In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

Keywords: HIV, translation initiation, Gag, IRES, HIV protease

Gag translation is driven by three IRES located within the coding region

We have previously shown that translation of the HIV-2 genomic RNA from the authentic AUG1 site (producing p57) occurs by internal entry of the ribosomes on an IRES sequence which has the unique ability to load ribosomes upstream of its core sequence (Herbreteau et al. 2005). This is rendered possible by a region of 50 nucleotides located just downstream from the first AUG (position +548) that forms a long range RNA interaction and was shown to be critical for initiation at the authentic initiation site (Herbreteau et al. 2005). However, the mechanism by which initiating ribosomes are able to access the two other downstream AUG sites (positions +746 and +899) remained to be determined as well as the role of the 5′-UTR in the overall translation mechanism of the wild type genomic RNA.

Thus, we have constructed a series of bicistronic RNAs that is depicted in Figure 1A, and which contains the neomycin gene as the first cap-dependent cistron and harbors the LacZ gene as the second cistron. Various regions of the HIV-2 genomic RNA including the 5′-UTR, the coding region encompassing AUG1 to AUG3, or a series of deletions, were inserted in the bicistronic RNA and translated in the rabbit reticulocyte lysate. As shown previously (Herbreteau et al. 2005), the 5′-UTR alone led to poor expression of the LacZ gene (Fig. 1B, lane 1) whereas efficient translation of the three β-Gal isoforms was observed when the 5′-UTR was followed by segments of the gag coding region spanning from AUG1 to AUG3 (Fig. 1B, lanes 2,3). Moreover, complete removal of the 5′-UTR did not affect IRES activity in agreement with our previous findings (Fig. 1B, lane 4; Herbreteau et al. 2005). Internal deletions within the gag coding region were then generated in the context of the bicistronic construct. Interestingly, 5′ deletions starting some 114 nucleotides downstream from the AUG1 site did not impair translation initiation at AUG2 and AUG3 (Fig. 1B, lane 5). Moreover, a further 5′ deletion starting downstream from AUG2 did not affect translation at the third AUG site (Fig. 1B, lane 6).

In order to confirm the in vitro results, capped and polyadenylated bicistronics RNAs containing either the entire Gag coding region from AUG1 to AUG3 or a series of 5′deletions between the Firefly and the Renilla luciferase coding region (Fig. 2A) were transfected in HeLa cells. As expected, the empty vector was poorly translated (Fig. 2C, “pFR-NoIRES”). Activity of bicistronics RNAs containing the entire Gag coding region or 5′ deletions (Fig. 2C) was also quite low but was still twofold to fourfold above the negative control. It should be noted that luciferase activity driven by monocistronic capped and polyadenylated RNAs containing the HIV-2 coding region was also very low (data not shown). We have expressed the 2A protease from poliovirus in HeLa cells to cleave eIF4G. Proteolysis of eIF4G by this protease inhibits cap-dependent translation whereas IRES-driven translation is maintained or stimulated (Ziegler et al. 1995; Ohlmann et al. 1997). Upon eIF4G cleavage by the virally encoded protease 2A (Fig. 2B), translation of the Renilla luciferase driven by the HIV-2 Gag coding region, or segments of it, was highly stimulated compared to the negative control (Fig. 2C). Interestingly, as observed in vitro, 5′ deletions of the Gag coding region starting downstream from AUG1 (pFR-662-AUG3) or downstream from AUG2 (pFR-748-AUG3) showed translational activities comparable to that of the entire Gag coding region (pFR-AUG1-3) (Fig. 2C). These data indicate that translation initiation occurs independently at each of the three AUG initiation sites both in vitro and ex vivo suggesting the presence of three distinct and independent IRES elements.

