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• W2040894241 abstract "RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription. RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription. RNA helicase A (RHA) 2The abbreviations used are: RHA, RNA helicase A; CREB, cAMP-response element-binding protein; CTE, constitutive transport element; HIV-1, human immunodeficiency virus, type 1; TAR, transactivation response element; RRE, Rev response element; LZ, leucine zipper; TAP, tandem affinity protein purification; nt, nucleotide(s); siRNA, small interfering RNA; RT, reverse transcription; CA, capsid; MA, matrix; GFP, green fluorescent protein; NC, nucleocapsid; MAGI, multinuclear activation of a galactosidase indicator. 2The abbreviations used are: RHA, RNA helicase A; CREB, cAMP-response element-binding protein; CTE, constitutive transport element; HIV-1, human immunodeficiency virus, type 1; TAR, transactivation response element; RRE, Rev response element; LZ, leucine zipper; TAP, tandem affinity protein purification; nt, nucleotide(s); siRNA, small interfering RNA; RT, reverse transcription; CA, capsid; MA, matrix; GFP, green fluorescent protein; NC, nucleocapsid; MAGI, multinuclear activation of a galactosidase indicator. is a member of the DEXH-box (where X can be any amino acid) family of proteins and is also termed DHX9 (1Lee C.G. Hurwitz J. J. Biol. Chem. 1992; 267: 4398-4407Abstract Full Text PDF PubMed Google Scholar, 2Lee C.G. Hurwitz J. J. Biol. Chem. 1993; 268: 16822-16830Abstract Full Text PDF PubMed Google Scholar). The DEXH-box proteins, together with the DEAD-box and the Ski2 family members, are referred to as RNA helicases that are able to rearrange the structures of RNA molecules (3Rocak S. Linder P. Nat. Rev. Mol. Cell. Biol. 2004; 5: 232-241Crossref PubMed Scopus (617) Google Scholar). RHA contains a helicase core domain consisting of seven motifs that are conserved for all RNA helicases. Within the N-terminal region of RHA there are two copies of type A double-stranded RNA binding domains. Together with an RGG-box domain located at the C terminus, double-stranded RNA binding domains regulate RNA binding as well as helicase activities of RHA (4Zhang S. Grosse F. J. Biol. Chem. 1997; 272: 11487-11494Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). RHA is a nuclear protein and shuttles between the nucleus and the cytoplasm with the assistance of a bidirectional nuclear transport domain consisting of 110 amino acids at the C terminus (5Tang H. McDonald D. Middlesworth T. Hope T.J. Wong-Staal F. Mol. Cell. Biol. 1999; 19: 3540-3550Crossref PubMed Scopus (55) Google Scholar). This function of the RHA nuclear transport domain is subject to regulation of arginine methylation catalyzed by PRMT1 (protein-arginine methyltransferase 1) (6Smith W.A. Schurter B.T. Wong-Staal F. David M. J. Biol. Chem. 2004; 279: 22795-22798Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). RHA is able to unwind double-stranded RNA or DNA with the energy derived from hydrolysis of NTPs by virtue of its NTPase activity (1Lee C.G. Hurwitz J. J. Biol. Chem. 1992; 267: 4398-4407Abstract Full Text PDF PubMed Google Scholar). This property enables RHA to participate in multiple cellular processes from RNA transcription to RNA processing to RNA nuclear export (7Zhang S. Grosse F. Acta Biochim. Biophys. Sin. (Shanghai). 2004; 36: 177-183Crossref PubMed Scopus (44) Google Scholar). These multiple functions underlie the vital role of RHA in the germ line proliferation and development of Caenorhabditis elegans (8Walstrom K.M. Schmidt D. Bean C.J. Kelly W.G. Mech. Dev. 2005; 122: 707-720Crossref PubMed Scopus (22) Google Scholar) and also account for the early embryonic lethality observed with RHA knock-out mice (9Lee C.G. da Costa Soares V. Newberger C. Manova K. Lacy E. