The host protein Staufen1 interacts with the Pr55Gagzinc fingers and regulates HIV-1 assembly via its N-terminus
© Chatel-Chaix et al; licensee BioMed Central Ltd. 2008
Received: 17 January 2008
Accepted: 22 May 2008
Published: 22 May 2008
The formation of new infectious human immunodeficiency type 1 virus (HIV-1) mainly relies on the homo-multimerization of the viral structural polyprotein Pr55Gag and on the recruitment of host factors. We have previously shown that the double-stranded RNA-binding protein Staufen 1 (Stau1), likely through an interaction between its third double-stranded RNA-binding domain (dsRBD3) and the nucleocapsid (NC) domain of Pr55Gag, participates in HIV-1 assembly by influencing Pr55Gag multimerization.
We now report the fine mapping of Stau1/Pr55Gag association using co-immunoprecipitation and live cell bioluminescence resonance energy transfer (BRET) assays. On the one hand, our results show that the Stau1-Pr55Gag interaction requires the integrity of at least one of the two zinc fingers in the NC domain of Pr55Gag but not that of the NC N-terminal basic region. Disruption of both zinc fingers dramatically impeded Pr55Gag multimerization and virus particle release. In parallel, we tested several Stau1 deletion mutants for their capacity to influence Pr55Gag multimerization using the Pr55Gag/Pr55Gag BRET assay in live cells. Our results revealed that a molecular determinant of 12 amino acids at the N-terminal end of Stau1 is necessary to increase Pr55Gag multimerization and particle release. However, this region is not required for Stau1 interaction with the viral polyprotein Pr55Gag.
These data highlight that Stau1 is a modular protein and that Stau1 influences Pr55Gag multimerization via 1) an interaction between its dsRBD3 and Pr55Gag zinc fingers and 2) a regulatory domain within the N-terminus that could recruit host machineries that are critical for the completion of new HIV-1 capsids.
Human immunodeficiency type 1 (HIV-1) assembly consists in the formation of new viral particles which is the result of the radial multimerization of approximately 1,400 to 5,000 copies of the viral polyprotein Pr55Gag (also named Gag) according to their quantification in mature or immature particles, respectively [1–3]. Pr55Gag is thought to contain most of the determinants required for viral assembly since the expression of Pr55Gag alone leads to the formation and release of virus-like particles (VLPs), structurally not really distinguishable from immature HIV-1 [4–6]. Pr55Gag is a modular protein that contains 6 domains: matrix (MA), capsid (CA), nucleocapsid (NC), p6 and two spacer peptides, p2 and p1. Each of these domains plays specific roles during HIV-1 life cycle. During assembly, the MA domain, through its myristylated moiety and its highly basic domain, anchors assembly complexes to membranes [4–6]. Whether assembly takes place at the inner leaflet of the plasma membrane or at the multivesicular bodies (or both) is still under debate [7–17].
Pr55Gag multimerization is likely initiated by NC/NC contacts [18, 19] probably when Pr55Gag is still in a cytosolic compartment [20–23]. The basic amino acid stretch present in NC is thought to non-specifically recruit RNA that serves as a scaffold for multimerizing Pr55Gag [24–26]. Indeed, mutations abrogating the global positive charge of this sub-domain compromise viral assembly [24, 25]. NC also possesses two zinc fingers that are important for the specific packaging of HIV-1 genomic RNA [27–29]. Recently, Grigorov et al. demonstrated the involvement of both NC zinc fingers in Pr55Gag cellular localization and HIV-1 assembly . Similarly, the first NC zinc finger was shown to be part of the minimal Pr55Gag sequence required for multimerization (called the I domain) [5, 6]. Since NC function during assembly can be mimicked by its substitution with a heterologous oligomerization domain [31, 32], NC/NC contacts probably serve as a signal for the higher order multimerization of Pr55Gag under the control of other domains. Indeed, the C-terminal third of the CA domain and the spacer peptide p2 are part of the I domain and have been shown by mutagenesis and structural analyses to be also very important players during HIV-1 assembly [26, 33–42].
