (Figure 6C). The presence of additional cleavage sites on PABP1 could also explain in part the variability observed among the degradation products rendered by HIV-1 and HIV-2 PRs. The relative conservation of the PABP1 cleavage sites of viral PRs supports the view that this protein is a genuine substrate for the HIV-1 and HIV-2 PRs. DISCUSSION A variety of viral products are responsible for cell damage that occurs during virus infection. Animal viruses encode PRs that proteolyze viral precursors to render the mature proteins. Apart from these viral polypeptides, a number of host proteins are also substrates of viral PRs. The hydrolysis of these host proteins alters different cellular functions. In this sense, PRs of several viruses cleave protein factors of the cellular translational machinery. In this way, there is a down-regulation of cellular mRNA translation, while the synthesis of viral proteins is facilitated. This is the case of the initiation factor eIF4G, which is cleaved by some picornavirus-encoded PRs, leading to the inhibition of translation directed by cellular capped mRNAs (Prevot et al., 2003). Moreover, bisection of eIF4G by picornavirus PRs enhances the translation of the virus mRNA genome that contains an IRES motif at its 5’ end. Recent reports have illustrated that eIF4G is targeted also by PRs of several retroviruses (Alvarez et al., 2003, Ohlmann et al., 2002, Ventoso et al., 2001). In this manner, retroviral PRs inhibit cap-dependent translation without affecting IRES-driven protein synthesis directed by retroviral mRNAs (Alvarez et al., 2003, Ohlmann et al., 2002, Perales et al., 2003, Ventoso et al., 2001). Moreover, PABP, like eIF4G, is cleaved by PRs of some picornaviruses and caliciviruses during infection (Joachims et al., 1999, Kerekatte et al., 1999, Kuyumcu-Martinez et al., 2004a, Kuyumcu-Martinez et al., 2002). In addition, PABP degradation may be implicated, together with eIF4G cleavage, in the inhibition of cellular translation that occurs during infection. Our present findings reveal that PABP cleavage is not exclusive to picornavirusor calicivirus-infected cells but it also takes place in HIV-infected cells. The fact that HIV-1, HIV-2 and MMTV PR hydrolyze PABP is of interest for a better understanding of the strategy used by several members of the Retroviridae family to regulate translation in infected cells. The cleavage of PABP by the poliovirus 3C occurs at the C-terminal domain of the protein. In this manner, the cell terminal portion, which is implicated in interaction with other proteins, is separated from the RNA-interacting N-terminal domain (Kuyumcu-Martinez et al., 2002). This cleavage may selectively inhibit poly(A)dependent translation (Kuyumcu-Martinez et al., 2002, Kuyumcu-Martinez et al., 2004b). The C-terminal domain of PABP interacts with several proteins that are implicated in mRNA translation (Cosson et al., 2002, Hoshino et al., 1999, Imataka et al., 1998, Kozlov et al., 2001, Le et al., 1997, Tarun et al., 1997). In this sense, bisection of PABP by 3C disrupts the interaction of PABP with eIF4B and eRF3, which may affect late events in translation, such as ribosome recycling. The identification of cleavage sites in PABP recognized by both HIV-1 and HIV-2 PRs reveals that these proteases act in a region located within the C-terminal domain. This location is close to the cleavage site recognized by poliovirus 3C. These results add evidence to the idea that this region is highly susceptible to proteolysis by proteases from different virus species. Apart from the processing at positions 410 and 477, HIV PR also hydrolyzes PABP1 at position 237, separating the two first RRM motifs, which may retain some functions of the N-terminal domain. In this sense, all RRM motifs of PABP possess RNA-binding ability, but the two first domains together (RRM1-RRM2) have the highest affinity for the poly(A) tail (Burd et al., 1991). Moreover, these studies have revealed that the RRM2 motif is involved in direct interaction with eIF4G (Imataka et al., 1998, Kessler & Sachs, 1998). The addition of both purified recombinant PABP RRM1-RRM2 or PABP RRM1-RRM4 fragments to PABP-depleted Krebs-2 cell extracts, rescue translation, although less efficiently than the addition of intact PABP (Kahvejian et al., 2005). The suggestion was advanced that the interaction of PABP with eIF4G is essential for efficient translation, while the binding of PABP C-terminal domain with other factors is necessary for a complete stimulation of translation (Kahvejian et