- Open Access
Mechanism of HIV-1 Tat RNA translation and its activation by the Tat protein
© Charnay et al; licensee BioMed Central Ltd. 2009
- Received: 4 March 2009
- Accepted: 11 August 2009
- Published: 11 August 2009
The human immunodeficiency virus type 1 (HIV-1) Tat protein is a major viral transactivator required for HIV-1 replication. In the nucleus Tat greatly stimulates the synthesis of full-length transcripts from the HIV-1 promoter by causing efficient transcriptional elongation. Tat induces elongation by directly interacting with the bulge of the transactivation response (TAR) RNA, a hairpin-loop located at the 5'-end of all nascent viral transcripts, and by recruiting cellular transcriptional co-activators. In the cytoplasm, Tat is thought to act as a translational activator of HIV-1 mRNAs. Thus, Tat plays a central role in the regulation of HIV-1 gene expression both at the level of mRNA and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of Tat can be made early on in HIV-1 infected cells to sustain its own synthesis. To address this issue we studied translation of the Tat mRNA in vitro and in human cells using recombinant monocistronic and dicistronic RNAs containing the 5' untranslated region (5'-UTR) of Tat RNA.
This study shows that the Tat mRNA can be efficiently translated both in vitro and in cells. Furthermore, our data suggest that translation initiation from the Tat mRNA probably occurs by a internal ribosome entry site (IRES) mechanism. Finally, we show that Tat protein can strongly stimulate translation from its cognate mRNA in a TAR dependent fashion.
These results indicate that Tat mRNA translation is efficient and benefits from a feedback stimulation by the Tat protein. This translational control mechanism would ensure that minute amounts of Tat mRNA are sufficient to generate enough Tat protein required to stimulate HIV-1 replication.
- Internal Ribosome Entry Segment
- Rabbit Reticulocyte Lysate
- HeLa Cell Extract
- Internal Ribosome Entry Segment Activity
- Internal Ribosome Entry Segment
The human immunodeficiency virus type 1 (HIV-1) encodes for the three canonical polyprotein precursors Gag, Pol, and Env, which are required for the formation of infectious viral particles by infected cells. In addition, HIV-1 encodes for six regulatory proteins, among which the Tat and the Rev factors are absolutely required for viral gene expression at the transcriptional and post-transcriptional levels in infected cells . HIV-1 Tat is a small basic protein that mainly localizes to the nucleus of infected cells, where it acts as a potent transcriptional activator that is indispensable for the synthesis of the full length viral RNA (reviewed in [2–4]). Transcriptional activation by Tat is mediated by multiple interactions between Tat and the nascent viral TAR RNA and between Tat and cellular factors involved  in transcription initiation and elongation such as P-TEFb [4–11]. In addition, Tat has been shown to stimulate translation of viral mRNAs [12–14]. Importantly, this cytoplasmic function of Tat seems to require a nuclear experience, since the RNA-protein complex formed between Tat protein and nuclear factors must be assembled in the nucleus in order to later exert its function in the cytoplasm [12–14]. Thus, the HIV-1 Tat protein plays a central role in the regulation of HIV-1 gene expression both at the level of transcription and protein synthesis. The requirement of Tat in these processes poses an essential question on how sufficient amounts of this viral protein can be made early on in HIV-1 infected cells to sustain its own synthesis. Soon after completion of viral DNA synthesis by reverse transcriptase and before its integration into the host genome, the viral DNA can be transcribed, but this generates only low levels of fully spliced viral mRNAs encoding Tat and Nef . These observations led us to hypothesize that Tat mRNA is translated even under conditions where it is present in minute quantities together with a high concentration of cellular mRNAs.
Translation of mRNA into protein represents an essential step in gene expression. The regulation of translation is a mechanism used to modulate gene expression in a wide range of biological situations including cell growth, development and the response to biological cues or environmental stresses such as viral infection [16–20]. During viral infection at least two general modes of translational control can be envisaged. The first represents a global control, in which the translation of most cellular mRNAs is regulated. This is evident during the infection of some members of the Picornaviridae [18–20] where global regulation mainly occurs by the modification of translation initiation factors. The second corresponds to a mRNA-specific control, whereby the translation of a particular mRNA or a defined group of mRNAs is modulated without affecting general protein biosynthesis or the translational status of the cellular transcriptome as a whole. Translational control of a specific mRNA is normally driven by regulatory protein complexes that recognize particular elements that are usually present in the 5' and/or 3' untranslated regions (UTRs) of the target mRNA [21–24]. It is well recognized that translation control of protein synthesis is mostly exerted at the initiation step.
