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Molecular and Cellular Biology, April 2004, p. 2637-2648, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2637-2648.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Staufen in the Human Immunodeficiency Virus Type 1 Gag Ribonucleoprotein Complex and a Role in Generating Infectious Viral Particles

Laurent Chatel-Chaix,1,2 Jean-Francois Clément,1,3 Catherine Martel,2 Véronique Bériault,1,4 Anne Gatignol,1,4,5 Luc DesGroseillers,2 and Andrew J. Mouland1,3,4,5,6*

Lady Davis Institute for Medical Research-Sir Mortimer B. Davis Jewish General Hospital,1 Departments of Biochemistry,2 Microbiology and Immunology, Université de Montréal,3 Departments of Microbiology and Immunology,4 Medicine,5 Division of Experimental Medicine, McGill University, Montréal, Québec, Canada6

Received 1 December 2003/ Accepted 7 January 2004


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ABSTRACT
 
Staufen is a host protein that is selectively incorporated into human immunodeficiency virus type 1 (HIV-1) particles in a poorly defined process that involves the selection of HIV-1 genomic RNA for encapsidation and the activity of its third double-stranded RNA-binding domain (dsRBD3). To better understand this, we characterized its interactions with pr55Gag, the principal mediator of HIV-1 genomic RNA encapsidation. Chimeric proviruses harboring wild-type or mutant forms of Staufen were expressed in 293T cells. Cell fractionation analyses demonstrated that Staufen cosedimented with pr55Gag within detergent-resistant, trypsin-sensitive complexes that excluded mature capsid and matrix proteins. Coimmunoprecipitation and bioluminescence resonance energy transfer assays demonstrated a specific and direct interaction between Staufen and the nucleocapsid domain of pr55Gag in vitro and in live cells. This interaction is shown here to be mediated by Staufen's dsRBD3, with a contribution from its C-terminal domain. Immunoprecipitation and reverse transcription-PCR analyses showed that the 9-kb genomic RNA was found within Staufen-containing immune complexes. Spliced HIV-1 RNAs were not detected in these Staufen complexes, indicating a preferential association of Staufen with the 9-kb species. These results substantiate that Staufen and pr55Gag interact directly during HIV-1 expression. Knockdown of Staufen expression by small interfering RNAs in HIV-1-expressing cells demonstrated that this cellular protein was important for the generation of infectious virus. These data show that Staufen, pr55Gag, and genomic RNA are part of the same intracellular complex and support a role for Staufen in pr55Gag function in viral assembly, genomic RNA encapsidation, and the generation of infectious viral particles.


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INTRODUCTION
 
Human immunodeficiency virus type 1 (HIV-1) assembly is an ordered series of steps that is characterized by the formation of intermediate assembly complexes of the viral precursor Gag or pr55Gag. While the expression of pr55Gag alone is sufficient to generate virus-like particles (9), viral assembly steps following pr55Gag synthesis are poorly defined. Each step of the assembly process appears to be regulated by distinct domains of pr55Gag. Since the minimal Gag domain for self-association was recently shown to be the nucleocapsid (NC) (47), oligomerization of pr55Gag mediated by the NC domain is thought to be one of the first events of the assembly process. NC's role in assembly has also been shown to depend on its nonspecific RNA binding activity, mediated by basic amino acid residues (8, 11).

The capsid (CA) (14, 30, 48), matrix (MA) (21, 32) and p2 (31) domains of pr55Gag all contribute to the highly ordered steps of Gag multimerization that likely lead to ultrastructural and morphological changes of intracellular assembly complexes. Although the MA domain is dispensable for multimerization, it is essential to target Gag to membranes via its amino-terminal myristylic acid, and it is also a critical determinant for particle formation (20, 37). Genomic RNA is selected for packaging by the NC domain of pr55Gag, and the p6 domain of pr55Gag is involved in virus budding and release (9). Following these steps, pr55Gag is processed by the viral protease to generate MA, CA, NC, p6 proteins, and two spacer peptides, p2 and p1, leading to structural rearrangements and the formation of a mature virus (9).

HIV-1 assembly also involves the activity of several cellular factors that, for the most part, interact with pr55Gag to mediate their incorporation. An ATP-binding protein, HP68, was found to colocalize and interact with pr55Gag and to participate in virion assembly and capsid formation (50). Tsg101 is an endosomal sorting protein that influences morphogenesis and budding events via an interaction with pr55Gag (17). The proteins VAN, EF-1{alpha}, and actin all interact with pr55Gag and are present in purified virus preparations but have poorly defined roles in assembly and morphogenesis, gene expression, and earlier events of the HIV-1 life cycle (6, 10, 28, 38, 45). These data underscore the importance of studying pr55Gag-host interactions during the late assembly steps of the HIV-1 life cycle because these interactions can potentially be exploited as new targets for therapeutic intervention (19).

We recently demonstrated that the double-stranded-RNA-binding protein Staufen is selectively packaged into HIV-1 virions and is associated with viral RNA in the cytoplasm and in the virus (34). Staufen incorporation in HIV-1 depends on the integrity of the principal double-stranded-RNA-binding domain, dsRBD3 (34), and it is likely mediated by its RNA-binding capacity (34). These data strongly favored a role for Staufen in the selection of genomic RNA for encapsidation and also inferred that it functioned somehow in the intravirion HIV-1 ribonucleoprotein or reverse transcription complex. While Staufen is proving to be involved in RNA trafficking in mammalian cells, especially in neuronal cells (24), details about its roles in HIV-1 replication remain obscure.

In this study, we provide new information on the virus-host interaction involving Staufen from in vitro experiments as well as in live cells. We show that Staufen cofractionates with pr55Gag within detergent-resistant cytosolic complexes and is a component of an intracellular HIV-1 ribonucleoprotein complex. Furthermore, we demonstrate that Staufen interacts specifically with the NC domain of pr55Gag in a direct and RNA-independent manner, as shown in vitro as well as in live-cell assays. We also show that the HIV-1 genomic RNA and not the 1.8- or 4-kb spliced RNA species selectively coimmunoprecipitates with Staufen. Finally, RNA interference experiments demonstrate that specific knockdown of Staufen gene expression results in a significant reduction in viral infectivity. The results presented here suggest that Staufen influences HIV-1 replication at steps that include viral assembly and that this is achieved via specific interactions with HIV-1 pr55Gag and genomic RNA.


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MATERIALS AND METHODS
 
Cell culture and transfection. 293T cells were grown at 37°C in Dulbecco's modified Eagle's medium containing 8% fetal bovine serum and antibiotics (Gibco-BRL, Invitrogen). All transfections were performed with the calcium phosphate-mediated coprecipitation method as reported (34) except that a lipofection technique was used for the silent interfering RNA (siRNA) experiments described below.