al., 2005). Thus, cleavage of PABP within the RRM3 motif by HIV PR may abolish in part, but not completely, the function of its N-terminal domain, since the RRM1-RRM2 region still retains the eIF4G binding site and the ability to bind RNA. The finding that HIV PR cleaves PABP, together with the eIF4G cleavage catalyzed by this protease, may account for the fact that acute infection by HIV-1 interferes with host protein synthesis (Ventoso et al., 2001). Infection on C8166 with HIV-1 led to a drastic decline in host translation coincident with the decrease of intact eIF4GI (Ventoso et al., 2001). Our present findings indicate that cleavage of both eIF4GI and PABP occurs with similar kinetics in vitro. Therefore, PABP hydrolysis may also contribute to the inhibition of host cell translation. Total proteolysis of eIF4GI does not suffice to block the translation of cellular mRNAs engaged in the translational machinery (Irurzun et al., 1995, Perez & Carrasco, 1992). The shut-off of host protein synthesis is coincident with the hydrolysis of eIF4GII in poliovirus and rhinovirus infected cells as well as in apoptotic cells (Gradi et al., 1998, Svitkin et al., 1999). The hydrolysis of PABP by poliovirus 3C fully inhibits the translation of endogenous mRNAs in HeLa extracts (Kuyumcu-Martinez et al., 2004b). Thus, cleavage of eIF4GI by HIV-1 or HIV-2 PR may not be sufficient to abrogate host mRNA translation. In conclusion, the cleavage of PABP by HIV PR, together with eIF4GI hydrolysis, could be responsible for the host shut-off observed in some cell lines upon HIV-1 infection (Ventoso et al., 2001). The observation that cellular protein synthesis diminishes concomitantly with the eIF4GI hydrolysis in HIV-1 infected cells, may be due, in fact, to the proteolysis of both eIF4GI and PABP produced by HIV PR. Curiously, several retroviral proteases such as those from MoMLV, HTLV-1 or SIV-1 hydrolyze only eIF4GI and eIF4GII, leading to the inhibition of protein synthesis in transfected COS-7 cells (Alvarez et al., 2003). Although HIV-1 and HIV-2 PRs both exhibit the ability to proteolyze eIF4GI and PABP, they cleave eIF4GII inefficiently (Alvarez et al., 2003, Ohlmann et al., 2002, Ventoso et al., 2001). These data suggest that, at least, two of these factors must be inactivated to efficiently inhibit the cap and poly (A) dependent translation. The HIV PR not only cleaves factors implicated in translation, but also other host proteins. Indeed, several cellular polypeptides have been identified as substrates of HIV PRs. (Shoeman et al., 1990, Tomasselli et al., 1991). The potential role of cleavage of cellular proteins in the cytotoxic effect inflicted by HIV has been pointed out by several laboratories. Proteolysis by HIV-PR of microtubule-associated proteins, cytoskeletal proteins and nuclear factor-κB, among others, has been described (Dunn, 1998, Wlodawer & Gustchina, 2000). Further investigation is needed as to the exact role that each of these proteolytic cleavages by HIV PR, including eIF4G and PABP, plays in the cytopathogenicity provoked by HIV infection. 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 (Agy et al., 1990, Somasundaran & Robinson, 1988). In HIV-1, the Vpr protein blocks proliferation of CD4+ T cells at the G2 cell cycle checkpoint (Brasey et al., 2003, Goh et al., 1998). At this stage, the initation of cap-dependent translation is suppressed, probably by both eIF4GI and PABP cleavage, while the initiation of IRES containing mRNAs ensures the synthesis of Gag and Gag-Pol. In this context, cleavage of both PABP and eIF4GI could facilitate viral gene expression and contribute to the inhibition of host protein synthesis. The finding that many retroviral and picornaviral PRs target eIF4G, and in some cases PABP, indicates that these viruses may share a common mechanism of translational control. ACKNOWLEDGEMENTSFinancial support from Grant BMC2003-00494 from the DGICYT and anInstitutional Grant to the Centro de Biología Molecular "Severo Ochoa" from theFundación Ramón Areces, are acknowledged. E.A. and A.C. held fellowships from theMinisterio de Educación y Ciencia. REFERENCESAdam, S. A., Nakagawa, T., Swanson, M. S., Woodruff, T. K. & Dreyfuss, G. 