Translation initiation of eukaryotic mRNAs mostly occurs by a scanning mechanism, whereby the 40S ribosomal subunit binds to the mRNA 5' cap structure and scans the RNA in the 5' to 3' direction until an initiation codon in a favourable 'Kozak' context is encountered . Translation initiation involves the recognition of the mRNA 5' cap structure by eIF4F, which is composed of eIF4E, which binds the 5' cap, eIF4A, and eIF4G, which links the mRNA 5' cap (via eIF4E)] and the 40S ribosomal subunit (via eIF3) [26, 27]. Studies on picornavirus protein synthesis led to the discovery of an alternative mechanism of translation initiation, via an internal ribosome entry segment (IRES) [28–30]. A major difference between cap-dependent versus IRES-mediated ribosome binding and initiation of translation is that the eIF4E component of the eIF4F complex is dispensable for most of the latter activity [31, 32]. At present IRESes are defined solely by functional criteria and cannot yet be predicted by the presence of characteristic RNA sequences or structural motifs [30, 33]. Despite these apparent experimental restraints, since the initial characterization of IRESes in Picornaviridae, viruses from other families including several members of the Retroviridae were found to initiate translation via an IRES ([34–41] reviewed in reference ). Indeed, internal ribosome entry has been described in alpha- (ASLV), gammaretroviruses (MoMuLV) and lentiviruses (SIV and HIV).
Based on these findings we wanted to study the mechanism by which the Tat mRNA is translated using recombinant monocistronic and bicistronic RNAs containing all or part of the 5' UTR of the Tat mRNA. In addition, we examined the mechanism by which translation of the Tat mRNA is controlled in vitro in rabbit reticulocyte lysates (RRL) and in human cells. Our results show that the Tat mRNA is efficiently translated in vitro and in cells, despite the presence of large amounts of cellular mRNAs. Moreover, we show that the Tat protein exerts a positive feedback on the translation of its cognate mRNA. Thus, Tat mRNA appears to be efficiently translated even under conditions where it is in minute amounts among highly abundant cellular mRNAs. Taken together our data explains how minute amounts of Tat mRNA can account for viral protein production required to kick-start HIV-1 replication.
Molecular cloning of the Tat mRNA sequences
Tat RNA versus globin RNA translation in vitro
The efficiency of Tat RNA translation was studied in the non-nuclease treated RRL (URRL) , because it contains a high concentration of endogenous globin mRNA (about 7 × 10-7 M). We examined translation of Tat1 and Tat2 RNAs expressing Rluc in the URRL (Fig. 2A) using conditions where the 5' cap Glob-Fluc was also present in excess (Fig. 2B; grey bars). Results show that under these stringently competitive conditions Tat1 RNA and Tat2 RNA at a concentration of 1 × 10-9 M were translated (Fig. 2B, white and black bars, respectively) and levels of Tat RNA translation linearly increased with increasing RNA concentrations (see white and black bars).
Taken together, these results show that the two HIV-1 Tat RNAs were efficiently translated in the URRL under conditions where both the endogenous globin mRNA and the recombinant Glob Fluc RNA were in vast excess. These findings also show that even at low concentrations the Tat mRNA can efficiently recruit ribosomes for its own translation.
Investigating Tat RNA translation in the rabbit reticulocyte lysate
Results reported in figure 3B show that translation in the RRL of the mono 5'Glob-RNA was 5' cap-dependent (see β-galactosidase levels in lanes 2 and 3), while that of the mono EMCV RNA was not (compare lanes 5 and 6). In agreement with this, β-galactosidase was synthesized in the context of the bicistronic Bi-EMCV RNA (lane 7) but clearly not synthesized when the Bi-Glob RNA was used as template (see B-Gal in lane 4). Results showed that for the The Tat RNAs the mono-Tat1 and mono-Tat2 RNAs (Fig 3C) were translated in RRL (Fig. 3D). Strikingly, in the monocistronic context, Tat1 and Tat2 RNA translation occurred independently from the 5' cap structure (Fig. 3D, compare lanes 1–2 and 4–5, respectively). In agreement with this observation, the two cistrons of the Bi-Tat RNAs were clearly expressed in the RRL (see Renilla and Tat in lanes 3 and 6) albeit Tat was synthesized about 2.5 fold less as compared with the monocistronic RNA (compare lanes 2–3 and 5–6 in Fig. 3D). Thus, data show that Tat can be synthesized in a cap-independent manner (Fig. 3C, lanes 1–2) and as the 3' cistron of a bicistronic mRNA (lane 3) while the globin 5' UTR was unable to direct β-galactosidase synthesis under the same experimental conditions (Fig. 3B, lane 4).