In order to confirm that internal initiation takes place at each of the three AUG initiation sites from monocistronic RNAs in vitro, we have used the L-protease from foot-and-mouth disease virus (FMDV) that also cleaves eIF4G. Thus, the RRL was pre-incubated for 10 min with increasing concentrations of in vitro generated L-protease in order to cleave eIF4G (see Supplemental Fig. 1). Then, the RRL was programmed with a set of different control RNAs in which the LacZ coding region is driven by the capped 5′-UTR of globin (Fig. 3, lanes 1–3) or the uncapped IRES of EMCV (Fig. 3, lanes 4–6). Capped and uncapped wild-type HIV-2 genomic RNA (Fig. 3, T7-WT HIV-2, lanes 7–12) and the HIV-2 genomic RNA devoided of its 5′-UTR and commencing directly at AUG1 (Fig. 3, T7-AUG1 HIV-2, lanes 13–18) were also translated in the RRL treated by the L-protease. As expected, cap-dependent Globin-LacZ translation was strongly inhibited by addition of the protease in a dose-dependent manner (Fig. 3, lanes 2,3) whereas EMCV IRES-driven translation was stimulated (Fig. 3, lanes 4–6). Interestingly, production of the three Gag isoforms p57, p50, and p44 from the wild-type RNA was not diminished by the addition of the L-protease whether this mRNA was capped or uncapped (Fig. 3, lanes 7–12). Translation of the leaderless HIV-2 RNA, which is more efficient than that from the wild-type RNA, was poorly affected by eIF4G cleavage showing a small inhibition only at high doses of L-protease (Fig. 3, lanes 13–18). Interestingly, the relative ratio of expression of the three gag isoforms was not affected by this proteolytic event.

To further confirm these results and to evaluate any contribution of the cap from the wild-type HIV-2 RNA, we carried out translation assays using a competitive untreated rabbit reticulocyte lysate (Fig. 4). We have recently shown that this system faithfully recapitulates the synergistic effect of the cap and the poly(A) tail and the selective advantage of IRES dependent translation (Soto Rifo et al. 2007). Therefore, the untreated RRL was programmed with increasing amounts of the polyadenylated capped and uncapped HIV-2 polyadenylated RNAs (Fig. 4, lanes 1–6). As observed in the nuclease treated lysate, translation of the uncapped and polyadenylated T7-WT was as efficient, if not more, as the capped version of the RNA, suggesting a poor contribution of the cap to overall Gag translation (Fig. 4, lanes 1–6). Furthermore, addition of the L protease also led to translation stimulation of the capped and polyadenylated T7-WT RNA, whereas endogenous globin and lipoxygenase translation was inhibited (Fig. 4, lanes 7–10). These experiments show that the synergy between the poly(A) tail and the cap exerts only a mild effect on overall translation from the HIV-2 wild-type genomic RNA, confirming the use of an internal initiation mechanism to produce the Gag polyproteins.

The three IRES are functionally independent

The next step was to determine whether each of the three AUGs could be used for initiation independently from each other. Therefore, the experimental approach consisted of the hybridization of short 2′-O-methyloligoribonucleotides that were annealed to different regions of the HIV-2 mRNA molecule. Annealing of the oligos to the RNAs was tested by electrophoresis of RNA–oligo duplexes on agarose gels (see Supplemental Fig. 2). As a control experiment to evaluate the effect of oligo hybridization on ribosomal scanning, a 2′-O-methyloligoribonucleotide complementary to the globin 5′-UTR was used and the resulting oligo–mRNA duplex was translated in the RRL (Fig. 5, lanes 1–4). This resulted in a drastic inhibition of translation of the globin–LacZ RNA showing that the oligo–mRNA duplex was stable enough to arrest most of the scanning 40 S ribosomes. Interestingly, when a 2′-O-methyloligoribonucleotide was annealed to the primary binding site (PBS), which lies in the middle of the HIV-2 5′-UTR, we could only observe a marginal decrease in translation from the first AUG site with no effect on the downstream AUGs (Fig. 5, lanes 5–8). Hybridization of an oligo targeting the RNA region including the AUG1 initiation site resulted in the pronounced inhibition of translation at the proximal site but with virtually no effect on the expression of p50 and p44 which are synthesized from initiation at AUG2 and AUG3, respectively (Fig. 5, lanes 9–12). In a similar manner, hybridization of a 2′-O-methyloligoribonucleotide at the second AUG site (AUG2) yielded some unexpected results (Fig. 5, lanes 13–16). First of all, it did not impair expression of p44 from the third AUG initiation site, suggesting that translation at AUG3 does not result from leaky scanning or read through from AUG2. Second, it can be clearly seen on the autoradiography that initiation occurred upstream of the AUG2 as judged by the slightly larger translation product made (Fig. 5, cf. lane 9 and lanes 10–12). This product (named p52) might result from translation initiation at a non-AUG codon since no AUG triplets (either in frame or out of frame) are located between AUG 1 and AUG2 and may correspond to an in-frame CUG codon that is present some 19 codons upstream of AUG2.