Construction of chimeric proviruses. The hemagglutinin (HA)-tagged Staufen cDNA was PCR amplified from pcDNA3-RSV-Staufen-HA (44) with Pfu polymerase (Stratagene) and sense (5'-GATGCTCGAGATGAAACTTGGAAAAAAACC-3') and antisense (5'-CACATCTAGATCATTTATTCAGCGGCCGCACTGAGCAGCGT-3') oligonucleotides. The resulting PCR products were digested with XhoI and XbaI and cloned in pNL4-3/PKR (4) to replace the XhoI and XbaI pkr fragment in the nef open reading frame. The same approach was used to introduce two Staufen mutants in the nef open reading frame: a full-length Staufen protein with a point mutation in dsRBD3 (StaufenF135A) and a C-terminally truncated mutant (dsRBD2-4) lacking the tubulin binding domain (TBD) and dsRBD5. HxBRU provirus (46) was used in the siRNA experiments described below.

Construction of Gag expressers. DNA fragments encoding full-length or different parts of HxB2 Gag polyprotein were PCR amplified with the Pfu Turbo DNA polymerase (Stratagene) and the Rev-independent Gag expresser plasmid pCMV55M1-10 (40) as the template. The primer pairs used in the PCRs are summarized in Table 1. PCR products were digested with KpnI and BamHI and inserted in the KpnI and BamHI cloning sites of pRluc-N1(h) (Packard BioScience/Perkin-Elmer Life Sciences) in frame with the Renilla reniformis luciferase coding sequence (1). To construct pCMV-Staufen/YFP, pcDNA3-RSV-Staufen-HA (44) was digested with NotI, treated with the Klenow fragment of DNA polymerase to blunt the extremities, and digested with HindIII. The resulting fragment was then inserted in the HindIII and SmaI sites of pCMV-GFP-Topaz (1) in frame with the yellow fluorescent protein (YFP) coding sequence.


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  		TABLE 1. Primer combinations used in PCR amplifications for Gag constructs based on HIV-1 HxB2 pr55Gag

Cell fractionation on sucrose gradients. 293T cells were homogenized for 1 min in 12.5% sucrose-10 mM HEPES (pH 7.3)-1 mM EDTA and then centrifuged at 1,000 x g for 5 min. The supernatant was layered onto a continuous 20 to 60% (wt/vol) sucrose gradient (in 10 mM HEPES [pH 7.3], 1 mM MgCl2) as reported previously (29). In other experiments, to increase stringency, cells were homogenized in a lysis buffer containing detergent (100 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.5% NP-40, pH 7.3) and layered onto a 10-ml continuous 10 to 60% (wt/vol) sucrose gradient. Both gradients were centrifuged at 31,000 x g for 2.5 h in an SW41 rotor (Beckman-Coulter). Fractions were collected from the top and analyzed by Western blotting for Staufen (with mouse monoclonal anti-HA or anti-Staufen antibodies), CA, MA (from Spearman, NIH AIDS Reference and Reagent Program), and ribosomal L7 (Novus Biologicals).

Immunoprecipitation and RT-PCR. 293T cells were either transfected with Staufen-expressing chimeric proviruses or cotransfected with cDNAs coding for Gag/Rluc and Staufen-HA fusion proteins. Forty hours posttransfection, cells were harvested, washed three times in phosphate-buffered saline (PBS), and lysed (100 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.5% NP-40, and protease inhibitor cocktail [Roche]). Staufen and Gag expression was monitored by Western blot analysis with mouse monoclonal anti-HA and anti-CA antibodies, respectively. For immunoprecipitation analysis, 3 to 5 mg of cell lysates was precleared with normal rabbit serum and 25 µl of a 50:50 slurry of protein A-Sepharose (Amersham Biosciences), incubated with rabbit polyclonal anti-CA antiserum (American BioTechnologies; dilution 1:200) for 90 min at 4°C, and with 25 µl of a 50:50 slurry of protein A-Sepharose for 1 h at 4°C.

To immunoprecipitate HA-tagged proteins, lysates were incubated with rat monoclonal anti-HA antibody covalently coupled with agarose beads (Roche Applied Bioscience) for 4 h at 4°C. In some cases, the stringency of the immunoprecipitation was increased by adjusting the concentrations of NP-40 and sodium dodecyl sulfate to 1% and 0.1%, respectively. The resulting immunoprecipitation pellets were washed four times with lysis buffer and once with PBS. Immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mouse monoclonal anti-HA and anti-CA antibodies to detect Staufen and Gag in Western blot analyses. The immunoprecipitation of endogenous Staufen was performed essentially as described above for anti-CA except that a mouse monoclonal antiserum was used (34). A rabbit antiserum was used to identify Staufen in Western analyses.

To identify Staufen-associated RNAs, Staufen-HA was first immunoprecipitated with a monoclonal anti-HA antibody as previously described (34). The immunoprecipitation pellets were incubated with 20 µg of RNase-free DNase I (Invitrogen) for 1 h at 37°C and then with 50 µg of proteinase K for 30 min at 42°C. Coprecipitated RNAs were extracted as described before (34), dissolved in diethylpyrocarbonate-treated water, and reverse transcribed with random primers and avian myeloblastosis virus reverse transcriptase with the First Strand cDNA synthesis kit (Roche Applied Bioscience). The resulting cDNAs were PCR amplified with Taq polymerase (Roche Applied Bioscience) and either of three primer pairs that discriminate between the three HIV RNA size classes. All three sets include the same sense primer (5'-CTGAGCCTGGGAGCTCTCTGGC-3' ) present in the TAR region of all HIV-1 RNA species (34).

Primer pair 1 specifically amplify a 430-bp fragment from the genomic HIV-1 RNA (9 kb) because the antisense primer (5'-TCCAGTGATTTTTTTCTCCATGCTTGCCCATACTATATGTTT-3') is located in the gag gene. Primer pair 2 preferentially amplify PCR fragments of 410 to 500 bp from the spliced 4-kb HIV-1 RNA species because the antisense primer (5'-TCATTGCCACTGTCTTCTGCTCT-3' ) hybridizes in the region of the vpu gene. This region is absent in spliced 1.8-kb HIV-1 RNA species. Primer pair 3 preferentially amplify 460- to 600-bp fragments from the spliced 1.8-kb RNA species, the antisense primer (5'-CCGCAGATCGTCCCAGATAAG-3') being localized in the second exon of the rev gene. Amplification from genomic and 4-kb spliced RNAs is unlikely in our conditions due to the length of the product. Reverse transcription (RT)-PCR products were analyzed on a 0.8% agarose gel. When necessary, and to enhance the sensitivity of detection, PCR products were subjected to two or five additional PCR cycles in the presence of 10 µCi of [{alpha}-32P]dCTP as described before (39). Labeled PCR products were resolved on 6% denaturing polyacrylamide gels and detected by autoradiography.