To further investigate Tat RNA translation in the RRL, the monocistronic RNAs encoding the Tat protein were translated in the presence of the 7methyl-GTP cap analog (Fig. 3E, bottom panel). The rational of this experiment relies on the competitive binding of initiation factor eIF4E to the m7Gppp cap analog, which has been added in excess to the in vitro reaction. Figure 3E (top panel, lane 2 and 3) recapitulates results presented in Figure 3B (lanes 2 and 3) where translation of the mono-Glob RNA is cap dependent. As expected, the 7m-GTP cap analog reduced by 7 fold the translation of the capped mono-Glob RNA in the RRL (lanes 2 and 3 in top and bottom panels) and had only a marginal effect on the uncapped mono-Glob RNA. The 7m-GTP cap analog did not affect translation from the mono-EMCV RNA (compare lanes 4–5, in top and bottom panels). In agreement with previous data (Fig. 3D), translation of the mono-Tat RNAs was not altered by the addition of the 7m-GTP cap analog (Fig. 3E, top and bottom panel, lanes 6–9).
Taken together, these results show that in the RRL the HIV-1 Tat mRNA can be translated by an IRES mechanism.
Tat mRNA translation in human HeLa P4 cells
To examine Tat mRNA translation in cells, we selected the human HeLa P4 cells because this cell line is known to support HIV-1 replication and is a convenient indicator system to monitor HIV-1 Tat-mediated transactivation of the viral LTR. In HeLa P4 cells the expression of the LacZ gene is under the control of the LTR. Therefore, Tat expression will trans-activate the viral LTR and turn on production of β-galactosidase (see methods). In this experimental setting, the expression of the β-galactosidase reporter is used as an indicator of Tat protein production. In a first series of DNA transfection assays, it was found that β-galactosidase was efficiently expressed upon transfection of the full length Tat1 and Tat2 DNAs (data not shown).
Results show that Rluc was expressed in a dose-dependent manner upon transfection of the three recombinant pdual DNAs (Fig. 4C). It is noteworthy that β-galactosidase was expressed at a high level following pdualTat1 and pdualTat2 transfection but was about 5–6 times less with pdualTatcod, the construct lacking the Tat 5'UTR (Fig. 4B). In these experiments, Tat expression from the pdualTatcod vector was considered as background due to the leakiness of the experimental system. These ex vivo data support our previous findings indicating that the Tat mRNA can be translated in the context of a bicistronic mRNA by a cap-independent mechanism.
These results indicate that in such a bicistronic context the 5' UTR sequences from the PBS to the Tat initiation codon are necessary for Tat protein synthesis. Taken together, the data presented in figures 3, 4, 5 strongly suggest that the Tat mRNA can be translated via an IRES-dependent mechanism both in vitro and in cell culture .
Trans-activation of Tat RNA translation by Tat in HeLa P4 cells
Translational control of specific mRNAs is normally driven by regulatory protein complexes that recognize particular elements that are usually present in the 5' and/or 3' untranslated regions (UTRs) of the target mRNA [21–24]. Because Tat binds with high affinity to the 5' TAR element, we wondered whether such a specific interaction would have an impact on the translation of the Tat mRNAs. Along this line, the Tat-TAR interaction has been described to have an impact on translation of the full length HIV-1 mRNA .
Results from a first series of experiments revealed that Tat was able to trans-activate the translation of the UTR-Tat and UTRg-RNAs (Additional file 2B), but not that of pRenilla (data not shown). In the next series of assays, we transfected low quantities of the Tat expressing DNA (from 2 to 20 ng) and monitored Rluc activities (Additional file 2B). Upon normalization to the same RNA copy number as assessed by RT-qPCR (see methods), the results showed that Tat was able to activate by 5–10 fold the translation of the viral Tat RNA and genomic RNA 5' UTRs (see figure 6B and Additional file 2A). This Tat-mediated activation of translation occurred for very low quantities of transfected Tat DNA (2 to 20 ng per 2.5 × 105 cells), and this was clearly less efficient with higher amounts of Tat DNA (40–200 ng per 2.5 × 105 cells) (Additional file 2B). It should also be noted that the 5' UTR of the HIV-1 genomic mRNA was about 3–4 fold less efficient than the Tat 5' UTR in promoting Rluc expression in HeLa cells, with or without Tat (Fig. 6B, compare top and bottom panels, first and last bars, respectively).