The HIV-1 assembly process within the cell appears to be tightly regulated in time and space and relies on the sequential acquisition and release of host proteins that are required for the cellular localization, multimerization and budding of new capsids [4, 43]. For instance, the ATP-binding protein ABCE1/HP68 is important for the completion of Pr55Gag multimerization via a transient interaction with the NC domain of Pr55Gag [44–47]. Adaptor proteins 1, 2, 3 (AP-1; AP-2; AP-3) are involved in Pr55Gag intracellular trafficking through their association with the MA domain of Pr55Gag [12, 48, 49]. Finally, endosomal sorting complex required for transport (ESCRT)-I and -III machineries are recruited by the p6 domain of Pr55Gag and are crucial for the budding and release of the neosynthesized viral particles .
Staufen1 (Stau1) is also a Pr55Gag-binding protein that influences HIV-1 assembly [51–53]. Stau1 belongs to the double-stranded RNA-binding protein family [54, 55] and is involved in various cellular processes related to RNA. Stau1 was first studied for its role in the transport and localization of mRNAs in dendrites of neurons . More recently, Stau1 was identified as a central component of a new mRNA decay mechanism termed Staufen-mediated decay . In addition to its functions in RNA localization and decay, Stau1 can also stimulate translation of repressed messengers containing structured RNA elements in their 5'UTR .
Stau1 is a host factor that is selectively encapsidated into HIV-1 . Stau1 co-purifies with HIV-1 genomic RNA and interacts with the NC domain of Pr55Gag [52, 53] suggesting that Stau1 assists NC's functions during the HIV-1 replication cycle. Stau1 levels in the producer cells are important for HIV-1 since both Stau1 overexpression and depletion using RNA interference affect HIV-1 infectivity [52, 53]. In addition to a putative role in HIV-1 genomic RNA packaging , we recently showed that Stau1 modulates HIV-1 assembly by influencing Pr55Gag multimerization . Indeed, using a new Pr55Gag multimerization assay relying on bioluminescence resonance energy transfer (BRET), we demonstrated that both Stau1 overexpression and depletion enhanced multimerization and consequently increased VLP production. Although Stau1 and Pr55Gag interact in both cytosolic and membrane compartments, this effect of Stau1 on Pr55Gag oligomerization was only observed in membranes, a cellular compartment in which Pr55Gag assembly primarily occurs. However, the mechanism by which Stau1 influences HIV-1 assembly at the molecular level remains unknown although it is likely that it relies on the Stau1 interaction with HIV-1 Pr55Gag.
Using co-immunoprecipitation and BRET assays, we showed that both Pr55Gag NC zinc fingers are involved in Stau1/Pr55Gag interaction as does the Stau1 dsRBD3 . Unexpectedly, we found that the binding of Stau1 to NC is not sufficient per se to fully enhance Pr55Gag multimerization. To determine which domain of Stau1 modulates the HIV-1 Pr55Gag multimerization process, we analyzed several Stau1 deletion mutants for their capacity to enhance Pr55Gag multimerization. Using the Pr55Gag/Pr55Gag BRET assay either in live cells or after cell fractionation, we showed that the first 88 amino acids at the N-terminal of Stau1 confer the capacity to enhance both Pr55Gag multimerization and VLP production. Although unable to enhance multimerization, this mutant was still able to interact with Pr55Gag. This study provides important new information about the molecular determinants required for Stau1 function in HIV-1 assembly.
Cell culture and reagents
Human embryonic kidney fibroblasts (HEK 293T) were cultured in Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with 10% cosmic calf serum (HyClone) and 1% penicillin/streptomycin antibiotics (Multicell). Transfections were carried out using either the calcium phosphate precipitation method or the Lipofectamine 2000 reagent (Invitrogen). For Western blots, mouse and rabbit HRP-coupled secondary antibodies were purchased from Dako Cytomation and signals were detected using the Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). Signals were detected with a Fluor-S MultiImager apparatus (Bio-Rad). Anti-Na-K ATPase antibodies were kindly provided by Dr. Michel Bouvier.