BRET analysis. For bioluminescence resonance energy transfer (BRET) analysis, 293T cells (2 x 106) were cotransfected with cDNAs coding for Staufen/YFP and different Gag/Rluc fusion proteins. Forty hours posttransfection, the cells were washed in PBS, collected in 1 ml of PBS containing 5 mM EDTA, and then diluted to 106 cells/ml. Coelenterazine (h; Molecular Probes) was added at a final concentration of 5 µM. Luminescence and fluorescence were quantitated with a Fusion {alpha}-FP apparatus (Perkin-Elmer-Canberra Packard BioScience). Three measures were obtained: first, light emitted at 475 to 480 nm by Renilla luciferase; second, emission fluorescence at 525 to 530 nm without excitation due to energy transfer from Renilla luciferase to YFP; third, emission fluorescence at 525 to 530 nm after excitation at 485 nm to measure total expression of YFP fusion proteins. The BRET ratio was defined as [(emission at 510 to 590 nm) - (emission at 440 to 500 nm) x Cf]/(emission at 440 to 500 nm), where Cf corresponds to (emission at 510 to 590 nm)/(emission at 440 to 500 nm) for Renilla luciferase-fused Gag mutants expressed alone in the same experiments, as previously described (1).

To ensure that the BRET ratios reflected a real interaction between fusion proteins, dose-response assays were performed for each protein tested. 293T cells were cotransfected with constant amounts of different Gag/Rluc fusion proteins and increasing concentrations of either Staufen/YFP or YFP alone. At each concentration, the BRET ratios were determined as described above and plotted as a function of the ratio of total YFP activity after excitation to total Renilla luciferase activity. These curves allowed us to compare BRET ratios obtained with Staufen/YFP (due to specific interaction with Gag/Rluc) and YFP alone (control) at the same expression levels. In these conditions, whereas BRET ratios increased linearly with YFP concentrations, they increased more rapidly and were eventually saturated as the expression level of Staufen/YFP increased, reflecting a specific interaction between Staufen and Gag.

siRNA knockdown: transfections, Western blots, and RT-PCR analysis. Four double-stranded 21-bp siRNAs to a selected region of Staufen cDNA were chosen and tested for Staufen knockdown efficiency in preliminary experiments. The duplex siRNA 3084 (5'-AAATAGCACAGTTTGGAAACT-3') was determined to produce the most significant knockdown of Staufen gene expression and was chosen for the experiments presented in this article (purchased from Qiagen-Xeragon, Germantown, Md.). A control nonsilencing siRNA (catalog no. 1022076; 5'-AATTCTCCGAACGTGTCACGT-3') was purchased from Qiagen-Xeragon and included in either mock or HxBRU (vpr+ vif+ nef- vpu-)-transfected cells. 293T cells were trypsinized and plated in six-well plates at 2 x 105 cells per well for 12 to 16 h before transfection. Transfections were performed with the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, Calif.) with siRNA at a final concentration of 10 nM. Cells were transfected with either nonsilencing siRNA (mock or HxBRU) or Staufen siRNA 3084. At 24 h, the cells were mock-transfected (KSII with nonsilencing siRNA) or transfected with HxBRU plus nonsilencing RNA or plus Staufen siRNA 3084; 24 h later, the cells were washed with ice-cold PBS and lysed in NP-40 buffer (100 mM NaCl, 10 mM Tris, 1 mM EDTA, 0.5% NP-40). Virus was pelleted by ultracentrifugation of supernatants.

Cytosolic extracts quantitated for protein by the micro-Bradford assay and for Staufen and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were assessed by Western blot analysis with a mouse monoclonal anti-Staufen antibody (9E9) as described above and a GAPDH antibody (Research Diagnostics, Inc.) Actin was also identified with an antiactin antibody (not shown). The other half of the cellular extract was treated with urea buffer and phenol-chloroform-isoamyl alcohol to extract cytosolic RNA (33). RNA was precipitated with ethanol and glycogen carrier (Roche). The RNA pellets were resuspended in diethylpyrocarbonate-treated water and quantitated by optical density measurement; 1 µg of RNA from each sample was used in RT-PCR analysis with the Superscript One-Step RT-PCR kit with Platinum Taq (Invitrogen). Either Staufen mRNA to produce a 337-bp PCR product (sense, 5'-aatctagaTTTACCAGGGCAGCTCCGAA-3'; antisense, 5'-aatctagaCAACTCAGACAGCAACTTTAAGATGT-3'; lowercase indicates restriction sites) or GAPDH RNA exactly as described in reference 23 was specifically amplified as described previously (34).

HIV-1 infectivity assay. 293T cells were treated with a nonsilencing siRNA (mock or HxBRU alone) or treated with Staufen siRNA 3084 as described above. Virions produced from 293T cells were collected, filtered, and pelleted by ultracentrifugation; 8 x 105 CEM-green fluorescent protein (GFP) cells (18) were infected with equivalent amounts (25 ng) of virus as determined by p24 enzyme-linked immunosorbent assay (ELISA) (3, 5) in the presence of 10 µg of Polybrene (Sigma-Aldrich) per ml and in a final volume of 200 µl of culture medium. At 3 h postinfection, CEM-GFP cells were resuspended in 10 ml of fresh medium (RPMI with 10% fetal bovine serum and 500 µg of G418 per ml) and incubated for 48 h. The cells were then pelleted, washed twice with PBS, and fixed in 1% formaldehyde in PBS. Then 106 viable cells were transferred to a 96-well plate. Fluorescence was measured with a Fusion-Alpha reader apparatus (Perkin-Elmer Life Sciences) with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Data are expressed as relative infectivity where wild-type HIV-1 infectivity (HxBRU treated with a nonsilencing siRNA) was arbitrarily set to a value of 1.


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RESULTS
 
Expression of Staufen from HIV-1 chimeric proviruses. To characterize a putative interaction between Staufen and HIV-1 proteins or RNA, we constructed HIV-1 chimeric proviruses in which Staufen cDNA was inserted into the nef open reading frame (Fig. 1A). In this context, Staufen should be expressed early during HIV-1 expression, since Nef is among the first viral proteins to be expressed. We also prepared two additional chimeric proviruses containing either the full-length Staufen protein with a point mutation in dsRBD3 that abolishes its RNA-binding capacity (StaufenF135A) or a C-terminally truncated mutant (dsRBD2-4) lacking the tubulin binding domain and the dsRBD5. To verify Staufen expression in this proviral context, expression levels were analyzed by Western blotting of extracts of transfected 293T cells. As shown in Fig. 1B, Staufen proteins were all expressed at comparable levels. In addition, Staufen expression in the context of the provirus did not affect Gag expression levels or Gag processing, since the same levels of pr55Gag, CA, and CAp25 were observed (Fig. 1B).