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13709-13713Crossref PubMed Scopus (72) Google Scholar).The regulation activity of RHA in RNA transcription is implicated by its presence within the RNA polymerase II holoenzyme complex. For example, RHA has been shown to bridge the interactions between RNA polymerase II and transcription co-activators such as CREB-binding protein and BRAC1 (breast cancer-specific tumor suppressor protein 1) (10Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 11Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (337) Google Scholar). RHA also directly interacts with the p65 subunit of NF-κB and stimulates NF-κB-mediated reporter gene expression (12Tetsuka T. Uranishi H. Sanda T. Asamitsu K. Yang J.P. Wong-Staal F. Okamoto T. Eur. J. Biochem. 2004; 271: 3741-3751Crossref PubMed Scopus (72) Google Scholar). Involvement of RHA in transcription is further indicated by the function of its homologue in Drosophila, named the maleless (MLE) gene, that increases gene expression from the male X chromosome (13Kuroda M.I. Kernan M.J. Kreber R. Ganetzky B. Baker B.S. Cell. 1991; 66: 935-947Abstract Full Text PDF PubMed Scopus (239) Google Scholar, 14Lee C.G. Chang K.A. Kuroda M.I. Hurwitz J. EMBO J. 1997; 16: 2671-2681Crossref PubMed Scopus (122) Google Scholar). RHA has been found as a component of prespliceosomes (15Hartmuth K. Urlaub H. Vornlocher H.P. Will C.L. Gentzel M. Wilm M. Luhrmann R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16719-16724Crossref PubMed Scopus (242) Google Scholar), although it is unclear at which step of splicing RHA functions. In regard to RNA nuclear export, RHA directly binds to the constitutive transport element (CTE) RNA of type D retroviruses, such as Mason-Pfizer monkey virus. Together with cellular factors Tap (Tip-associated protein) and HAP95 (helicase A-binding protein 95), RHA promotes export of CTE-containing RNA molecules (16Tang H. Gaietta G.M. Fischer W.H. Ellisman M.H. Wong-Staal F. Science. 1997; 276: 1412-1415Crossref PubMed Scopus (130) Google Scholar, 17Tang H. Wong-Staal F. J. Biol. Chem. 2000; 275: 32694-32700Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Yang J.P. Tang H. Reddy T.R. Wong-Staal F. J. Biol. Chem. 2001; 276: 30694-30700Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar).In light of its multiple functions in cellular RNA metabolism, it is not surprising that RHA also regulates the activity of human immunodeficiency virus type 1 (HIV-1) RNA. For instance, RHA stimulates transcription of HIV-1 RNA (19Fujii R. Okamoto M. Aratani S. Oishi T. Ohshima T. Taira K. Baba M. Fukamizu A. Nakajima T. J. Biol. Chem. 2001; 276: 5445-5451Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The underlying mechanism involves binding of RHA to the transactivation response element (TAR) that is located at the very 5′ end of HIV-1 RNA. This interaction is mediated by the second double-stranded RNA binding domain of RHA, reminiscent of that taking place between TAR and TAR-binding protein (20Gatignol A. Buckler-White A. Berkhout B. Jeang K.T. Science. 1991; 251: 1597-1600Crossref PubMed Scopus (327) Google Scholar). It is perceived that, through binding to TAR, RHA either modulates the transactivation activity of Tat or facilitates the transcription activity of RNA polymerase II. RHA also enhances Rev/Rev response element (RRE)-mediated gene expression (21Li J. Tang H. Mullen T.M. Westberg C. Reddy T.R. Rose D.W. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 709-714Crossref PubMed Scopus (126) Google Scholar). Since RHA exhibits weak interaction with RRE and thus unlikely plays a direct role in the export of HIV-1 full-length RNA, RHA may promote the release of unspliced HIV-1 RNA from spliceosome. In addition to its involvement in HIV-1 gene expression, we now show that RHA associates with the Gag protein and is recruited into virus particles. These results suggest a novel activity of RHA in HIV-1 assembly.EXPERIMENTAL PROCEDURESPlasmid DNA Construction—The Gag-TAP (tandem affinity protein purification) plasmid DNA was generated by replacing the GFP DNA segment with the TAP sequence in the context of Gag-GFP DNA that was described previously (22Guo X. Hu J. Whitney J.B. Russell R.S. Liang C. J. Virol. 