The construction of pcDNA3-RSV-Stau155-HA3, pcDNA3-RSV-Stau1F135A-HA3, pcDNA-RSV-Stau1ΔNt88-HA3, pCMV-Stau155-YFP, pCMV-Stau1F135A-YFP, pCMV-Stau1ΔNt88-YFP, pCMV-Stau1ΔdsRBD3-YFP, pCMV-Pr55Gag-Rluc, pCMV-Pr55Gag-YFP, pCMV-NC-p1-YFP and pCMV-CA-p2-NC-p1-Rluc was reported before [51–54, 59]. The HxBRU PR-provirus and the Rev-independent Pr55Gag expressor were described before [51, 53, 60].
To construct pcDNA-RSV-Stau1ΔNt37-HA3, a polymerase chain reaction (PCR) was performed using pcDNA3-RSV-Stau155-HA3 as template, sense (5'-ATCAGGTACCATGGGTCCATTTCCAGTTCCACCTTT-3') and anti-sense (5'-CACATCTAGATCATTTATTCAGCGGCCGCACTGAGCAGCGT-3') oligonucleotide primers and the Phusion DNA polymerase (New England Biolabs). The PCR product was purified and digested with KpnI and XbaI restriction enzymes (Fermentas) and then cloned into the KpnI/XbaI cassette of pCDNA3-RSV.
To generate pcDNA-RSV-Stau155-Flag plasmid, oligonucleotides (5'-GGCCTTGATTACAAGGATGACGATGACAAG-3' and 5'-GGCCCTTGTCATCGTCATCCTTGTAATCAA-3') were hybridized and then inserted into the NotI sites of pcDNA-RSV-Stau155-HA3 in replacement of the HA-tag. For the construction of pcDNA-RSV-Stau1ΔNt88-Flag, the EcoRI fragment of pcDNA-RSV-Stau1ΔNt88-HA3 that contained the mutated Stau1 sequence, was cloned into EcoRI-digested pcDNA-RSV-Stau155-Flag plasmid.
The expressors of NC-p1-YFP and Pr55Gag-YFP mutants were PCR amplified using the PCR all-around technique  to generate the following mutations: the C15S mutation was introduced with the primer pair 5'-AAGAGTTTCAATTGTGGCAAA-3' and 5'-GAAACTCTTAACAATCTTTCT-3'; the C49S mutation was generated with the primer pair 5'-GATAGTACTGAGAGACAGGCT-3' and 5'-AGTACTATCTTTCATTTGGTG-3'; R7S, R10S and K11S mutations (R7 mutant) were introduced with the primer pair 5'-TTTAGCAACCAAAGCTCGATTGTTAAGTGTTTC-3' and 5'-AATCGAGCTTTGGTTGCTAAAATTGCCTCTCTG-3'. PCR reactions were carried out with the Phusion enzyme (New England Biolabs) at 95°C for 50 s, 55°C for 60 s and 72°C for 90 s, for 18 cycles. Resulting products were incubated with 10 units of DpnI enzyme (Fermentas) and then transformed into competent bacteria. Positive clones containing the mutation(s) were screened by restriction and sequencing analyses. The double zinc fingers mutant expressors (pCMV-Pr55Gag C15–49S-YFP and pCMV-NC-p1C15–49S-YFP) were generated by PCR with the oligonucleotide primer pair for the C49S mutation using the corresponding plasmids that contain the C15S mutation.
Membrane flotation assays and S100-P100 fractionation
Forty hours post-transfection, cell extracts were prepared by passing the cells 20 times through a 23G1 syringe in TE (10 mM Tris pH7.4, 1 mM EDTA pH 8) containing 10% sucrose and proteases inhibitors (Roche). Nuclei were removed by centrifugation at 1,000 × g. Resulting cytoplasmic extracts were separated using the membrane flotation assay as previously described . Membrane-associated complexes were collected (fractions 2 and 3). Membranes were solubilized by treating these complexes with 0.5% Triton X-100 at room temperature for 5 minutes and samples were subjected to S100/P100 fractionation as previously described  by ultracentrifugation at 100,000 × g for 1 h at 4°C. Supernatants (S100 fractions) and pellets (P100 fractions) were collected and analyzed by Western blotting using anti-CA, anti-HA and anti-Na-K ATPase mouse antibodies.