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Structural and biochemical studies of retroviralproteases. Biochim Biophys Acta 1477, 16-34. FIGURE LEGENDSFigure 1. PABP cleavage in HIV-1-infected cells. (A) MT-2 cells were infected withHIV-1 (moi 1). At 3 dpi the integrity of PABP was determined by Western blot (upperpanel). The corresponding Western blot against eIF4A (lower panel) is shown. Mock,mock-infected cells; HIV-1, cells infected with HIV-1. Cleavage product derived fromPABP in infected cells is indicated as c.p. An unknown anti-PABP-reactive band is alsodenoted by an asterisk. Mr, molecular mass. (B) Western blots against eIF4GI (upperpanel) and eIF4GII (lower panel) at 3 dpi (C) Western blot against p24 at 3 dpi. Theposition of the intact initiation factors is also indicated in the respective panel. Figure 2 Cleavage of PABP in cells electroporated with recombinant SVs thatexpress heterologous proteases. (A) Cells were electroporated with transcriptionbuffer (BHK), wt SV, SV-PR or SV-2A RNAs and incubated in the presence or absence of 12 μM saquinavir. At 16 hpe, cell extracts were analyzed by western blotting using specific antibodies against human PABP1. Cleavage product derived from PABP inSV-PR-infected cells is indicated as c.p.; SQ, saquinavir; Mr, molecular mass. (B)Detection of eIF4GI cleavage products by Western blotting using a mixture of antiseraagainst its Nand C-terminal regions. c.p., cleavage fragments. The amount of hydrolyzed eIF4GI for each transfection experiment is indicated below eachcorresponding lane. (C) Detection of eIF4GII cleavage products by Western blottingusing an antisera against the C-terminal region. Ct, C-terminal fragments of eIF4GII.The amount of hydrolyzed eIF4GII for each transfection experiment is indicated beloweach lane. (D) Cleavage kinetics of PABP, eIF4GI and eIF4GII by HIV-1 PR. BHK cells were electroporated as described in materials and methods and cell lysates wereobtained at the indicated times. The values were obtained by densitometric scanning ofthe corresponding intact protein band at each time indicated. Figure 3. Cleavage of PABP, eIF4GI and eIF4GII in transfected cells. (A) COS-7cells were transfected with the empty pTM1 vector or with pTM1 carrying insertscontaining the PR gene of several retroviruses or poliovirus 2A. At 16 hpt, equalamounts of protein extract were loaded in a 15 % polyacrylamide gel and analyzed byWestern blotting using a monoclonal antibody against PABP. Data are referred tocontrol experiments carried out with the empty pTM1 vector, and the values were obtained by densitometric scanning of the protein band of ≈70 kDa corresponding to PABP. (B) Western blot against eIF4GI. (C) Western blot against eIF4GII. Theestimated amount of cleaved protein in cell extracts obtained from transfections withdifferent constructs is indicated below each panel. Figure 4. Cleavage of PABP in vitro by purified recombinant HIV-1, HIV-2 andMoMLV PRs. 50 μg of crude HeLa S10 extracts were incubated with 3 ng/μl of eachrecombinant PR in a total volume of 20 μl for 90 min and subjected to Western blottinganalysis by using: (A) a polyclonal antisera against 100-kDa subunit of eIF3 factor, (B)an antibody against PABP, (C) an antibody against eIF4A or (D) a monoclonal antibodyraised against eIF4E factor. Figure 5. Cleavage kinetics of PABP, eIF4GI and eIF4GII in vitro by purifiedrecombinant HIV-1 and HIV-2 PRs. 50 μg of crude HeLa S10 extracts were incubatedwith 3 ng/μl of HIV-1 PR (A) or HIV-2 PR (B) in a total volume of 20 μl during theindicated times and subjected to Western blotting analysis by using a monoclonalantibody against PABP (upper panel), a mixture of antisera against N-terminal and Cterminal regions of eIF4GI (middle panel) or an antisera against the C-terminal regionof eIF4GII (lower panel). Degradation kinetics of PABP, eIF4GI and eIF4GII in HeLacell extracts incubated with HIV-1 PR (C) and HIV-2 PR (D). Determinations wereobtained by densitometric scanning of the corresponding intact protein band at theindicated times. Figure 6. Identification of HIV-1 PR and HIV-2 PR cleavage sites on PABP1. (A) 5μg of recombinant GST-PABP1 protein were incubated with 100 ng of recombinantHIV-1 PR, HIV-2 PR or MoMLV PR in a total volume of 20 μl as described in materialsand methods. Cleavage products were separated by SDS-polyacrylamide gelelectrophoresis and the hydrolysis fragments were stained by Coomassie blue. (B)Aminoacid sequence of the PABP1 cleavage sites identified using HIV-1 and HIV-2PRs. (C) Diagram showing the functional domains found in PABP1 based on theavailable data, including the position of mapped cleavage sites for HIV-1 and HIV-2PRs. The RNA binding domains (RRM) and the regions involved in the binding to othertranslation factors are shown. PABP, poly(A)-binding protein. Sequence numberingrefers to the human PABP1 isoform.