2004; 78: 551-560Crossref PubMed Scopus (16) Google Scholar). The TAP sequence was amplified using primers 5′-GCACCTGCAGAAGCGACGATGGAAAAAGAATTC-3′/5′-GCACGATATCTTATT CTTTGTTGAATTTGTTATC-3′ (underlined nucleotides represent the PstI and EcoRV restriction sites) and was inserted into Gag-GFP following digestion with PstI and EcoRV to generate Gag-TAP. Construction of Gag-LZ-TAP involved substituting the leucine zipper (LZ) sequence from yeast transcription factor GCN4 for the NC (nucleocapsid) region of Gag within the Gag-TAP DNA. PCR was performed with primers 5′-GCACACGGGCCCATGAAGCAGCTCGAGG-3′/5′-GCACATCTGCAGTTGTGACGAGGGGTCGTTGCC-3′ (underlined nucleotides represent restriction sites for ApaI and PstI) using BH10-LZ as DNA template (23Guo X. Liang C. Virology. 2005; 335: 232-241Crossref PubMed Scopus (12) Google Scholar). The amplified LZ DNA fragment was digested with ApaI and PstI, followed by insertion into Gag-TAP to engineer Gag-LZ-TAP.To generate the hGag-TAP DNA construct, the hGag sequence was first amplified from vector pVRC3900 (kindly provided by Dr. Gary Nabel) with primers 5′-GCATGATATCATGGGCGCCC GCGCCAGCGTG-3′/5′-GCATCTCGAGTTGTGACGAGGGGTCGCTGC-3′ (underlined nucleotides represent EcoRV and XhoI restriction sites), followed by insertion into pcDNA3-C-TAP (kindly provided by Drs. Nahum Sonenberg and Anne-Claude Gingras). DNA constructs D449-TAP, D408-TAP, D390-TAP, and D378-TAP were generated through inserting into pcDNA3-C-TAP the PCR products obtained with the sense primer 5′-GCATGATATCATGGGCGCCCGCGCCAGCGTG-3′ (the underlined nucleotides represent the EcoRV restriction site) together with the following antisense primers: 5′-GCATCTCGAGAAAATTCCCTGGCCTTCCCTTG-3′, 5′-GCATCTCGAGCCAGCAGCCCTTCTTGCGGGG-3′, 5′-GCATCTCGAGCTTGCCGCAGTTGAAGCACTT-3′, 5′-GCATCTCGAGCATGATGGTGGCGCTGTTGG-3′ (the underlined nucleotides represent the XhoI site).The D337/D9664 plasmid DNA was generated with the PCR-based strategy. The 337GAG339 DNA fragment was removed in PCR using primer pair 5′-GTACTTCAAGAACTGCTGACATCCTTGCTACAAGGGACTTTC-3′/5′-GAGCTCCCAGGCTCAGATCTG-3′. The resultant PCR products were used as megaprimers in a second round of PCR together with primer 5′-GCATGTTAACTGGAAGGGCTAATTCACTCCCAAC-3′. The final PCR products were digested with HpaI and BglII, followed by insertion into BH10 to generate BH-D337. Similarly, DNA fragment 9664CTCT9667 was removed in PCR using primers 5′-GTGCCCGTCTGTTGTGTGAGGTAACTAGAGATCCCTCAG-3′/5′-GCTTCTTCCAAGTACTTCTGCAG-3′. The resulting PCR products were used as megaprimers in a second round of PCR together with primer 5′-GTGGGAGCAGCATCTCGAGACCTG-3′. The final PCR products were digested with XhoI and StuI prior to insertion into BH-D337 to generate the D337/D9664 mutated DNA construct.Purification of Gag Protein with the TAP Procedure—The following protocol is adapted from the TAP method (24Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1415) Google Scholar). The 293T cells were transfected with DNA constructs expressing Gag-TAP fusion proteins. Subsequent to washing with ice-cold phosphate-buffered saline, cells were lysed in a buffer containing 50 mm Hepes (pH 8.0), 100 mm KCl, 2 mm EDTA, 0.1% Nonidet P-40, 2 mm dithiothreitol, 10 mm NaF, 0.25 mm NaOVO3,5nm okadaic acid, 5 nm calyculin A, 50 mm glycerolphosphate, 10% glycerol, and protease inhibitors (Roche Diagnostics). Following clarification at 10,000 × g, the supernatants were incubated with IgG beads (Amersham Biosciences) at 4 °C overnight. After washing with lysis buffer and TEV buffer (10 mm Hepes (pH 8.0), 150 mm NaCl, 0.1% Nonidet P-40, 0.5 mm EDTA, and 1 mm dithiothreitol), the Gag-bound IgG beads were incubated with TEV protease (Invitrogen) in TEV buffer at 16 °C for 2 h. The released Gag-TAP proteins were further incubated with calmodulin beads (Amersham Biosciences) in calmodulin-binding buffer (10 mm Hepes (pH 8.0), 150 mm NaCl, 1 mm MgOAc, 1 mm imidazole, 0.1% Nonidet