293T cells were transfected in 6-well plates with constant amounts of the Rluc-fused energy donor expressor (25–75 ng), increasing amounts of YFP-fused acceptor expressor (0.25–2 μg) and Stau1-HA3-expressing plasmid (1–1.5 μg) when indicated. 48 hours post-transfection, cells were collected in PBS-EDTA 5 mM and diluted to approximately 2 × 106 cells/mL. BRET assays were performed as described before [51, 52] using a Fusion α-FP apparatus (Perkin-Elmer). In this interaction assay, an X-Rluc fusion protein is used as an energy donour whereas a Y-YFP fusion protein is an energy acceptor. When the two fusion proteins are in close proximity (< 100Å), non-radiative resonance energy is transferred from X-Rluc to Y-YFP which in turn emits measurable fluorescence. This can be quantified by the calculation of the BRET ratio which allows detection of protein-protein interactions. The BRET ratio was defined as [(emission at 510 to 590 nm)-(emission at 440 to 500 nm) × Cf]/(emission at 440 to 500 nm), where Cf corresponds to (emission at 510 to 590 nm)/(emission at 440 to 500 nm) when Rluc fused protein is expressed alone. The total YFP activity/Rluc activity ratio reflects the relative levels of the two fusion proteins in the cells. The BRET ratio increases with the total YFP activity/Rluc activity ratio since more YFP-fused molecules bind to Rluc-fused proteins. For Pr55Gag multimerization assays, in order to avoid misinterpretation due to variations in relative levels of the Pr55Gag fusion proteins, changes in the Pr55Gag/Pr55Gag BRET ratios following Stau1 overexpression were always analyzed at similar total YFP activity/Rluc activity ratio.
When Pr55Gag/Pr55Gag BRET assays were performed following membrane flotation assays, the Rluc substrate coelenterazine H (NanoLight Technology) was added to 90 μL of each fraction and BRET ratio was determined as in live cells. BRET ratios in fractions 1, 3, 4, 5 and 6 were not considered because luciferase activity was too low in these fractions and hence, did not lead to the determination of a reliable BRET ratio.
For CA-p2-NC-p1-Rluc/Stau1-YFP and Stau155-Rluc/NC-p1-YFP interaction assays, BRET ratios were always compared at similar total YFP activity/Rluc activity ratio. The BRET ratio determined in the context of the expression of the unfused YFP protein (YFP) corresponds to non specific interactions between the energy donor and the YFP. Hence, this background BRET ratio was always subtracted from all BRET ratios and was set to 0%. The BRET ratio determined following co-expression of the energy donor and the wild type energy acceptor was set to 100%.
For dose-response Pr55Gag/Pr55Gag BRET assays, 293T cells were transfected with fixed amounts of pCMV-Pr55Gag-Rluc and pCMV-Pr55Gag-YFP and increasing amounts (0.25–2 μg) of different Stau1-HA3 expressors. BRET assays were performed 48 hours post-transfection as described above.
293T cells were transfected with Stau155-flag and Gag expressors using Lipofectamine 2000 (Invitrogen). Twenty hours post-transfection, cells were collected in lysis buffer (150 mM NaCl, 50 mM Tris pH 7.4, 1 mM EDTA, 1% Triton X-100) containing proteases inhibitors (Roche). Each cell lysate (1.5 mg of proteins) was pre-cleared with IgG-agarose (Sigma-Aldrich) for 1 h at 4°C and then subjected to immunoprecipitation using 15 μL of anti-Flag M2 affinity gel (Sigma-Aldrich) for 2 h at 4°C. Immune complexes were washed 3 times during 5 minutes with cold lysis buffer, eluted with the Flag peptide (Sigma-Aldrich), resolved in SDS-containing acrylamide gels and analyzed for their content in Stau1 and Gag proteins by Western blotting using mouse monoclonal anti-Flag (Sigma-Aldrich), anti-GFP (Roche) and anti-CA antibodies.