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  		FIG. 1. Construction and characterization of Staufen-expressing HIV-1 chimeric proviruses. (A) Schematic representation of chimeric proviruses. cDNAs coding for either wild-type Staufen protein, full-length Staufen with a point mutation in the RNA-binding domain dsRBD3 (StaufenF135A), and a C-terminally truncated mutant containing dsRBD2, -3, and -4 (dsRBD2-4) were inserted in the nef open reading frame of pNL4-3/PKR. Staufen proteins were tagged with an HA epitope. (B) Expression of chimeric proviruses. 293T cells were transfected with an empty vector (lane M), pNL4-3 (lane 1), pNL4-3/Staufen-HA (lane 2), pNL4-3/StaufenF135A-HA (lane 3) or pNL4-3/dsRBD2-4-HA (lane 4), and cell extracts were analyzed by Western blotting with anti-HA and anti-CA antibodies to monitor expression of both Staufen-HA and Gag proteins. Asterisks, nonspecific bands.

Staufen and HIV-1 pr55Gag cofractionate on sucrose gradients. We first studied the subcellular distributions of Staufen and HIV-1 Gag during HIV-1 replication. These studies showed that Staufen and pr55Gag partially colocalized in the cytoplasm of HIV-1/Staufen-expressing cells (L. Chantal-Chaix, L. DesGroseillers, and A. J. Mouland, data not shown). We therefore performed sucrose gradient sedimentation assays in order to determine the relationships between cytosolic Staufen and HIV-1 pr55Gag or CA. This technique allows the separation of cellular compartments or organelles according to their buoyant density. Cytoplasmic cell extracts were prepared from pNL4-3- and pNL4-3/Staufen-HA-expressing cells and separated on sucrose gradients (20 to 60%, wt/vol; Fig. 2A and B). Each fraction was analyzed by Western blotting for the presence of Staufen and HIV-1 proteins. In cells that expressed pNL4-3 only, endogenous Staufen was found in fractions 10 to 13, with a peak in fraction 13. Ribosomal protein L7, a marker of whole ribosomes, was also found in these Staufen fractions (Fig. 2A), as shown previously (29). When anti-CA was used to identify Gag proteins, unprocessed pr55Gag, like Staufen, was principally found in fractions 11 to 14 with a peak in fraction 13 (indicated in the shaded box under Fig. 2B). In contrast, the processed forms of pr55Gag, CA p24 and p25, sedimented mainly in fractions 6 to 12, likely corresponding to budding viruses. Approximately 50% of the total CA was detected in the low-density fraction 15, likely representing lysed viruses. Identical results were obtained for the distribution of Staufen-HA in cells expressing pNL4-3/Staufen-HA (Fig. 2B) such that Staufen cofractionated with pr55Gag and not with the processed forms of pr55Gag. This set of experiments demonstrated that while the distributions of Staufen and pr55Gag were not identical, both proteins cosedimented in sucrose gradients and peaked in the same fraction. It also shows that the expression of Staufen in the context of pNL4-3/Staufen did not affect the cytoplasmic distribution of HIV-1 proteins, and moreover, HIV-1 gene expression did not have any apparent effects on that of Staufen.



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  		FIG. 2. Staufen and pr55Gag cofractionate on sucrose gradients. (A) 293T cells were transfected with pNL4-3, and cell extracts were separated on a 20 to 60% sucrose gradient. Fifteen fractions were collected and analyzed by Western blotting with mouse monoclonal anti-Staufen, anti-CA, and anti-ribosomal protein L7 antibodies as indicated. Inputs from mock (M) and transfected (T) cells are shown on the right. (B) Same experiment as in panel A except that 293T cells were transfected with pNL4-3/Staufen-HA. Staufen was detected with a mouse monoclonal anti-HA antibody. (C) 293T cells were transfected with pNL4-3/Staufen-HA and lysed in a buffer containing 0.5% NP-40. Cell lysates were separated on a 10 to 60% sucrose gradient containing 0.5% NP-40. Fractions were analyzed by Western blot analyses with anti-HA, anti-CA, and anti-L7 antibodies as indicated.

To further characterize these complexes, we repeated these sucrose gradient analyses in the presence of nonionic detergent (Fig. 2C) with a 10 to 60% sucrose gradient. This treatment disrupts weak interactions and releases membrane-associated complexes. Under these conditions, Staufen and pr55Gag cosedimented with a similar profile in intermediate density fractions 7 to 13. Because a 10 to 60% gradient was used in this analysis, we expected that the sedimentation profiles of Staufen and pr55Gag profiles would be slightly different, which is what we observed. The data show that detergent treatment did not significantly change the sedimentation profile of either Staufen or pr55Gag. These proteins sedimented in fractions 7 to 13 (34.5 to 24% sucrose) after detergent treatment (Fig. 2C) or mainly in fractions 10 to 13 (34.5 to 25% sucrose) without detergent (Fig. 2A or 2B). In contrast, the processed forms of Gag such as CA p25 and p24 and MA (not shown) were no longer present in high-density fractions (6 to 12 in Fig. 2A and B) but were found at the top of the gradient (mainly in fractions 14 and 15) following detergent treatment, likely corresponding to detergent-solubilized viral particles. Therefore, Staufen and pr55Gag are found in detergent-resistant complexes, consistent with previous Gag data (26, 27). When cell extracts were treated with trypsin before gradient analysis, neither Staufen nor pr55Gag was detectable in sucrose gradients, indicating that both proteins are sensitive to trypsin digestion and are mainly in a cytosolic compartment (Chatel-Chaix et al., data not shown). In contrast, CA p25/p24 levels and distribution were not affected because they are most likely protected from trypsin by their association with membranes. Our results now show that Staufen cofractionates with pr55Gag and is likely part of the same HIV-1 detergent-resistant complex, a complex that was previously shown to mainly contain pr55Gag and to correspond to cytoplasmic virion assembly intermediates (26, 27).