P-40, 2 mm CaCl2, and 10 mm β-mercaptoethanol). After washing with calmodulin-binding buffer and calmodulin rinsing buffer (100 mm Hepes (pH 8.0), 75 mm NaCl, 1 mm MgOAc, 1 mm imidazole, and 2 mm CaCl2), the Gag proteins were eluted with a buffer containing 200 mm Hepes (pH 8.0), 75 mm NaCl, 1 mm MgOAc, 1 mm imidazole, and 25 mm EGTA. The eluted proteins were separated on SDS-polyacrylamide gels followed by Coomassie Blue staining. The visible protein bands were excised, in-gel-digested with trypsin, and subject to liquid chromatography/mass spectrometry analysis performed at McGill University and Genome Quebec Innovation Centre.Purification of Virus Particles—293T cells were transfected with HIV-1 cDNA. Virus particles in the culture supernatants were first pelleted at 100,000 × g and further purified in a second ultracentrifugation (100,000 × g) through a 15%/65% sucrose step gradient. Virus particles were collected at the 15%/65% sucrose interface and further pelleted at 100,000 × g. The purified virus particles were loaded on the top of a 20-70% continuous sucrose gradient, followed by centrifugation at 100,000 × g for 16 h at 4 °C. Twelve 1-ml fractions were collected, and the materials were subject to Western blots. In experiments to characterize the association of cellular proteins with viral RNP complex within virus particles, purified viruses were treated with 1% Triton X-100 at room temperature for 5 min in TN buffer containing 10 mm Tris-HCl (pH 8.0) and 100 mm NaCl. The treated samples were spun at 10,000 × g, and the pellets were washed three times with TN buffer (25Welker R. Hohenberg H. Tessmer U. Huckhagel C. Krausslich H.G. J. Virol. 2000; 74: 1168-1177Crossref PubMed Scopus (165) Google Scholar).Viral RNA Analysis—RNA was prepared from purified virus particles and characterized either in RNase protection assays or in native Northern blots (26Russell R.S. Hu J. Beriault V. Mouland A.J. Laughrea M. Kleiman L. Wainberg M.A. Liang C. J. Virol. 2003; 77: 84-96Crossref PubMed Scopus (61) Google Scholar). In RNase protection assays, viral RNA was incubated with excessive amounts of 32P-labeled viral RNA probes (nt positions -164 to 321) at 42 °C overnight followed by digestion with single-strand RNA-specific RNases (Ambion Inc., Austin, TX). The protected RNA fragments were separated on 5% polyacrylamide, 8 m urea gels and visualized through exposure to x-ray films. As for native Northern blots, viral RNA first underwent electrophoresis on 0.9% native agarose gels followed by transfer onto nylon membranes (Amersham Biosciences). Hybridization was performed with 32P-labeled HIV-1 DNA probes (nt positions 1-2000). Viral RNA signals were visualized through exposure to x-ray films. To assess expression of viral RNA within 293T cells that had been transfected with small interfering RNA (siRNA) oligonucleotides and the BH10 viral DNA construct, total cellular RNA was extracted with Trizol reagent (Invitrogen) and examined in Northern blots.Immunofluorescence Staining—293T cells were transfected with the Gag-GFP DNA construct and then fixed with 4% paraformaldehyde at room temperature for 20 min. Following treatment with 0.2% Triton X-100 at room temperature for 10 min, the cells were incubated with anti-RHA rabbit antibodies (kindly provided by Dr. Jeffery D. Parvin) at a dilution of 1:200 for 2 h at 37°C. After washing with phosphate-buffered saline, a second incubation with goat anti-rabbit IgG Alexafluor 647-conjugated secondary antibodies (Invitrogen) was performed for 1 h at 37°C. The stained cells were examined using the LSM 5 PASCAL confocal microscope. The images were recorded from layers of 0.9 μm in thickness.Knockdown of RHA with siRNA Oligonucleotides—siRNA oligonucleotides 5′-GAAUGACCUGGGAAGCCAAdTdT-3′ (sense)/5′-UUGGCUUCCCAGGUCAUUCdTdT-3′ (antisense) were designed to target RHA mRNA sequence at nt 2929-2947. Control siRNA oligonucleotides (catalogue number 4611 or 4613) were purchased from Ambion Inc. Lipofectamine 2000 (Invitrogen) was used to transfect siRNA oligonucleotides into 293T cells. Levels of endogenous RHA were measured in Western blots using anti-RHA antibodies. The same membranes were probed with anti-β-actin antibodies (Sigma) to measure levels of β-actin as an internal control.Primer Extension (27Cen S. Niu M. Kleiman L. Retrovirology. 