Taken together, these results indicate that there are three distinct and functionally independent internal entry sites located entirely in the gag coding region: IRES 1 lies downstream from its AUG entry site as described previously, the second IRES is located between AUG1 and AUG2, and the third IRES spans between AUG2 to AUG3. Interestingly, each of these three IRES has its own initiation site.

The HIV-2 protease regulates the pattern of expression of Gag isoforms

We next went on to investigate the molecular determinants that could control HIV-2 translation. In particular, it was of interest to study the effects of the viral HIV-2 protease on the translation of its cognate mRNA. This protease was previously shown to play a role in translation by cleaving the initiation factor eIF4G (Ventoso et al. 2001; Ohlmann et al. 2002). Such a proteolytic event yields an N-terminal fragment which contains the eIF4E binding site and a carboxy-terminal domain identical to the L-protease generated eIF4G fragment except that it lacks a small 40 aa RNA binding domain previously described as being critical for ribosomal scanning (Prevot et al. 2003a). As a result, this C-terminal eIF4G fragment resulting from HIV-2 protease cleavage, which harbors the eIF4A and eIF3 binding site, is not competent to support ribosomal scanning. Thus, the RRL was pretreated with increasing amounts of the HIV-2 protease and then programmed with Globin-LacZ, EMCV-LacZ, capped and uncapped wild-type HIV-2 (T7-WT HIV-2), or the capped and uncapped leaderless HIV-2 (T7-AUG1 HIV-2) together with the Hepatitis C virus (HCV) mRNAs whose translation is not dependent on the integrity of eIF4G (Fig. 6). The cleavage of eIF4G was monitored by Western blot (see Supplemental Fig. 3). Addition of the recombinant HIV-2 protease did not affect HCV IRES translation (Fig. 6, lanes 8,9), indicating that no damage to the general translational machinery was caused by addition of the retroviral enzyme. As expected, expression of Globin-LacZ was virtually abolished (Fig. 6, lane 3) whereas translation of EMCV-IRES containing mRNA was affected to a lesser extent (Fig. 6, lane 6).

In contrast, production of p57, p50, and p44 Gag resulting from translation of T7-WT at the three AUG sites was only marginally impaired by addition of the HIV-2 enzyme (Fig. 6, lanes 11,12,14,15) whereas expression of the leaderless construct was inhibited by about 60% (Fig. 6, lanes 17,18,20,21). It is interesting to note that capping of the mRNAs (either T7-WT or the leaderless) did not change the pattern of protein expression. More importantly, and unlike the situation with the L-protease (see Figs. 3, 4), the pattern of expression of p57, p50, and p44 from the wild-type or the leaderless constructs was modified by addition of the HIV-2 protease. For instance, synthesis of p57 was inhibited from the WT RNA whereas production of p50 and p44 remained virtually similar to the control (Fig. 6, cf. lane 10 and lanes 11,12). In contrast, initiation at the third AUG site was almost abolished from the leaderless construct (Fig. 6, cf. lane 13 and lanes 14,15).

Taken together, these results show that expression of the HIV-2 protease has quantitative and qualitative impacts on the translation of its cognate mRNA and these effects are affected by the presence or absence of the 5′-UTR. This suggests that the level of expression of p57, p50, and p44 can vary according to changes in the physiological conditions used and that the 5′-UTR can affect the mechanism by which the ribosomes are recruited to the Gag coding region, thus explaining the different results obtained from the wild-type and the leaderless RNAs.

The Gag polyprotein controls its own translation

During the early steps of virus assembly, the Gag polyprotein plays a critical role in HIV-2 RNA packaging by binding to the mRNA, which allows the resulting ribonucleoprotein complex to be addressed to the plasma membrane (Kaye and Lever 1999). Interestingly, this process occurs at times when the genomic viral RNA is translated and, thus, most probably interferes with the process of protein synthesis. Curiously, the effect of the Gag polyprotein on translation has rarely been directly investigated by using in vitro translational assays. Thus, we have used recombinant HIV-1 Gag polyprotein that was added to translation assays programmed with different constructs including mRNAs in which LacZ production was driven by the Globin 5′-UTR or the EMCV IRES. The capped HIV-2 T7-WT and T7-AUG1 HIV-2 RNAs were also translated in the presence of increasing concentration of recombinant Gag polyprotein. It should be noted that the HIV-1 Gag protein was shown to be able to bind to the HIV-2 RNA packaging signals with a similar affinity to the HIV-1 RNA signals (Kaye and Lever 1998); however this was confirmed in our experiment setting by Far Western and dot blot (data not shown). The relative molar ratio of Gag recombinant protein to RNA used ranged from 0 to 82 and the resulting Gag–RNA ribonucleoprotein complex was translated in the RRL under the conditions described in Materials and Methods. Results presented in Figure 5 show that, at low concentration, Globin-LacZ expression was only partly affected by the addition of Gag (Fig. 7, lanes 1–9). Translation driven by the EMCV IRES seemed to be more sensitive to the addition of the Gag polyprotein as production of β-Gal was gradually diminished in a dose dependent manner (Fig. 7, lanes 10–18).

