The Interaction between HIV-1 Gag and Human Lysyl-tRNA Synthetase during Viral Assembly
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m301840200
ISSN1083-351X
AutoresHassan Javanbakht, Rabih Halwani, Shan Cen, Jenan Saadatmand, Karin Musier‐Forsyth, Heinrich G. Göttlinger, Lawrence Kleiman,
Tópico(s)HIV Research and Treatment
ResumoHuman lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag·LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208–259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys. Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag and LysRS that are required for this interaction. In vitro reactions between wild-type and truncated HIV-1 Gag and human LysRS were monitored using GST-tagged molecules and glutathione-agarose chromatography. Gag/LysRS interaction in vivo was detected in 293FT cells cotransfected with plasmids coding for wild-type or mutant HIV-1 Gag and LysRS, either by monitoring Gag·LysRS complexes immunoprecipitated from cell lysate with anti-LysRS or by measuring the ability of LysRS to be packaged into budded Gag viral-like particles. Based on these studies, we conclude that the Gag/LysRS interaction depends upon Gag sequences within the C-terminal domain of capsid (the last 54 amino acids) and amino acids 208–259 of LysRS. The latter domain includes the class II aminoacyl-tRNA synthetase consensus sequence known as motif 1. Both regions have been implicated in homodimerization of capsid and LysRS, respectively. Sequences falling outside these amino acid stretches can be deleted from either molecule without affecting the Gag/LysRS interaction, further supporting the observation that LysRS is incorporated into Gag viral-like particles independent of its ability to bind tRNALys. The life cycle of HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; LysRS, lysyl-tRNA synthetase; IleRS, isoleucine-tRNA synthetase; ProRS, proline-tRNA synthetase; GlnRS, glutamine-tRNA synthetase; ArgRS, arginine-tRNA synthetase; TrpRS, tryptophan-tRNA synthetase; MetRS, methionine-tRNA synthetase; TyrRS, tyrosine-tRNA synthetase; AsnRS, asparagine-tRNA synthetase; Gag, HIV-1 precursor protein containing sequences coding for HIV-1 structural proteins; Gag-Pol, HIV-1 precursor protein containing sequences coding for retroviral structural proteins and retroviral enzymes; VLP, viral-like-particle; Z, zipper; GST, glutathione S-transferase; TNE, Tris sodium chloride EDTA. has been intensely studied (for recent review see Ref. 1Swanstrom R. Wills J.W. Coffin J. Hughes S. Varmus H. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 263-334Google Scholar). Upon infection of a cell by HIV-1, the viral RNA genome is copied into a double-stranded cDNA by the viral enzyme reverse transcriptase. tRNALys3 is required to initiate reverse transcription (2Mak J. Kleiman L. J. Virol. 1997; 71: 8087-8095Crossref PubMed Google Scholar). The resultant viral DNA is translocated into the nucleus of the infected cell where it integrates into the host cell DNA and codes for viral mRNA and proteins. Proteins comprising the viral structure include both the glycosylated envelope proteins (glycoproteins 120 and 41) and mature proteins resulting from the processing of the large precursor protein, Gag (Pr55gag): matrix (MAp11), capsid (CAp24), and nucleocapsid (NCp7). Gag also contains C-terminal sequences for the p6 protein, which is believed to play a role in viral budding from the cell. The three viral enzymes used in the HIV-1 life cycle result from the processing of another precursor Gag-Pol (Pr160gag-pol) and are protease (PRp11), reverse transcriptase (RTp66/p51), and integrase (INp32). Both Gag and Gag-Pol are translated from the same full-length viral RNA, and this RNA, which also serves as the viral genomic RNA, is packaged into assembling virions via binding to nucleocapsid sequences in Gag (3Berkowitz R. Fisher J. Goff S.P. Krausslich H.G. Morphogenesis and Maturation of Retroviruses.Vol. 214. Springer-Verlag New York Inc., New York1996: 177-218Google Scholar, 4Geigenmüller U. Linial M.L. J. Virol. 1996; 70: 667-671Crossref PubMed Google Scholar). The in vivo interaction of Gag with Gag-Pol has also been well documented (5Park J. Morrow C.D. J. Virol. 1992; 66: 6304-6313Crossref PubMed Google Scholar, 6Smith A.J. Cho M.I. Hammarskjöld M.L. Rekosh D. J. Virol. 1990; 64: 2743-2750Crossref PubMed Google Scholar, 7Smith A.J. Srivivasakumar N. Hammarskjöld M.