2004; 1: 33Crossref PubMed Scopus (22) Google Scholar)—The tRNA3Lys-viral RNA template was extracted from purified virus particles, and the relative amount of viral RNA template was determined in slot blot experiments. The tRNA3Lys primer was extended for 6 bases (CTGCTA) from the same amounts of viral RNA templates by HIV-1 reverse transcriptase (RT) in a cell-free reaction containing 50 mm Tris-HCl (pH 7.8), 100 mm KCl, 10 mm MgCl2, 10 mm dithiothreitol, 0.2 mm dCTP, 0.2 mm dTTP, 5 μCi of [α-32P]dGTP, and 0.05 mm ddATP (used to terminate the reaction at the 6-base stage). The products were fractionated in a 5% polyacrylamide denaturing gel and were visualized through exposure to x-ray films.Determination of Viral cDNA Production—The D337/D9664 virus was collected from transfected 293T cells, treated with DNase I to remove plasmid DNA, and purified through the 15%/65% sucrose step gradient. Purified virus particles equivalent to 1 μg of viral capsid (CA) (p24) antigen were incubated at 37 °C for 6 h in a buffer containing 50 mm Tris-HCl (pH 8.0), 2 mm dithiothreitol, 2 mm MgOAc, 0.4 mm dNTPs, 1 mm n-octyl glycoside, 60 mm KCl, 10 mm NaCl, and 0.04% Triton X-100. Reactions were terminated with buffer containing 1% SDS, 50 mm EDTA, and 0.4 m NaCl. Following protease K digestion and phenol/chloroform extraction, newly reverse transcribed viral cDNA was precipitated with 2.5 volumes of 95% ethanol and further assessed in PCR and real time PCR performed with primer pairs 5′-GTACTTCAAGAACTGCTGACATCGAG-3′/5′-GTCTGAGGGATCTCTAGTTACCAGAG-3′ or 5′-CTGGTTAGACCAGATCTGAGCCTG-3′/5′-GTCTGAGGGATCTCTAGTTACCAGAG-3′ Real time PCR was conducted with the LightCycler FastStart DNA Master SYBR Green 1 kit (Roche Diagnostics) in the light cycler machine (Roche Diagnostics).Measurement of Virus Infectivity—An amount of viruses equivalent to 2 ng of viral CA protein was used to infect 5 × 105 MT-2 cells. Virus growth was monitored through determining levels of RT activity in the culture fluids at various time points. Virus infectivity was also determined by performing the MAGI assays (28Kimpton J. Emerman M. J. Virol. 1992; 66: 2232-2239Crossref PubMed Google Scholar). HeLa cells, that express CD4 and are stably transfected with HIV-1 long terminal repeat-β-galactosidase reporter DNA, were infected with viruses equivalent to 10 ng of viral CA protein. Forty-eight hours after infection, cells were fixed and stained using the β-GAL staining kit (Invitrogen). The number of blue cells was scored to determine the infectivity of HIV-1.RESULTSHIV-1 Gag-associated Cellular Factor Candidates Revealed by the Results of Mass Spectrometry—The TAP method has been widely used in yeast proteomic studies and was recently adapted to study protein-protein interactions in mammalian cells (24Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1415) Google Scholar). To identify cellular factors that associate with HIV-1 Gag protein, we have appended the IgG-binding units of protein A from Staphylococcus aureus and the calmodulin-binding peptide to the C terminus of Gag and generated an HIV-1 cDNA clone named Gag-TAP (Fig. 1A) (24Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Seraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1415) Google Scholar). These two tags allow isolation of Gag protein from transfected cells to high purity using the IgG beads and the calmodulin beads in a two-step affinity purification procedure. The purified protein samples were subject to mass spectrometry analysis (Fig. 1). When N-TAP protein alone was purified from transfected 293T cells and the eluted samples were separated on SDS-PAGE, two protein bands were visualized