Staufen interacts with HIV-1 pr55Gag in an RNA-independent manner. In preliminary studies, yeast two-hybrid analyses revealed that full-length Staufen and pr55Gag interacted (34) to a level similar to that found for pr55Gag/pr55Gag (2). Our immunofluorescence results also suggested that at least a proportion of Staufen and pr55Gag are in close proximity and could be components of the same HIV-1 assembly complex (data not shown). To address this possibility, we first determined whether endogenous Staufen interacted with pr55Gag during proviral expression. To determine this, cells were mock transfected or transfected with pNL4-3 alone. Extracts from these cells were immunoprecipitated with a monoclonal anti-CA antibody. A cell extract was analyzed for endogenous Staufen prior to immunoprecipitation with a rabbit polyclonal antibody used previously (34) (Fig. 3A, top left panel). This antibody reacts with two isoforms of Staufen, 55 and 63 kDa (34). pr55Gag was then immunoprecipitated from equal quantities of extracts, and the immunoprecipitates were analyzed for the presence of endogenous Staufen protein (Fig. 3A, top right panel) and immunoprecipitated Gag products (Fig. 3A, lower panel). Staufen was found to be present in the HIV-1 Gag immunoprecipitates during HIV-1 expression but not in the immunoprecipitate from mock-transfected cell lysates. There was a specific immunoprecipitation with the 55-kDa Staufen isoform (Fig. 3A, top right panel). A 63- to 65-kDa protein signal was obtained in both conditions and is not related to the 63-kDa Staufen isoform. These results suggest that endogenous Staufen is physically recruited to pr55Gag-containing complexes during proviral gene expression.



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  		FIG. 3. Staufen interacts with pr55Gag. (A) 293T cells were transfected with either an empty vector (M) or pNL4-3 (1), and the cell lysates were immunoprecipitated with anti-CA antibodies. Endogenous Staufen was identified in cellular lysates (A, top left panel). Following immunoprecipitation with the anti-CA antibody, Staufen (A, top right panel) and Gag products (A, lower panel) were identified by Western analyses in the immunoprecipitates. (B and C) 293T cells were transfected with an empty vector (lane M), pNL4-3 (lane 1), pNL4-3/Staufen-HA (lane 2), pNL4-3/StaufenF135A-HA (lane 3), or pNL4-3/dsRBD2-4-HA (lane 4), and cell extracts were immunoprecipitated with anti-CA (B) or anti-HA (C) antibodies. Proteins in the pellets were analyzed by Western blotting with anti-HA and anti-CA as indicated. (D) 293T cells were transfected with an empty vector (lane M), pNL4-3 (lane 1), or pNL4-3/Staufen-HA (lane 2). Cell lysates were incubated in the presence (+) or absence (-) of RNases A and T1 for 30 min prior to immunoprecipitation analysis. The asterisk shows nonspecific immunoglobulin G labeling. Data shown are representative of four independent experiments.

In order to map the determinants in the Staufen protein that are important for this interaction, 293T cells were transfected with the chimeric Staufen proviruses shown in Fig. 1, followed by immunoprecipitation and Western analysis. Cell lysates were immunoprecipitated with anti-CA antiserum, and Staufen-HA was identified in the immunoprecipitates by Western blotting. From cells transfected with pNL4-3/Staufen-HA provirus, a 60-kDa band corresponding to Staufen-HA was detected with an anti-HA antibody in the Gag immunoprecipitate, whereas Staufen was not detected in Gag immunoprecipitates from pNL4-3/StaufenF135A-HA- or dsRBD2-4-HA-expressing cells (Fig. 3B). An approximately equal amount of Gag (pr55Gag, pr41Gag, or CA p24 and p25) was immunoprecipitated in all conditions. These results demonstrate that Staufen's interaction with pr55Gag in HIV-1-expressing cells requires contributions from both an intact dsRBD3 and C-terminal domain.

In the reverse experiment, cells were transfected with pNL4-3 or the pNL4-3/Staufen proviruses, and Staufen-HA was immunoprecipitated with anti-HA. Immunoprecipitated proteins were then analyzed by Western blot analysis with monoclonal anti-CA. Anti-HA was included to identify Staufen-HA proteins at the same time. This analysis shows that pr55Gag coimmunoprecipitated with wild-type Staufen-HA but not with either of the Staufen mutants (Fig. 3C). We were not able to detect processed forms of pr55Gag, CA, and NC in the immunoprecipitates (data not shown), consistent with our gradient analysis, in which we showed that these do not cofractionate with Staufen (Fig. 2A and B). These data strongly suggest that Staufen preferentially interacts with the Gag precursor pr55Gag.

Since a single point mutation in Staufen's dsRBD3 abolishes both its RNA-binding activity (29) and the Staufen/pr55Gag interaction (Fig. 3B), we investigated whether RNA contributes to this interaction, as it does for the pr55Gag/EF-1{alpha}, pr55Gag/pr55Gag, and pr55Gag/Gag-Pol (or pr160Gag/Pol) interactions (8, 10, 11, 22). Cell lysates were mock digested or digested with RNases A and T1 prior to immunoprecipitation with anti-CA antiserum. As shown in Fig. 3D, Staufen remained associated with Gag-containing complexes in the presence of RNase, demonstrating that this interaction is maintained even if the cellular RNA is removed. However, we cannot exclude the possibility that bridging RNA may be protected from RNase digestion by the Staufen/pr55Gag complex with this assay, and these results, while reproducible, should be interpreted with care.

Staufen/pr55Gag interaction requires the HIV-1 NC domain. Coimmunoprecipitation assays were also used to map the pr55Gag domain that interacts with Staufen. Full-length pr55Gag as well as four deletion mutants containing different Gag subdomains (MA-CA, CA, CA-p1, and CA-p6) were fused in-frame with R. reniformis luciferase (Fig. 4A). The CA subdomain was included in each mutant in order to allow immunoprecipitation by our anti-CA antibody. 293T cells were cotransfected with the Gag/Rluc and Staufen-HA expressors. In the cell lysates, the Gag/Rluc deletion mutants and Staufen-HA were all expressed at comparable levels except for pr55Gag/Rluc, which was consistently expressed at low levels (Fig. 4B). Following immunoprecipitation with anti-CA antibodies, coprecipitated proteins were analyzed by Western blotting with anti-HA to detect Staufen-HA (Fig. 4C). Although each Gag mutant was immunoprecipitated at comparable levels (Fig. 4C, bottom panel), Staufen-HA was only detected in the immunoprecipitates when the NC subdomain was expressed (lanes 1, 4, and 5). These results show that Staufen interacts with pr55Gag with a major contribution from the NC domain. Negligible amounts of Staufen-HA coprecipitated with CA/Rluc and MA-CA/Rluc. Moreover, these results suggest that the interaction does not require the expression of additional viral components.



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  		FIG. 4. Staufen binds pr55Gag via the NC domain. (A) Schematic representation of pr55Gag and deletion mutants fused to Renilla luciferase (Rluc), labeled 1 to 5. (B and C) 293T cells were cotransfected with these constructs (labeled 1 to 5 in panel A and corresponding to lanes 1 to 5 in panels B and C) and a Staufen-HA expresser. Cell lysates were analyzed by Western blotting with anti-CA and anti-HA antibodies to detect expression of the proteins (B) or immunoprecipitated (IP) with anti-CA antibodies (C). Coimmunoprecipitated proteins were analyzed by Western blotting with anti-HA and anti-CA (as a control) antibodies. Data shown are representative of two independent experiments.