Interestingly, deletion of the TAR-polyA sequences (p5'UTR2-Tat Renilla) had two effects, leading to a higher level of Rluc translation and no influence of Tat (Fig. 6C) as compared with the p5'UTR-Tat Rluc construct (compare Fig. 6B and 6C). These observations confirm that the HIV-1 5'UTR restricts HIV-1 mRNA translation and suggest that the Tat-TAR interaction relieves the translational repression imposed by the leader structure [13, 46, 47]. As expected, Tat had no effect on the expression of Rluc using the pRenilla construct (Fig. 6C, top panel).
Analysis of Tat-mediated activation of Tat RNA translation in the RRL
Several studies show that Tat protein requires other cellular factors to exert the translational activation of the full length HIV-1 mRNA [12–14]. Studies in Xenopus laevis oocytes show that the HIV-1 RNA-Tat protein complex must be assembled in the nucleus in order to facilitate translation in the cytoplasm . In agreement with these observations, the above findings show that Tat protein exerts a translational control on viral mRNA translation from the 5'UTR. Furthermore, data show that this phenomenon occurs even when low quantities of the Tat plasmid are used (Fig. 6B). Since Tat has potent RNA binding and chaperoning activities  and stimulates translation from the viral mRNA, we sought to evaluate if the Tat-TAR interaction was responsible for the activation of viral RNA translation and to establish if translational control by Tat required other cellular factors. This possibility was investigated in vitro in the RRL and URRL using a recombinant version of the Tat (1–86) protein , under different experimental conditions.
Firstly, Tat was added to the RRL or URRL followed by either one of the viral RNA, namely UTR-Tat or UTRg-RNA expressing Rluc. Under these conditions, Tat was found to have no, or at best a modest, positive effect on viral RNA translation in vitro (data not shown). Secondly, Tat was mixed with the RNA in vitro, and then the mix was added to the RRL/URRL translation mixture. Under these conditions translation of RNA containing either the complete 5' UTR of the Tat RNA or of the genomic RNA was decreased up to 3–4 fold upon addition of Tat (Additional file 3). At the same time, Tat only slightly decreased the translation of the Rluc RNA and that of a 5' UTR-Tat RNA where the TAR-polyA has been deleted (Additional file 3). Thirdly, Tat synthesized in the RRL and the Tat/RRL mixture was added to either one of the viral Rluc RNAs and to the control Rluc RNA. Under these conditions, increasing quantities of Tat/RRL were found to strongly inhibit Rluc translation from the viral 5'UTR and only slightly inhibit that of the control Rluc RNA (data not shown).
Taken together these results show that the recombinant Tat protein was not capable of exerting a positive effect on the translation of its cognate mRNA. Furthermore, data suggest that the Tat-TAR interaction inhibited protein synthesis. We therefore reasoned that Tat-mediated translational activation of the HIV-1 RNA might require post-translational modifications  and/or cellular cofactors that are absent from the rabbit reticulocyte lysate. To examine this possibility, URLL was supplemented with HeLa cell extracts. The rationale of using these extracts relies on reports showing that HeLa cell extracts support translation of the full length HIV-1 RNA  and that supplementation of RRL with cytoplasmic HeLa extracts allowed efficient translation from the HIV-1 genomic 5' UTR [40, 50, 51]. The addition of increasing amounts of HeLa cell extracts, up to 0.2 μg/μl, to the URRL prior to RNA translation did not modify the pattern of Rluc translation using the viral RNAs or the control RNA. Addition of recombinant Tat (see materials and methods) to the cell extract before translation had a slightly inhibitory effect on viral and control Rluc RNA translation (data not shown).
Taken together these results favor the notion that Tat requires post-translational modifications to be fully active as a translational activator of its own mRNA. Alternatively, Tat needs to interact with cellular factors, most probably in the nucleus, in order to be able to activate translation of the HIV-1 Tat and full-length RNAs in the cytoplasm [12–14]. This last possibility stems from the fact that HeLa cell extracts were incapable of assisting Tat-associated translational activation when directly mixed with the viral protein.
Tat mRNA translation appears to rely on an IRES mechanism, in a manner similar to that found for the HIV-1 full-length RNA [39, 40]. This finding is not without precedent since the Env mRNA of the gammaretrovirus MoMuLV was shown to be translated by an IRES mechanism, in a manner similar to that of the full-length RNA coding for Gag and Gag-Pol . In the case of the HIV-1 genomic IRES, Brasey et al.  showed that the IRES overlaps the primer binding site (PBS), the DIS, the splice donor (SD) and the major Psi packaging signal that are located upstream from the Gag initiation codon (Fig. 1 and 8). Evidence was also provided showing that sequences encompassing the PBS, the DIS and the SD had an IRES activity (Figure four in ). In agreement with these observations, we found that the TAR and polyA stem-loops were not necessary for the IRES activity of the Tat RNA (Fig. 5). Moreover, the Tat1 IRES activity was found to be clearly more active than its genomic counterpart (Fig. 6).