with Coomassie Blue staining that contained tubulins and ribosomal protein S4, respectively (Fig. 1B). This finding supports the conclusion that the majority of the proteins shown in Fig. 1A were purified as a result of interaction with Gag either in a direct or indirect manner. Among these protein candidates are a group of cellular RNA helicases, including RHA, DEAD-box protein 18 (DDX18), DEAD-box protein 24 (DDX24) and RNA helicase Gu(α). Results of immunoprecipitation and Western blots showed that both RHA and RNA helicase Gu(α) interacted with Gag, yet only RHA was detected to associate with virus particles (data not shown). DDX18 and DDX24 were not tested in these latter experiments due to the lack of antibodies. In this study, we further investigated the potential role of RHA in production of infectious virus particles.FIGURE 1Cellular factors associated with HIV-1 Gag. A, results of mass spectrometry revealing the cellular proteins that were co-purified with HIV-1 Gag protein. Domain structure of the Gag-TAP fusion protein is shown at the top. The calmodulin binding peptide (CBD), TEV protease cleavage site, and protein A (ProtA) sequences are linked to the C terminus of Gag in the context of BH10 (protease-negative) HIV-1 cDNA clone. Following transfection of 293T cells with Gag-TAP, cell lysates were subjected to a two-step affinity purification procedure (available on the World Wide Web at www.mc.vanderbilt.edu/vumcdept/cellbio/gould/). The purified proteins were fractionated on SDS-polyacrylamide gels and visualized by Coomassie Blue staining. The protein bands were analyzed with trypsin in-gel digestion and mass spectrometry. Identity of proteins within each band is shown on the right side of the gels. B, control experiments performed with the N-TAP vector. N-TAP contains the protein A and calmodulin binding peptide sequences.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RHA Associates with HIV-1 Gag Protein in an RNA-dependent Manner—To characterize Gag-RHA interaction, we first examined the Gag complex isolated from transfected 293T cells in Western blots using anti-RHA antibodies. The results showed that RHA was co-purified with Gag but not in the control experiments using the TAP sequence alone (Fig. 2A). When Gag was immunoprecipitated from the lysates of transfected cells using anti-CA antibodies, RHA was also detected in the immunoprecipitated materials in Western blots using anti-RHA antibodies (data not shown). Furthermore, pull-down of the FLAG-RHA protein using anti-FLAG antibody-coated agarose beads led to detection in the purified materials of Gag protein that was expressed from the BH10-PR-DNA co-transfected into 293T cells with the FLAG-RHA DNA construct (Fig. 2B). Interestingly, when the NC domain was replaced with the leucine zipper motif from yeast transcription factor GCN4 (29Accola M.A. Strack B. Gottlinger H.G. J. Virol. 2000; 74: 5395-5402Crossref PubMed Scopus (251) Google Scholar), the resultant Gag mutant named Gag-LZ-TAP lost the interaction with RHA (Fig. 2A). To validate the essence of NC in Gag-RHA interaction, we tested several Gag mutants that lack various lengths of peptide sequences from the C terminus (Fig. 2C). These Gag mutants were generated using the hGag sequence that contains modified RNA sequence but the same amino acid sequence as the Gag from HIV-1 cDNA clone HXB2 (30Huang Y. Kong W.P. Nabel G.J. J. Virol. 2001; 75: 4947-4951Crossref PubMed Scopus (105) Google Scholar). When the isolated Gag mutants were examined in Western blots using anti-RHA antibodies, similar levels of RHA were observed to associate with the full-length hGag and the hGag mutant D449 that lacked the p6 sequence (Fig. 2C). Further deletion of the zinc finger sequence in mutants D408 and D390 led to substantially decreased amounts of RHA that could be co-purified. In the case of hGag mutant D378 that was missing the entire NC region, interaction with RHA was completely lost.FIGURE 2HIV-1 Gag interacts with RHA within cells in an RNA-dependent manner. A, RHA associates with wild-type HIV-1 Gag but not the Gag mutant Gag-LZ. The mutated Gag-LZ had NC replaced with the leucine zipper sequence from the yeast transcription factor GCN4 (29Accola M.A. Strack B. Gottlinger H.G. J. Virol. 