Staufen and HIV-1 NC domain of pr55Gag interact directly in live cells. To determine whether the characterized Staufen/pr55Gag interaction above was not an artifact that resulted from the preparation of detergent-solubilized cellular extracts, we performed protein interaction assays in live cells by BRET analysis (1). For this method, a BRET is achieved when two proteins interact directly with a physical separation of approximately <=50 Å. Energy from the donor luciferase molecule is transferred to the red-shifted GFP (YFP) molecule acceptor, which then produces a fluorescent light emission to produce a positive BRET signal, calculated as described in Materials and Methods. Staufen was fused to YFP (acceptor), and all of the Gag mutants were fused to Renilla luciferase (donor) as shown in Fig. 5A. 293T cells were cotransfected with Staufen-YFP and each of the Gag/Rluc expressers, and BRET ratios were determined in live cells at 40 h posttransfection. Because full-length pr55Gag/Rluc was poorly expressed (Fig. 4B), it was not included in this set of experiments.



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  		FIG. 5. Staufen directly binds the HIV-1 NC domain of pr55Gag in living cells. (A) 293T cells were cotransfected with Staufen/YFP and different Renilla luciferase-fused truncated Gag expressers. BRET ratios were defined as described in Materials and Methods. n is the replicate number, and the error bars represent the standard error of the mean calculated from at least three experiments for each interaction set. (B) 293T cells were cotransfected with constant amounts of pCMV-CA-p1/Rluc and various amounts of either Staufen/YFP or YFP expressers. The graph presented here is a representative example of the saturation studies performed to provide evidence for a specific interaction between the proteins. BRET ratios were plotted as a function of the excited YFP activity to total Renilla luciferase activity ratio, allowing comparison of BRET ratios between Staufen-YFP and YFP when expressed at the same levels.

As shown in Fig. 5A, positive BRET ratios were always observed when Staufen-YFP was coexpressed with Gag mutants containing the NC domain (CA-p1/Rluc, CA-p6/Rluc, p2-p1/Rluc, and NC/Rluc). In contrast, negligible BRET ratios were obtained when the Gag fragments lacked the NC domain (MA-CA/Rluc, CA/Rluc, MA/Rluc, and p6/Rluc). Positive BRET ratios were consistently two- to threefold higher when NC was expressed in the form of a precursor (CA-p1 or CA-p6/Rluc) than when it was expressed as a mature protein (NC/Rluc). Dose-response assays with cells transfected with Staufen/YFP or YFP alone provided further evidence that this association in live cells was specific (Fig. 5B and Materials and Methods). These data confirm the results obtained in the coimmunoprecipitation analyses and further show that Staufen and pr55Gag interact directly in live cells. We mapped the pr55Gag-interacting domain to the NC domain, and the data also suggest that Staufen has higher affinity for pr55Gag than for the mature Gag proteins.

HIV-1 genomic RNA is found in the Staufen immune complex. We previously documented a direct correlation between the level of expression of Staufen and the abundance of genomic RNA encapsidated into virus particles (34). This suggested that Staufen was implicated in the selection of genomic RNA for encapsidation. While the NC domain of pr55Gag is the principal mediator of this process (49), which depends on an interaction between pr55Gag and genomic RNA (13), the relationship between Staufen- and pr55Gag-mediated genomic RNA encapsidation was not clear. Therefore, to further characterize the Staufen-pr55Gag complex and the relationship between Staufen and HIV-1 RNA, we attempted to identify the Staufen-associated HIV-1 RNA species in coimmunoprecipitation assays with our Staufen proviral constructs. 293T cells were transfected with pNL4-3/Staufen-HA and mutant proviruses, and cell extracts were immunoprecipitated with anti-HA. The identification of the coimmunoprecipitated HIV-1 RNA species was determined by RT-PCR with three primer pairs that recognized either the genomic RNA or the 4- or 1.8-kb spliced RNAs, as previously described (39) and as illustrated in Fig. 6A.



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  		FIG. 6. Selective association of HIV-1 genomic RNA with Staufen-containing complexes. (A) Schematic representation of the three different primer pair combinations. Each primer pair is specific for amplification of either the 9-kb genomic RNA (primer pair 1) or the spliced 4-kb (primer pair 2) or 1.8-kb (primer pair 3) RNA species. (B, C, and D) 293T cells were transfected with empty vector (lane 1), pNL4-3 (lane 2), pNL4-3/Staufen-HA (lane 3), pNL4-3/StaufenF135A-HA (lane 4), or pNL4-3/dsRBD2-4-HA (lane 5), and cell lysates were immunoprecipitated with anti-HA antibodies. Coimmunoprecipitated RNAs were RT-PCR amplified with the primer pairs described above. PCR products obtained with primer pairs 1 (B), 2 (C), and 3 (D) were analyzed on 0.8% agarose gels. Negative controls included RT-PCR amplification in the absence of RNA (lane 6) or reverse transcriptase (lane 7) and amplification of RNA from mock-transfected cells (lane 8). Positive controls included RT-PCR amplification of RNA from pNL4-3-transfected cells before immunoprecipitation (lane 9). Amplification of RNA from cell extracts in the absence of reverse transcriptase did not yield a PCR signal (not shown). (E and F) PCR products obtained with primer pairs 2 (E) and 3 (F) were analyzed on 6% denaturing acrylamide gels after two or five additional PCR cycles in the presence of [32P]dCTP. Products from spliced RNA species are indicated on the right.

With primer pair 1 for genomic RNA, we detected a specific DNA product of the expected size in the immunoprecipitates isolated from HIV-1/Staufen-HA lysates (Fig. 6B, lane 3). The amplification was specific because there was no signal when the avian myeloblastosis virus reverse transcriptase was omitted (Fig. 6B, lane 7). In contrast, RT-PCR from HIV-1 alone (Fig. 6B, lane 2) or from the pNL4-3/StaufenF135A-HA or pNL4-3/dsRBD2-4-HA immunoprecipitates did not lead to a detectable PCR product with the primer pair for genomic RNA (Fig. 6B, lanes 4 and 5). With the same immunoprecipitated RNA extracts, RT-PCR was repeated with primer pairs specific for spliced HIV-1 RNAs. In both cases, there was no evidence of a PCR product after 35 cycles (Fig. 6C and 6D). To further rule out the presence of spliced HIV-1 RNAs following the RT reaction, PCR was performed and the last two or five PCR cycles were performed in the presence of [32P]dCTP. PCR products were resolved on 6% polyacrylamide gels. As shown in Fig. 6E and F, we did not detect an association with the 4- and 1.8-kb RNA species even in these conditions.