Virus-like particle purification
293T cells were transfected with Stau155-HA3 and Gag expressors using Lipofectamine 2000 (Invitrogen). Twenty hours post-transfection, supernatants were collected and cleared through a 0.45 μm filter. VLPs were pelleted through a sucrose cushion (20% in Tris-NaCl buffer) by ultracentrifugation during 1 hour at 220,000 × g. VLPs were resuspended in Tris-NaCl buffer and analyzed by Western blotting using anti-CA antibodies. Pr55Gag signals in the VLPs and the cell extracts were quantitated using the Quantity One (version 4.5) software (Bio-Rad).
Both NC zinc fingers mediate Stau1/Pr55Gaginteraction
This suggests that the integrity of at least one NC zinc fingers is required for Stau1/NC interaction.
We used a second technique to confirm the involvement of both zinc fingers in Stau1-NC interaction in the context of full-length Pr55Gag. We generated a Pr55Gag-YFP-expressing plasmid in which both zinc fingers were mutated (Pr55Gag C15–49S-YFP)(see below). As shown in Figure 2C, this mutant was expressed to the same level as the wild-type Pr55Gag-YFP and migrated in SDS-containing acrylamide gels at the expected molecular weight (80 kDa). Following co-expression of Flag-tagged Stau155 with wild type or mutated Pr55Gag-YFP in 293T cells (Figure 2D, upper panel), Stau155-Flag-containing complexes were immunoprecipitated using anti-Flag antibodies. Immunopurified material was analyzed by Western blot using monoclonal anti-GFP and anti-Flag antibodies (Figure 2D, lower panel). As expected, Pr55Gag-YFP successfully co-precipitated with Stau155-Flag. In contrast, despite similar levels of expression in the cell (Figure 2D, upper panel), the Pr55Gag C15–49S-YFP mutant was not efficiently co-immunoprecipitated with Stau155-Flag as compared to wild type (Figure 2D, lower panel) suggesting that the association between this mutant and Stau155 is impaired. Pr55Gag C15S-YFP and Pr55Gag C49S-YFP mutants retained some association with Stau155-Flag although they displayed lower binding capability than the wild type Pr55Gag (not shown), consistent with the BRET assay. Altogether, these results show that the two zinc fingers within the NC domain of Pr55Gag mediate its association with Stau1. Moreover, this suggests that Stau1 influences those assembly processes that depend on NC zinc fingers.
Mutations in the NC zinc fingers severely compromises Pr55Gagmultimerization and release
Then, we determined whether mutations in the zinc fingers affect Pr55Gagmultimerization. Using the BRET assay in live cells, we tested the capacity of Pr55Gag C15–49S-YFP to dimerize with Pr55Gag-Rluc, the wild-type Pr55Gag-YFP being used as control (Figure 3A). As shown in Figure 3C, Pr55Gag homo-dimerization was readily detectable with a BRET ratio of 0.09 at saturation. In contrast, Pr55Gag C15–49S-YFP failed to interact with Pr55Gag-Rluc in the BRET assay since its saturation curve was similar to the one obtained with the monomeric Gag mutant MA-CAWM184–185AA-YFP. Altogether, these results clearly show that, in the context of VLP assembly, Pr55Gag zinc fingers are important for multimerization and release. This suggests that Stau1, through its binding to the NC zinc fingers could influence crucial processes that are controlled by these motifs during HIV-1 assembly.
The N-terminal domain of Stau1 is required for the Stau1-mediated enhancement of Pr55Gagmultimerization
Stau1ΔNt88still interacts with HIV-1 Gag
The ability of Stau1ΔNt88 to associate with Pr55Gag was confirmed in co-immunoprecipitation assays (Figure 5E). Pr55Gag and flag-tagged Stau1 or Stau1ΔNt88 were co-expressed in 293T cells (Figure 5E, left panel) and proteins in the cell extracts were immunoprecipitated using anti-flag antibody. Western blot analyses of the immune complexes showed that Pr55Gag successfully co-precipitated in a specific manner with both Stau155-flag and Stau1ΔNt88-FLAG (Figure 5E, right panel). These results show that, although Stau1ΔNt88 is unable to stimulate Pr55Gag multimerization, its interaction with Pr55Gag was maintained. This result suggests that Stau1 association to Pr55Gag is not sufficient to influence HIV-1 assembly and that Stau1 N-terminus contains a regulatory element that is important for its function during this process.