However, in sharp contrast, translation of HIV-2 WT construct was markedly inhibited by preincubation of the mRNA even at low concentration of the recombinant Gag protein (Fig. 7, lanes 22,23) and translation was actually abolished at higher concentrations (Fig. 7, lanes 24 and above). Moreover, a significant change in the pattern of expression of the different Gag isoforms could also be observed at low concentrations of Gag added with expression of the longest p57 Gag isoform being more affected (Fig. 7, cf. lane 19 and lanes 20,21, and see the quantification panel). Interestingly, translation of the leaderless HIV-2 RNA (T7-AUG1) was also inhibited by recombinant Gag addition (Fig. 7, lanes 28–36), but this was not accompanied by drastic changes in the pattern of expression of the three Gag isoforms. Overall, translation from this construct was more resistant to Gag addition than the 5′-UTR containing wild type HIV-2 genomic RNA. It is noteworthy that the addition of a recombinant RNA binding protein such as the Lupus autoantigen (La) failed to induce any quantitative or qualitative variation on the expression of Gag isoforms ruling out any nonspecific effect (data not shown). Furthermore, translation of a chimeric RNA which contains the 5′-UTR of human β-globin driving the synthesis of the HIV-2 Gag coding region was also monitored (Globin∷Gag). Interestingly this showed a translational profile which was similar to that of the globin-LacZ RNA and the leaderless HIV-2 RNA (Fig. 7, lanes 37–45). In fact, translation from this RNA was not really sensitive to addition of the recombinant Gag protein and no significant change in the use of initiation codons could be observed. These experiments suggest that Gag polyprotein plays a regulatory role in the translation of its cognate mRNA and this regulation appears to be exerted via an interaction with the viral 5′-UTR.

This work and previous data have shown that translation of the HIV-2 genomic RNA can take place in the complete absence of 5′-UTR from a leaderless construct (Herbreteau et al. 2005). However, results presented herein suggest that the 5′-UTR may exert an inhibitory role in the presence of the Gag polyprotein by acting as a dock for Gag recruitment near the AUG1 codon, thus inhibiting p57 translation. Indeed, the 5′-UTR of lentiviruses harbors RNA signals that are the preferential Gag binding site for RNA packaging in the course of viral assembly. To investigate the role of the 5′-UTR–Gag interactions in the process of translation initiation, two different RNA competitors were independently preincubated with the Gag protein at a high protein to RNA ratio (83:1). These RNA competitors correspond to the whole 546-nt-long HIV-2 5′-UTR (from +1 to nucleotide 546) or a 546-nt-long unspecific RNA sequence taken from the unrelated β-Galactosidase (β-Gal) gene. These RNA competitors were added in a 1:1 molar ratio with the T7-WT and T7-AUG1 RNAs.

The results presented in Figure 8 show that translation of T7-WT and T7-AUG1 drive the synthesis of Gag p57, p50, and p44 together with another intermediate isoform that was observed previously (Fig. 5; data not shown) and which might result from alternative translation initiation at a non-AUG codon located between AUG1 and AUG2. Addition of an excess amount of recombinant Gag protein (83 molar excess over the RNA concentration) results in the complete inhibition of protein synthesis from both the wild type (T7-WT) and the leaderless (T7-AUG1) RNAs (Fig. 8, lanes 2,8). Interestingly, in the case of the wild-type construct, addition of the β-Gal unspecific RNA competitor was able to rescue translation of the p50 and p44 isoforms but not that of the p57 longest protein (Fig. 8, lane 4). The expression of the longest Gag isoforms was only completely restored when the HIV-2 5′-UTR was used as a competitor RNA (Fig. 8, lane 6). Interestingly, addition of the 5′-UTR or the unspecific RNA competitor was of similar efficiency to fully restore translation from the leaderless construct (Fig. 8, T7-AUG1 HIV-2, lanes 10,12). Taken together, these data suggest that the Gag polyprotein regulates translation of its cognate RNA by binding to its 5′-UTR. This results in the overall modulation of translation efficiency together with changes in the selection of the AUG initiation site.