The expected pattern of spliced RNAs was obtained with cell extracts from pNL4-3-expressing cells with this strategy (Fig. 6E and F) (39). These results demonstrate that there is selectivity for genomic RNA by Staufen in HIV-1-producing cells, supporting a role for Staufen in the selection of genomic RNA for encapsidation. This immunoprecipitation/RT-PCR analysis was also performed for endogenous Staufen during the expression of HIV-1, and the results also demonstrate that HIV-1 genomic RNA selectively coimmunoprecipitates with Staufen (data not shown). Furthermore, similar analyses were performed with the related double-stranded-RNA-binding protein TRBP. Whereas Staufen exhibited selectivity for the 9-kb RNA as described above, TRBP was found to coimmunoprecipitate with all HIV-1 RNA species (data not shown).

Staufen knockdown by RNA interference generates HIV-1 with compromised infectivity. Staufen expression was targeted by 21-bp siRNA duplexes as described in Materials and Methods. Cells were either mock transfected (with a nonsilencing control RNA duplex) or transfected with HxBRU (plus a nonsilencing control RNA duplex) or with HxBRU/Staufen (plus Staufen siRNA 3084) following the procedure described in Materials and Methods. Staufen siRNA 3084 knocked down Staufen protein expression by 80% (Fig. 7A) and Staufen mRNA levels by almost 60%, as shown in RT-PCR analyses (Fig. 7B). Staufen siRNA 3084 did not affect either GAPDH protein and RNA levels (Fig. 7A and B) or those of actin (data not shown), However, when HIV-1 infectivity was assessed with equal quantities of virus, as determined in a p24 ELISA (3, 5), Staufen siRNA 3084 resulted in a twofold drop in viral infectivity (Fig. 7C). In our previous work, Staufen overexpression also caused a significant decrease in viral infectivity (34). These data demonstrate that an optimal amount of Staufen expression is required to produce fully infectious viral particles.



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  		FIG. 7. siRNA-mediated knockdown of Staufen expression results in decreased HIV-1 infectivity. (A)Western blot analysis of Staufen and HIV-1 structural proteins levels in 293T cells 48 h posttransfection. Plasmid HxBRU (2 µg) was transfected in the presence of a nonsilencing (N-S) siRNA and with 10 nM Staufen siRNA 3084 directed against Staufen. The blots were probed with monoclonal antibodies to Staufen (9E9) and anti-GAPDH to control for protein loading. (B) RT-PCR analysis of intracellular Staufen mRNA levels 48 h after cotransfection of HxBRU and siRNA. RNA was purified from a portion of the samples described for panel A; 1 µg of RNA from each sample was reverse transcribed and amplified with oligonucleotides designed for Staufen and GAPDH to control the amount of RNA. Western blot and RT-PCR quantification of intracellular Staufen protein or mRNA seen in panels A and B was done with a Canberra Packard Alpha-Imager system. The average level of Staufen protein or mRNA present in each sample is given below each lane as a percentage relative to the level found in cells treated with HxBRU and a nonsilencing siRNA. Percent standard errors (SE) are given based on the average of three independent experiments. (C) GFP-based infectivity assay. The infectivity of virus preparations was determined by infecting CEM-GFP indicator T cells as described in Materials and Methods. Data are expressed as relative infectivity, where the infectivity of HxBRU treated with a nonsilencing siRNA was arbitrarily set to a value of 1. The standard error (SE) is given based on the average of five independent experiments.


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DISCUSSION
 
In this report, we show that the double-stranded-RNA-binding protein Staufen interacts with the NC domain of HIV-1 pr55Gag during HIV-1 gene expression. This interaction was demonstrated in in vitro assays with both proviral and expression constructs and in live cells by BRET analysis, indicating that it is direct and does not require other viral components. Cellular Staufen/pr55Gag complexes contain HIV-1 genomic RNA but not the spliced HIV-1 RNA species. They are resistant to membrane solubilization by detergent but are sensitive to trypsin proteolysis, two characteristics of cytoplasmic assembly detergent-resistant complexes (26, 27). Taken together, these results suggest that Staufen plays a role during virus assembly and genomic RNA encapsidation. Consistently, our earlier work showed that Staufen overexpression resulted in decreased virus infectivity but at the same time resulted in enhanced genomic RNA packaging in virions (34). The siRNA results shown in Fig. 7 further support a role for Staufen in virus assembly or at another level that impacts significantly on the infectious potential of virions. More recent work supports the notion that Staufen acts during assembly in that Staufen appears to affect virion morphogenesis (Mouland et al., unpublished data). It will be interesting to determine at what stage Staufen is acting in the assembly process, since other cellular proteins such as HP68 have been shown to be associated with several Gag assembly intermediates (50).

Confocal imaging analysis of cells transfected with pNL4-3/Staufen-HA provirus did not reveal any apparent changes in Staufen's cellular distribution compared to cells expressing Staufen alone (not shown). This is in contrast to the dramatic nuclear relocalization of Staufen when influenza virus NS1 protein is expressed (15). Partial colocalization was found between HIV-1 Gag and Staufen, suggesting that a proportion of Staufen is associated with pr55Gag at any time (data not shown). This interaction was substantiated by several assays, including yeast two-hybrid (34), in vitro coimmunoprecipitation, and BRET analyses in live cells (Fig. 3 to 5). The interaction appeared to be direct and was consistently found to be of the same order of magnitude as that found for Gag-Gag in these assays, indicating that this may reflect a transient association that is only required for specific steps in the viral replication cycle and/or to allow a fine-tuned regulation of assembly steps (Fig. 8). Indeed, dynamic and transient formation and modification of ribonucleoprotein complexes is a major hallmark of RNA transport and localization (41), and this also appears to be the case in the formation of an HIV-1 assembly complex (43, 50).



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  		FIG. 8. Proposed model for Staufen's involvement in the posttranscriptional steps of the HIV-1 life cycle. Following pr55Gag synthesis on polysomes, pr55Gag interacts with Staufen (step 1). The Staufen-pr55Gag-HIV-1 genomic RNA complex is released from ribosomes and migrates towards the assembly complexes (step 2). The Staufen-pr55Gag-genomic RNA ternary complexes are assembled as virion particles at the plasma membrane (step 3). Following viral protease activation, most of Staufen is excluded from the assembly complexes, leading to encapsidation of only 2 to 10 Staufen molecules per virion (step 4) (34). See the text for additional discussion of this model.