The above prompted us to look for a possible secondary structure of the 5' UTR of the Tat RNA using a bioinformatic approach (see methods) [52, 53]. A putative consensus secondary structure  is proposed in figure 8 where the most conserved secondary structures in the 5' UTR of Tat1 and Tat2 RNAs, which are not found in the 5' UTR of the genomic RNA [ and ref. therein], are (i) an interaction between the PBS sequence and the 5' part of Tat exon 2, (ii) a small non-structured segment rich in guanine and adenine residues 5' to the Tat AUG codon, and (iii) a small stem-loop next to the Tat AUG codon. In contrast, the TAR and polyA stem-loops remain as individual structures present in the 5' UTR of both the Tat and genomic RNAs, but are not required per se for the IRES activity (, and Fig. 5). The single-stranded segments present in the Tat 5' UTR could function as a landing pad for the binding of ribosomes near or at the Tat initiator codon (Fig. 8). This possibility is presently under investigation. Moreover, the Tat IRES appears to actively recruit the translation initiation complex to the benefit of Tat synthesis, a process absolutely required for the sustained expression of the viral DNA in the unintegrated or integrated forms [1–3, 15]. Cap-dependent translation is suppressed during the G2/M phase of the cell cycle . Interestingly, HIV-1 full length RNA synthesis is highly stimulated during the viral induced G2/M arrest . It is tempting to speculate that this IRES activity ensures Tat protein synthesis during the G2/M phase of the cell cycle [40, 58]. Synthesis of Tat during G2/M would be partially responsible for the high degree of transcriptional activity from the viral promoter observed during this phase of the cell cycle.
Next we studied the influence of Tat on the translation of its cognate mRNA. Results showed that Tat was able to trans-activate the translation of Tat RNA, by up to tenfold in HeLa cells (Fig. 6A, B), but not the translation of the Rluc RNA (Fig. 6C, top panel). In addition, the 5' terminal TAR-polyA sequences are required for the activation of Tat RNA translation (Fig. 6C, lower panel). The Tat-mediated activation of translation also most probably benefits the other viral RNAs, notably the genomic RNA (Fig. 6B, lower panel), which is in agreement with the finding of Leibowitz . We did not succeed in fully reconstituting the translational activation of the viral RNAs by Tat in the RRL systems (Fig. 7 and data not shown). A likely explanation for these observations is that the recombinant Tat protein used here conserves the RNA binding activity but is incapable of recruiting cellular proteins required for the Tat-mediated activation of translation. Results obtained with HeLa and Tat-HeLa cell extracts are however in agreement with what has been described in other experimental systems [12–14] and favour the notion that Tat needs to be post-translationally modifed or needs to contact cellular factors in order to be able to activate translation from the viral mRNA. An interesting possibility that also stems from our findings is that Tat expression in cells might transactivate cellular mRNAs coding for proteins required for Tat to function as a translational activator of its cognate mRNA. Tat is a basic protein with nucleic acid binding and chaperoning activities , and thus appears to behave as a scaffolding protein for both viral transcription and translation [1–11]. Yet, cellular factors recruited by Tat for the translational activation of the viral RNA remain to be determined , and this recruitment is presently under investigation.
This study shows that the Tat mRNA can be efficiently translated under unfavourable conditions, suggesting that only minute concentrations of mRNA are required to assure the Tat protein concentration needed to stimulate viral mRNA synthesis and translation. Moreover, we show that the viral protein Tat exerts a translational control over its cognate mRNA.
Human HeLa P4 cells, which express the CD4 receptor and the bacterial LacZ gene under the control of the HIV-1 LTR, were maintained in complete Dulbecco's Modified Eagles's Medium with Glutamax (DMEM, Gibco, Life Technologies Corporation, Carlsbad, California, USA), supplemented with 10% FCS and penicillin and streptomycin antibiotics.