Stau1ΔNt88-HA3 does not enhance the assembly of membrane-associated Pr55Gagcomplexes
The inability of Stau1ΔNt88-HA3 to modulate Pr55Gag multimerization was then tested in the context of provirus-driven immature HIV-1 production. We had previously shown that Stau1-mediated increase in Pr55Gag multimerization correlated with a partial resistance to mild detergent treatment of membrane-associated Pr55Gag complexes . Stau1 expressors and the protease-defective provirus HxBRU PR-were cotransfected in 293T cells. Forty hours post-transfection, membrane flotation assays on cytoplasmic extracts were performed and fractions containing membrane-associated complexes were collected. The resulting complexes were then subjected to ultracentrifugation at 100,000 × g for 1 hour (Figure 6C). In this assay, insoluble or high-density complexes are found in the pellet (P100) whereas proteins that are soluble or components of small complexes are retained in the supernatant (S100). Resulting P100 and S100 fractions were analyzed by Western blot. As previously shown , Pr55Gag as well as the membrane marker, sodium potassium (Na-K) ATPase, were primarily found in the P100 fraction because Pr55Gag was membrane-associated (data not shown). This was observed whether Stau1 proteins were over-expressed or not (data not shown). To separate Pr55Gag complexes from membranes, membranes were solubilized with 0.5% Triton-X100. These experiments were done at room temperature to also solubilize lipid rafts and other membrane compartments that are detergent-resistant at 4°C. As reported before, membrane solubilization prior to S100/P100 fractionation resulted in a complete shift of Pr55Gagcomplexes and Na-K ATPase into the S100 fraction (Figure 6D). Stau155-HA3 overexpression led to a partial resistance of 33% of Pr55Gag complexes to Triton-X100 treatment, likely as a consequence of enhanced Pr55Gag multimerization (Figure 6D). In contrast, Stau1ΔNt88-HA3, as well as Stau1F135A-HA3 used as control [51, 52] failed to increase the density of Pr55Gag complexes. Altogether, these results support the conclusion that the N-terminus of Stau1 is required for Stau1-mediated increase of Pr55Gag assembly during HIV-1 assembly.
Amino acids 26 to 37 of Stau155 are important for its function in Pr55Gagmultimerization
Effect of Stau1 N-terminal mutants on VLP production
We previously reported that Stau1 participates in HIV-1 assembly by influencing Pr55Gag multimerization . However, very little is known about the molecular mechanisms underlying this process. In this report, we show that, in addition to Pr55Gag-binding via its dsRBD3 [51, 52], Stau1's effect on Pr55Gag multimerization depends on amino acids located within its N-terminus. Moreover, we show important contributions from both NC zinc fingers for the Stau1/Pr55Gag interaction suggesting that Stau1 influences processes that depend on these NC sub-domains.
HIV-1 Gag mutants whose zinc fingers were disrupted, failed to interact with Stau1 as seen in BRET and co-immunoprecipitation assays (Figure 2). This suggests that Stau1 directly makes contact with these structures although we cannot rule out the possibility that these combined mutations affect the structure of other sub-domains of NC and potentially a Stau1-binding motif. Interestingly, viruses that harbour these mutations do not encapsidate Stau1 . Although this phenotype could be attributed to the loss of HIV-1 genomic RNA packaging and to Stau1 RNA-binding activity, this strongly suggests that Stau1 encapsidation into HIV-1 may be achieved by the formation of a Stau1-Gag-RNA ternary complex where Stau1 interactions with both Pr55Gag and genomic RNA contribute to this process [51, 52].