2000; 74: 5395-5402Crossref PubMed Scopus (251) Google Scholar). The Gag-TAP proteins were isolated from transfected 293T cells by affinity purification using the IgG beads and calmodulin beads, followed by analysis in Western blots using either anti-CA or anti-RHA antibodies. N-TAP represents the TAP peptide expressed from the pcDNA3-N-TAP vector and serves as a control in the purification experiments. The lysates of untransfected 293T cells were included in Western blots as a positive control for RHA. B, the BH10-PR-DNA was co-transfected into 293T cells with the FLAG-RHA DNA. The FLAG-RHA protein was precipitated with anti-FLAG antibody-coated agarose beads (Sigma), and the pull-down materials were assessed in Western blots using either anti-CA or anti-FLAG antibodies. C, Gag mutant lacking the NC region fails to bind RHA. The C-terminal truncated Gag mutants are illustrated, with the shaded boxes denoting the two zinc finger motifs of NC. Association of RHA with isolated Gag proteins was examined in Western blots using anti-RHA antibodies. D, association of Gag and RHA is sensitive to RNase treatment. The 293T cells were transfected with the Gag-TAP DNA and lysed in a buffer containing 100 μg/ml RNase A. This RNase treatment effectively depleted cellular RNA as shown by the degradation of rRNA. Gag was isolated using the TAP procedure, and levels of associated RHA were measured in Western blots using anti-RHA antibodies. E, colocalization of RHA and Gag within cells. 293T cells were transfected with the Gag-GFP DNA that had the GFP gene appended to the C terminus of Gag in the context of the HIV-1 cDNA clone BH10 (22Guo X. Hu J. Whitney J.B. Russell R.S. Liang C. J. Virol. 2004; 78: 551-560Crossref PubMed Scopus (16) Google Scholar). Endogenous RHA was stained with anti-RHA antibodies and Alexa-fluor 647-conjugated secondary antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The essence of NC for Gag and RHA association suggests an RNA-dependent nature of this interaction event. To test this, RNase A was added to cell lysates during Gag purification. The efficiency of RNase A treatment was monitored by the extent of ribosomal rRNA degradation (Fig. 2D). The results of Western blots revealed that RNase A treatment drastically diminished the levels of RHA that could be co-purified with Gag. This indicates that Gag-RHA interaction is RNA-dependent.Colocalization of RHA and Gag within Cells—RHA is a nuclear protein and shuttles between the nucleus and the cytoplasm (5Tang H. McDonald D. Middlesworth T. Hope T.J. Wong-Staal F. Mol. Cell. Biol. 1999; 19: 3540-3550Crossref PubMed Scopus (55) Google Scholar). HIV-1 Gag is generally believed to locate within the cytoplasm. It is thus intriguing to determine to which extent RHA and Gag co-localize within cells such that they can interact with each other. Toward this end, 293T cells were transfected with the Gag-GFP construct followed by immunofluorescence staining using anti-RHA antibodies, and images of a few cells are shown in Fig. 2E. The results revealed that RHA was mainly seen within the nucleus with a small detectable amount within the cytoplasm. Notably, a substantial portion of RHA was re" @default.
• W2040894241 created "2016-06-24" @default.
• W2040894241 creator A5008130890 @default.
• W2040894241 creator A5008995173 @default.
• W2040894241 creator A5040023643 @default.
• W2040894241 creator A5065705941 @default.
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• W2040894241 date "2006-05-01" @default.
• W2040894241 modified "2023-10-18" @default.
• W2040894241 title "Association of RNA Helicase A with Human Immunodeficiency Virus Type 1 Particles" @default.
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