Construction of the Tat DNA sequences
DNA oligonucleotides used in the present study
Sequence and position on Tat mRNA
LeaderR sense EcoRI
5' GAATTCGGTCTCTCTGGTTAGACCAGATC 3'
(exon1 sense for Tat1 et Tat2: 1→)
5' TATTCTGCTATGTCGACACCCAATTCAGTCGCCGCCCCTCG 3'
(exon1 reverse for Tat1:← 290)
5' GGATCTCTGCTGTCCCTGCAGTCGCCGCCCCTCG 3'
(exon1 reverse for Tat2:← 290)
5' AATTGGGTGTCGACATAGCAGAATAGGCG 3'
(exon2 sense for Tat1:290→ and for Tat2:343→)
5'GGGATTGGGAGGTGGGTTGCTTTGATAGAGAAGCTTGATGAGTCTGACTG3' (exon2: reverse for Tat1:← 558 and for Tat2:← 611)
5'CAGGGACAGCAGAGATCCAGTTTGGAAAGGACCAGCAAAGCTCCTCTGGAAAG 3' (exon':sense for Tat2:290→)
5' CTGCTATGTCGACACCCAATTCTTTCCAGAGGAGCTTTGCTG 3'
(exon':reverse for Tat2:← 343)
5' ACCCACCTCCCAATCCCGAG 3'
(exon3: sense for Tat1:558→ and for Tat2: 611→)
5' TAATAATGCGGCCGCAGTACAGGCAAAAAGCAGCTGCTTATATGC 3'
(exon3: reverse for Tat1:← 1718 and for Tat2:← 1770)
5' TATATTAGAATTCGTGTGCCCGTCTGTTGTGTGACT 3'
(exon1: sense for Tat1 and for Tat2:104 →)
5' ATATAAGAATTCCGAGGGGCGGCGACTG 3'
(exon1: sense for Tat1 and for Tat2:274→)
5' TATAATAGAATTCATGGAGCCAGTAGATCCTAGACTAGAG 3'
(exon2: sense for Tat1:343 → and for Tat2:396 →)
5' TAATATACCATGGGGTCTCTCTGGTTAGACCAGATC 3'
(exon1: sense for Tat1 and for Tat2:1→)
5' TATATACCCGGGAGTACAGGCAAAAAGCAGCTGCTTATATGC 3'
(exon3: reverse for Tat1:← 1718 and for Tat2:← 1770)
5' ATATATTCTAGAGGTCTCTCTGGTTAGACCAGATC 3'
(exon 1:for Tat1 et Tat2:1→)
5' TAATAATTCTAGAAGTACAGGCAAAAAGCAGCTGCTTATATGC 3'
(exon3 reverse for Tat1:← 1718 and for Tat2:← 1770)
5' TATATACCATGGGGCACACACTACTTTGAGCACTCAAGG 3'
5' TATATTACCATGGTTCTTGCTCTCCTCTGTCGAGTAACG 3'
(exon2: reverse for Tat1:← 342 and for Tat2:← 395)
5' ATATATACCATGGCTCTCTCCTTCTAGCCTCCGC 3'
(reverse: 5' to AUG of the RNAg)
5' TAATATACCATGGGTGTGCCCGTCTGTTGTGTGACT 3'
(exon1: sense for Tat1 and for Tat2:104 →)
5' TAATATACAGCTGTGGAAGGGCTAATTTGGTCCC 3' (sense for U3)
5' TATATTACAGCTGAGTACAGGCAAAAAGCAGCTGC 3' (reverse for U3)
The Tat DNA constructs also contain either the 5' LTR for ex vivo expression, or the T7 RNA polymerase promoter for in vitro RNA synthesis (see below). In addition, it was necessary to omit the 3' R sequence to prevent frequent recombination reactions during amplification.
The Tat exons were independently PCR amplified (Fig. 1B) using the designed ODNs (Table 1 and Additional file 1) and the Vent polymerase (New England Biolabs, Ipswich, MA, USA). Then, reconstitution of the Tat1 and Tat2 DNA fragments was carried out (Fig. 1B): minus strand ODNs were designed in such a way that their 5' extremity was complementary to the 5' extremity of the plus strand of the next exon (Table 1). Thus, by means of a "hybridization PCR" procedure, each exon was linked to the next one and the resulting DNA was subsequently amplified by PCR using additional ODNs (Table 1).
Plasmid DNA construction
(i) Plasmid DNA with Tat sequences. The complete Tat1 and Tat2 DNA fragments (Fig. 1B) and the deleted Tat1 and Tat2 DNA fragments were cloned into pD2EGFP-N1 (Clontech Mountain View, CA, USA) at the EcoRI/NotI sites, in place of the eGFP gene. The Tat DNA fragments in pD2EGFP-N1 were recovered upon EcoRI/XbaI digestion and cloned into the pdualuc bicistronic plasmid , cleaved by EcoRI/XbaI.