Immunoelectron microscopy experiments revealed cytoplasmic assembly intermediates representing small Gag oligomers of about 10 molecules (36), likely corresponding to the previously described cytoplasmic detergent-resistant complexes (26, 27) and the complexes identified immediately following pr55Gag synthesis (43). Our results favor the idea that Staufen is recruited early by pr55Gag in this cytoplasmic assembly detergent-resistant complex to participate in the first events of NC-mediated Gag multimerization. Consistently, the Staufen complexes identified in our fractionation studies had all the characteristics of cytoplasmic detergent-resistant complexes (Fig. 2). Furthermore, the Staufen/pr55Gag interaction does not require translocation to the membrane, since the interaction of Staufen with Gag occurred when the MA domain was absent (Fig. 4 and 5). Since Staufen likely functions in mRNA trafficking in the cytoplasm (24), it could participate in the translocation of the pr55Gag assembly complexes and/or play a role in the recruitment of genomic RNA to sites of viral assembly.

The results presented in this article point to a role of Staufen in the posttranscriptional steps of the HIV-1 life cycle (Fig. 8). Staufen is likely to be recruited early during viral assembly by newly synthesized pr55Gag via the NC domain (Fig. 8, step 1). Staufen's association with ribosomes (29) and the cosedimentation with ribosomes in sucrose gradients (Fig. 2) indicate that a Staufen-pr55Gag-ribosome complex could represent an early assembly complex intermediate in the HIV-1 replication cycle. Cellular mRNAs and/or HIV-1 genomic RNA may then compete with ribosomes to release Staufen-Gag complexes (Fig. 8, step 1). Consistently, a molecular competition is proposed to exist between ribosomal protein L18 and double-stranded RNA for the double-stranded-RNA-binding protein PKR (25), and another that was proposed to exist between ribosomes and genomic RNAMLV for Moloney murine leukemia virus Gag that may result in the encapsidation of ribosomes instead of genomic RNAMLV (35). Staufen could also contribute to the oligomerization of pr55Gag by recruiting RNA, a component that was shown to be a necessary cofactor for this (8, 11), or likewise promote the association of genomic RNA with pr55Gag (Fig. 8, step 2).

Because Staufen is a cytoskeleton-associated protein and is involved in the transport of ribonucleoprotein complexes (24), Staufen may mediate the transport of this assembly complex intermediate toward the plasma membrane, where a ternary assembly complex of Staufen, pr55Gag, and genomic RNA is maintained to lead to genomic RNA encapsidation (Fig. 8, step 3). While RNA does not appear to be an important cofactor for the Staufen/pr55Gag association (Fig. 3D), genomic RNA is necessary for Staufen incorporation because NC and packaging signal proviral mutants generate virus particles that are devoid of Staufen (34). Because a single point mutation in the Staufen dsRBD3 abolishes both the genomic RNA and pr55Gag association, a dual role for this domain is suggested, as demonstrated for several other related double-stranded-RNA-binding proteins such as PKR and TAR RNA-binding protein (12). Knocking down Staufen expression by siRNA will likely disrupt early or subsequent steps of assembly through Staufen's interactions with pr55Gag and RNA and contribute to the generation of poorly infectious virus particles (Fig. 7). The contributions of each type of interaction of Staufen during the assembly process will require further analysis.

Sucrose gradient, coimmunoprecipitation, and BRET analyses strongly support a preferential association of Staufen with pr55Gag and not with mature Gag proteins (Fig. 2 to 5). This dictates that during Gag maturation by the viral protease, Staufen may be progressively excluded from Gag assembly complexes by modulation of its affinity for Gag (Fig. 8, step 4). Structural changes in Gag during maturation were previously proposed to explain the partial exclusion of cyclophilin A from HIV-1 (7, 42).

Staufen expression levels appear to be important for HIV-1 replication. For example, Staufen overexpression not only enhances the Staufen copy number within virus particles, but also correspondingly enhances the number of HIV-1 genomic RNA copies packaged into the virion (34). It also results in morphological changes in virus particles (Mouland et al., unpublished data), and this could be due to Staufen that is overrepresented in the Staufen-pr55Gag-genomic RNA ternary complex during assembly. Staufen knockdown (Fig. 7) could also disrupt the composition of this ternary complex at this step of the assembly process. While proof that Staufen and HIV-1 genomic RNA interact directly has been difficult to show experimentally, it is tempting to speculate that genomic RNA dimerization during HIV-1 assembly may play a role in this switch of Staufen to associate with genomic RNA, especially since Drosophila Staufen's association with bicoid mRNA requires RNA dimerization (16). Staufen's selective association with a complex in which HIV-1 genomic RNA is found (Fig. 6), especially compared to the absence of specificity exhibited by TRBP, is indicative of a role of Staufen in the fate of this RNA species during HIV-1 expression.

If Staufen participates in the selection of genomic RNA by NC for encapsidation in the context of this Staufen-pr55Gag-genomic RNA complex, for instance, we can anticipate that the fine-tuned regulation of the amounts of Staufen associated with fully assembled viral particles could represent a mechanism by which the appropriate number of genomic RNA molecules is packaged into HIV-1 to generate fully infectious viral particles. The experiments presented here and previously (34) support this model while current investigations are under way to study the other putative roles of Staufen during HIV-1 replication.


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ACKNOWLEDGMENTS
 
We thank Kristi Bangs for originally identifying efficient siRNA targets for Staufen, Sylvie Bannwarth for performing the TRBP gradient analysis, Damian Purcell for DNA constructs and for providing advice on the RT-PCR analysis, Éric A. Cohen for generous supply of materials and antibodies, Michel Tremblay for ELISA reagents and advice, Michel Bouvier and Billy Breton for constructs and advice on BRET assays, and the NIH AIDS Reference and Reagent Program and Spearman and Jacques Corbeil for the anti-MA antibody and CEM-GFP cell line, respectively. We thank Johanne Mercier for technical contributions to some of these studies and Hugo Dilhuydy for confocal imaging analyses.

J.-F.C. and C.M. were supported by studentships from the Fonds pour la recherche en santé du Québec (FRSQ) and the Natural Sciences and Engineering Research Council of Canada (NSERC). A.J.M. is a New Investigator of the Canadian Institutes of Health Research (CIHR) and a Scholar of the FRSQ. This work was supported by grants from the CIHR, the Canadian Foundation for Innovation, and the Canadian Foundation for AIDS Research to A.J.M. and from NSERC to L.D.


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FOOTNOTES
 
* Corresponding author. Mailing address: HIV-1 RNA Trafficking Laboratory, Lady Davis Institute for Medical Research, Room 323A, Sir Mortimer B. Davis Jewish General Hospital, 3755 Côte-Ste-Catherine Rd., Montréal, Québec, Canada H3T 1E2. Phone: (514) 340-8260. Fax: (514) 340-7537. E-mail: amouland{at}microimm.mcgill.ca. Back


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Molecular and Cellular Biology, April 2004, p. 2637-2648, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2637-2648.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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