NC zinc fingers are known to play important roles during several steps of the HIV-1 replication cycle such as genomic RNA packaging and reverse transcription [27–29, 63]. In addition, they have been shown to be involved in Pr55Gag assembly [5, 6, 29, 30, 46, 64]. Our results with the BRET assay that monitors direct interaction between wild type and mutated Pr55Gag proteins are consistent with these results. Whereas some studies reported dramatic defects in assembly and particle production only when both zinc fingers were disrupted [29, 46], other showed that mutations in either of the NC zinc fingers (especially the C-terminal one) impaired HIV-1 assembly . It is possible that the loss of one zinc finger can be compensated by the intact one and would explain why, in certain studies, single mutation within these motifs have no major effects on HIV-1 assembly. Interestingly, mutations in both zinc fingers are required for a complete loss of interaction between Stau1 and Pr55Gag, mutation in one zinc finger causing only a partial reduction. Therefore, through its interaction with the zinc fingers, Stau1 most likely influences steps of assembly that are controlled by the NC zinc fingers. In addition, we do not exclude the possibility that Stau1 also participates in other steps of HIV-1 life cycle that depend on NC .
Although both Stau1 and the Gag-NC zinc fingers are engaged in specific interactions with the genomic HIV-1 RNA, we do not have any evidence that Stau1 interacts with NC via the genomic RNA since the Stau1-Pr55Gag association was shown to be resistant to RNase treatment . Moreover, although the C15/49S NC mutant retains its ability to bind RNA nonspecifically through its basic amino acids [24–26], this mutant is unable to recruit Stau1 supporting the idea that Stau1/Gag-NC interaction is not bridged by RNA. Nevertheless, it is possible that Stau1-Gag-NC interactions favour recruitment of the genomic RNA and its subsequent trafficking and encapsidation. Indeed, mutating the conserved CCHC residues of the NC zinc fingers drastically impairs genomic RNA packaging in newly formed virions and that of Stau1 . We previously showed that Stau1 association with HIV-1 genomic RNA is required for its subsequent encapsidation into the viral particle , our results now suggest that Stau1/Gag-NC interaction is also a critical determinant for this process. In addition, whether Stau1 interacts first with NC or with the genomic RNA to recruit the other partner or independently interacts with each of them through different pathways during HIV-1 assembly and RNA packaging processes is still unknown. The identification of a Stau1 mutant that retains its ability to associate with genomic RNA but is defective for Pr55Gag binding will help answer these questions.
We identified the first 88 amino acids of Stau1 as a regulatory motif of its activity during HIV-1 assembly. Indeed, whereas Stau1ΔNt88-HA3 mutant is still able to interact with Pr55Gag, it fails to enhance Pr55Gag multimerization as seen by BRET, fractionation and VLP release assays. These results strongly suggest that Stau1-binding to Pr55Gag is not sufficient to influence HIV-1 assembly. They eliminate the possibility that the observed increase in the Pr55Gag/Pr55Gag BRET ratio upon Stau155-HA3 over-expression was the result of non-specific changes in the proximity of Rluc and YFP tags due to Stau1 recruitment towards assembly complexes. Similarly, the possibility of major overall structural changes in Stau1ΔNt88-HA3 can be ruled out. Indeed, the Stau1ΔNt88-HA3 mutant retains its capacity to homo-dimerize, to enhance translation of repressed mRNAs, to bind ribosomes and to associate with membranes (unpublished data). Consequently, this sequence probably confers highly specific functions to Stau1 that are advantageous for HIV-1 assembly. It will be interesting to study Stau1ΔNt88 encapsidation into HIV-1 as a means to determine whether the Stau1-mediated enhancement of Pr55Gag multimerization is a prerequisite for its intraviral packaging or whether it relies on its association with HIV-1 genomic RNA and on the control of RNA selection for encapsidation .