Protease, antibodies, recombinant Gag protein, and other reagents

The L-protease from FMDV was generated by in vitro translation as described previously (Prevot et al. 2003a). The recombinant HIV-1 Gag protein was prepared as described (Datta et al. 2007) and solubilized in a buffer containing 20 mM Tris HCl pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM PMSF, and a protease inhibitors cocktail (Roche Inc.). These conditions are taken in consideration for translation experiments performed in the presence of this protein to maintain equivalent levels of salt, pH, DTT, and anti-protease in each reaction tube.

Hybridization of 2′-O-methyloligoribonucleotides

Antisense 2′-O-methyloligoribonucleotides spanning positions nucleotides 306–327 (antisense to the PBS), nucleotides 548–566 (antisense to AUG1), nucleotides 747–765 (antisense to AUG2) of the HIV-2 RNA were annealed to RNA in 20 mM Hepes/KCl (pH 7.6) and 100 mM KCl for 3 min at 65°C followed by 20 min incubation at room temperature.

An antisense 2′-O-methyloligoribonucleotide spanning positions nucleotides 22–42 (antisense to Globin 5′-UTR) was annealed to Globin-LacZ as described above and used as a control for measuring ribosomal scanning inhibition.

Plasmid construction

Standard procedures were used for plasmid DNA construction, purification, and linearization. Details of the constructs used in this study are given below.

In vitro transcription and translation

The Neo-Lac Z plasmid DNAs were linearized at the SspI site thus producing a 46 kDa truncated version of β-Galactosidase. The Globine∷Gag pcDNA3.1 plasmid DNA was linearized at the EcoRI site. The pFR plasmid was linearized at the EcoRI just after the synthetic poly(A) tail. RNAs were synthesized in vitro as previously described (Prevot et al. 2003b). The integrity of the RNAs was checked by electrophoresis on nondenaturing agarose gels and their concentration was quantified by spectrophotometry at 260 nm using a Nanodrop (Nanodrop Technologies).

In vitro transcribed RNAs were translated in 10 μL of either the Flexi Rabbit Reticulocyte System (Promega Co.) or the supplemented untreated RRL 50% (v/v) each (as described in Soto Rifo et al. 2007) in the presence of 75 mM KCl, 2.5 mM MgCl₂, 20 μM of each amino acid (minus methionine), and 0.6 mCi/mL of [³⁵S]-methionine (GE Healthcare Life Sciences). Translation was carried out for 30 min at 30°C and stopped by the addition of 2× SDS-loading buffer. Translation products were resolved by 15%SDS-PAGE, gels were dried and subjected to autoradiography using Biomax films (Eastman Kodak Co.). Densitometric analyses were performed by phosphorimaging with a Storm 850 PhosphorImager.

T7 RNA polymerase transcription of PCR DNA fragments

The DNA sequence corresponding to the coding region of HIV-2 gag (pROD10), were amplified by PCR using a 3′ oligonucleotide starting at the end of the HIV-2 gag, and a 5′ to 3′ sense oligonucleotide starting with the T7 promoter sequence and complementary to the +1 region of the 5′-UTR, or to the AUG1 start codon, to generate T7-WT and T7-AUG1, respectively. Following purification of the PCR fragments, in vitro transcription using the T7 bacteriophage polymerase was conducted as described above. It should be noted that due to the much higher translational efficiency of the leaderless construct as shown previously (Herbreteau et al. 2005), the latter was utilized at a lower RNA concentration than the wild-type HIV-2 construct.

RNA transfection

HeLa cells were transfected using the TransIT RNA transfection kit (Mirus Bio Corporation) as described (Dieterich et al. 2007).

Measure of luciferase activities from HeLa cells transfected with bicistronics RNAs

HeLa cells were plated at a density of 150,000 cells per well (24 well plate) and transfected with 100 ng of the p2A RNA coding for the protease from poliovirus (Ronfort et al. 2004). After 2 h, the cells were transfected again with 0.156 pmol of the corresponding capped and polyadenylated bicistronic RNA. Three hours post transfection, cells were lysed and luciferase activities were measured using the dual reporter luciferase assay (Promega) on a Veritas luminometer (Turner Biosystems) following the manufacturer's protocol.

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