The same Tat DNA fragments were also cloned into the p0pRenilla vector at the XbaI restriction site, next to the Renilla Luciferase gene.
The HIV-1 U3 promoter/enhancer region was PCR amplified as above using the HIV-1 pNL4.3 DNA template and cloned into p0pRenilla at the PvuII site, generating the pRenilla plasmid. We used this new DNA construct to insert at the NcoI site, 5' to the Renilla gene, each one of the DNA fragments corresponding to the complete 5' UTR of Tat1 and Tat2 RNAs, to the deleted 5' UTR (delta R-U5) of Tat1 and Tat2 RNAs, to the 5' UTR of the viral genomic RNA, and to the 5' first 111 nt of Tat RNA.
The Tat1 and Tat2 DNA fragments were also cloned into pRenilla at the NcoI/SmaI sites, in place of the Renilla Luciferase gene.
(ii) Other plasmid DNA. Plasmids pAB300-UTRGlobin and pAB300-UTREMCV contain the 5' UTR of the globin and EMCV RNA, respectively, at the NheI site just before the LacZ gene in pAB300.
Plasmids pBis-UTRGlobin and pBis-UTREMCV contain the 5' UTR of the globin and EMCV RNA, respectively, at the NheI site in the intercistronic region of the pBis plasmid .
HeLaP4 cells were plated at 250 000 cells per well in six-well plates in complete medium and DNA was transfected using Lipofectamine and Plus reagent (Invitrogen Life Technologies Corporation, Carlsbad, California, USA). 48 hours post transfection, HeLa P4 cells were lysed with 250 μl of lysis buffer per well and the Renilla Luciferase and β-Galactosidase activities were monitored with the 'Renilla Luciferase Assay system' (Promega Corporation, Madison, WI, USA) and 'β-gal Reporter Gene Assay' (Roche Molecular Systems, INC., Branchburg, NJ, USA), respectively. All measurements were performed with a Promega luminometer by substrate injection. All results were normalized for the same amount of total proteins in the HeLa cell extracts.
Reverse transcription and quantitative PCR reactions
RNA was extracted from cells using the Trizol reagent (Invitrogen), according to the manufacturer's instructions. Two μg of total cellular RNA were used per reverse transcription reaction using the SuperScript II reverse transcriptase (Invitrogen), and 1 μM of the given ODN (Table 1).
The mixture was heated for 5 min at 65°C and then kept on ice. Next it was incubated for 2 min at 42°C and RT was added. The reaction was for 50 min at 42°C.
To quantitatively assess RNA levels by cDNA amplification, we used the 'LighCycler FastStart DNA Master SYBR Green kit (Roche).
In vitroRNA synthesis and translation
The DNA templates of interest were linearized, purified by a phenol/chloroform extraction, and ethanol precipitated. 2 μg of DNAs were used per transcription reaction. In vitro RNA synthesis was performed as previously described [38, 44] for 1 h 30 min at 37°C in 50 μl final volume. For capped RNA synthesis, m7Gppp was added at the beginning of the reaction at 1 mM final concentration.
40 μl of LiCl (7.5 M, 75 mM EDTA) were added to the transcription reaction, which was kept for 30 min at -20°C. Then RNAs were recovered by centrifugation at 14000 g, 4°C for 30 min. The RNA pellets were washed with 120 μl of 70% ethanol, dissolved in 30 μl of pure water and kept at -20°C.
RNA translation in the rabbit reticulocyte lysate system
In vitro synthesized RNAs (5–100 ng) were translated in 10 μl of either 25% Flexi® Rabbit Reticulocyte System (Promega, USA) or the supplemented untreated RRL 50% (v/v) (as described in ) in the presence of 75 mM KCl, 0.5 mM MgCl2, 20 μM of each amino acid (minus cysteine) and 0.6 mCi/ml of [35S]-cystein (GE Healthcare Life Sciences Piscataway, NJ, USA).
Tat protein added to the in vitro translation reactions was chemically synthesized as described in  (CNRS, Immunologie et Chimie Thérapeutiques, UPR 9021- Strasbourg).