How does this region control Stau1 activity? It is likely that Stau1, in conjunction with its Pr55Gag-binding activity, attracts host factors to Pr55Gag complexes that are crucial for assembly. NC-associated proteins that are also important for the transition between specific Pr55Gag assembly intermediates such as ABCE1 [44, 45, 47] are good candidates. Although ABCE1/Pr55Gag interaction depends on NC basic amino acids , the disruption of both NC zinc fingers resulted in the loss of ABCE1/Pr55Gag association by 80% . Thus, it will be important to elucidate the functional relationship between Stau1 and NC-associated proteins to determine if their respective acquisition by assembly intermediates and functions during HIV-1 assembly are simultaneous or sequential.
Within the first 88 N-terminal amino acids of Stau1, a region of 12 amino acids (M26RGGAYPPRYFY37) controls Stau1 functions in regard to Pr55Gag assembly. Post-translational modifications are very common among RNA-binding proteins such as hnRNPs and RNA helicase A and are known to regulate their cellular proteome, function and localization [66–69]. It is then conceivable that post-translational modification of Stau1's N-terminal region controls the recruitment of protein partners and/or modulates Stau1 function during HIV-1 assembly. Alternatively, the N-terminal sequence may recruit ubiquitin ligase through two potential ESCRT targeting domains (PPRY and YPFPVPPL) . In most retroviruses (except HIV-1), the PPXY motif in Gag recruits a ubiquitin ligase and is required for virus budding and release [50, 70, 71]. Resulting ubiquitination allows targeting of the PPXY-containing protein to the ESCRT machinery located to the multivesicular bodies. Similarly, the YPX(n)L domain that is also present in some retroviruses (including HIV-1) recruits the AIP1/ALIX protein that also targets the cargo to the ESCRT [50, 72, 73]. Although Stau1ΔNt88-HA3 associates with membrane as efficiently as Stau155-HA3 (data not shown), it is conceivable that these signals control the localization of Stau1 to specific membrane compartments to support HIV-1 assembly. Interestingly, Popov et al. recently showed that AIP1/ALIX, through its Bro1 domain, is able to bind the NC domain of Pr55Gag in addition to p6 . Strikingly, this interaction requires the integrity of both zinc fingers and is RNA-independent, as observed for Stau1/Pr55Gag association. When over-expressed, AIP1/ALIX rescues the release defect of late domain-mutated HIV-1. Although AIP-1/ALIX over-expression has no effect on wild type HIV-1 release , in contrast to Stau1 , it is possible that Stau1 and AIP-1/ALIX, through their simultaneous or sequential association with the NC zinc fingers, cooperate during HIV-1 assembly. The putative interplay between AIP1/ALIX, ESCRTs and Stau1 during both wild type and L-domain-mutated virus assembly will be very interesting to explore in future studies.
Finally, our work highlights the modular nature of the Stau1 protein in the following ways. The double-stranded RNA binding domain (dsRBD3) interacts with Pr55Gag  whereas the N-terminus controls its activity during HIV-1 assembly. This is supported by the fact that Stau1Δ88-HA3 still associates with HIV-1 Gag (Figure 5) but fails to enhance its multimerization (Figures 4, 6, 8). Strikingly, Drosophila Stau1 orthologue, dStau, has also been shown to function as a modular protein . The third dsRBD of dStau for example is involved in dStau association to oskar RNA whereas the second dsRBD is important for the microtubule-dependent transport of the mRNP and the fifth dsRBD is involved in the derepression of oscar mRNA translation, once localized .
In this study, we provide important new information about the determinants in both Stau1 and Pr55Gag that impact on HIV-1 assembly.
We thank Louise Cournoyer, Alexandre Desjardins, Alexandre Ben Amor and Céline Fréchina for technical assistance, Miroslav Milev for critical comments on the manuscript, Dr. Éric Cohen for HxBRU PR-provirus, Dr. George Pavlakis for Rev-independent Pr55Gag expressor and Dr. Michel Bouvier for anti-Na-K ATPase.
KB is a recipient of the Fonds de la Recherche en Santé du Québec studentship. AJM is a recipient of a Canadian Institutes of Health Research (CIHR) New Investigator award. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to LDG (41596-04), grants from the CIHR to LDG (MOP-62751) and AJM (MOP-38111) and a New Opportunities Grant from the Canadian Foundation for Innovation to AJM.
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