Reactions were at 30°C for 45 min and stopped by the addition of 90 μl of buffer (0.1 mM DTT; 35% glycerol; 0.2 M Tris-HCl pH 6.8; 1% SDS; 0.5% bromophenol blue). 10 μl were loaded onto a 15% polyacrylamide-SDS gel (PAGE-SDS). After protein resolution, the gel was fixed in a solution containing 30% methanol and 10% acetic acid for 30 min, and subjected to autoradiography using Biomax films (Eastman Kodak, USA). Densitometric analyses were performed by Phospho Imaging with a Storm 850 phosphoimager. To evaluate the translation level of RNA encoding Renilla Luciferase, we monitored the Renilla luciferase activity directly from the translation reaction. Reactions were stopped with 40 μl of lysis buffer from the " Renilla Luciferase assay system" and 20 μl of that mixture were used to quantify the Renilla luciferase activity by luminometry.
Cytoplasmic extract of HeLa P4 cells
HeLa P4 cells (1 × 107) were washed with PBS, trypsinized and transferred in a 15 ml tube, and then centrifuged at 1500 rpm at 4°C for 5 min. All subsequent steps were carried out on ice. The cell pellet was washed twice with 10 ml of PBS, 2% FCS and centrifuged at 1500 rpm for 5 min. The cell pellet was resuspended in two volumes of hypotonic buffer (HEPES-KOH 10 mM, pH 7.6, potassium acetate 10 mM, MgOAc 0.5 mM, DTT 1 mM, protease inhibitors, and RNasin (40 U/ml)), and cells were lysed by passing through a needle. The cellular lysate was centrifuged at 14000 g for 10 min and the supernatant was analysed for its total protein content and kept at -80°C.
Two methods have been used to directly assess Tat expression in HeLa P4 cells.
Firstly, a Hybond-P membrane (GE Healthcare) was activated by means of a methanol-air treatment and rinsed in 20% methanol, 25 mM Tris pH 8 and 192 mM Glycine. Then, 0.5, 1, 2 and 5 μg of total or cytoplasmic extracts from HeLa P4 cells expressing HIV-1 Tat were carefully spotted onto the membrane. Next, the membrane was incubated during 1 h at 20°C in TBS-T (Tris-HCl pH 8, 50 mM, NaCl 0.15 M, 0.5% Tween 20) containing 5% milk powder, and then for 12–14 hours at 4°C in the presence of the mouse monoclonal anti-Tat antibody (antiTat7S directed againts the basic region of Tat) (a kind gift from Michel Leonetti, the CEA, France). Then, the membrane was extensively rinsed three times in TBS-T, and incubated with an anti-mouse IgG antibody (Dako). The membrane was rinsed three times in TBS-T, and incubated 5 min in the presence of the peroxydase substrate (Supersignal West Pico Chemiluminescent kit, Perbio).
Secondly, classical western blotting was carried out as above except that the cellular extracts (15 μg) were run over a 15% SDS PAGE gel. Detection of the chemoluminescent signals was carried out by autoradiography, as before.
A putative secondary structure for the 5' UTR of Tat1 and Tat2 RNAs was determined by means of bioinformatic analyses.
Firstly, 27 divergent sequences were selected in the HIV-1 genomic RNA database (Los Alamos database) http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html. Reconstitution of the complete Tat1 and Tat2 RNA sequences was carried out by manual splicing. Then, the sequences were aligned with the ClustalW software , and the alignments were manually edited. The obtained alignments were used to establish a consensus secondary structure for the Tat1 and Tat2 RNAs, using the RNAalifold software of the Vienna RNA package . Variability in the aligned RNA sequences, with special emphasis on co-variant sites, together with the consensus structure, was used to infer putatively conserved secondary structures in Tat1 and Tat2 RNAs. Apart from the TAR stem-loop, the most conserved structural features in the 5' UTR of Tat RNAs are: (i) an interaction between the PBS sequence and the 5' part of Tat1 exon 2 which is conserved in all sequences of the Los Alamos database; (ii) a small, non-structured segment rich in guanine and adenine 5' to the Tat AUG codon; and (iii) a small stem-loop next to the Tat AUG codon (Fig. 8). These features were used as constraints in Mfold  for the folding of the pNL4.3 Tat RNA sequences.
Work supported by Grants from the ANRS, Sidaction and INSERM to JLD, grant FONDECYT 1060655 to MLL, and Grant ECOS-CONYTYT C05 S01 to JLD, TO, and MLL. MLL is member of the Jeune Equipe Associée à l'IRD (LVMEIE) and the Instituto Milenio de Inmunología e Inmunoterapia (NMII). NC was supported by Sidaction. Thanks are due to Sylviane Müller (CNRS Strasbourg, FRANCE) for providing the Tat protein in a highly pure form, and to Michel Léonetti (CEA, Saclay, France) for the monoclonal anti-Tat 7S and 11S antibodies.
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