-L. Rekosh D. J. Virol. 1993; 67: 2266-2275Crossref PubMed Google Scholar, 8Srinivasakumar N. Hammarskjöld M.-L. Rekosh D. J. Virol. 1995; 69: 6106-6114Crossref PubMed Google Scholar), and Gag-Pol is carried into the assembling Gag particle by its interaction with Gag protein, probably through intermolecular interactions between homologous Gag sequences. The Gag and Gag-Pol proteins assemble at the cell membrane, and during budding from the cell, the viral protease, PRp11, is activated and cleaves these two precursor precursors into the proteins found in the mature virion. The major tRNALys isoacceptors in mammalian cells, tRNALys1,2 and tRNALys3, are also selectively packaged into the virion during its assembly (9Jiang M. Mak J. Ladha A. Cohen E. Klein M. Rovinski B. Kleiman L. J. Virol. 1993; 67: 3246-3253Crossref PubMed Google Scholar). Gag protein is capable of forming extracellular Gag viral-like particles (VLPs), which are made by transfecting cells with a plasmid coding only for the Gag protein, but the additional presence of Gag-Pol is required for the packaging of tRNALys into either Gag VLPs or into HIV-1 (10Mak J. Jiang M. Wainberg M.A. Hammarskjold M.-L. Rekosh D. Kleiman L. J. Virol. 1994; 68: 2065-2072Crossref PubMed Google Scholar). Increasing the amount of tRNALys3 incorporated into HIV-1 results in a viral population with increased levels of tRNALys3 annealed to the viral RNA genome and increased infectivity (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (57) Google Scholar). In addition to the tRNALys isoacceptors, human lysyl-tRNA synthetase (LysRS), the enzyme that aminoacylates tRNALys, is also selectively packaged into HIV-1 during its assembly (12Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar, 13Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar) and is a strong candidate for being the signal by which viral proteins recognize and selectively package the tRNALys isoacceptors. The packaging of LysRS into HIV-1 appears to be quite selective. Published work (12Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar, 13Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar) indicates that human IleRS, ProRS, and TrpRS are not detected in the virion, whereas other work in one of our laboratories 2R. Halwani and L. Kleiman, unpublished work. indicates the additional absence of human ArgRS, GlnRS, MetRS, TyrRS, and AsnRS. In addition, Rous sarcoma virus, which uses tRNATrp as a primer tRNA for reverse transcription, contains TrpRS but not LysRS (13Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar). An HIV-1 population contains, on average, ∼20–25 molecules of LysRS/virion (13Cen S. Javanbakht H. Kim S. Shiba K. Craven R. Rein A. Ewalt K. Schimmel P. Musier-Forsyth K. Kleiman L. J. Virol. 2002; 76: 13111-13115Crossref PubMed Scopus (69) Google Scholar) similar to the average number of tRNALys molecules/virion (14Huang Y. Mak J. Cao Q. Li Z. Wainberg M.A. Kleiman L. J. Virol. 1994; 68: 7676-7683Crossref PubMed Google Scholar). Our current hypothesis for the formation of a tRNALys-packaging complex includes a Gag·Gag-Pol complex interacting with a tRNALys·LysRS complex with Gag interacting with LysRS and Gag-Pol interacting with tRNALys. In addition to the reports cited above that provide evidence for an interaction between Gag and Gag-Pol, evidence supporting this model includes the following. 1) Whereas the incorporation of tRNALys into viruses requires Gag-Pol (10Mak J. Jiang M. Wainberg M.A. Hammarskjold M.-L. Rekosh D. Kleiman L. J. Virol. 1994; 68: 2065-2072Crossref PubMed Google Scholar), the incorporation of LysRS into HIV-1 occurs independently of tRNALys packaging, i.e. it is also packaged efficiently into Gag VLPs (12Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar), which do not selectively package tRNALys (10Mak J. Jiang M. Wainberg M.A. Hammarskjold M.-L. Rekosh D. Kleiman L. J. Virol. 1994; 68: 2065-2072Crossref PubMed Google Scholar). 2) Overexpression of LysRS in the cell results in a near doubling of the incorporation of both tRNALys and LysRS into HIV-1 (11Gabor J. Cen S. Javanbakht H. Niu M. Kleiman L. J. Virol. 2002; 76: 9096-9102Crossref PubMed Scopus (57) Google Scholar). 3) The ability of tRNALys to interact with LysRS is required for the incorporation of tRNALys into the virion (15Javanbakht H. Cen S. Musier-Forsyth K. Kleiman L. J. Biol. Chem. 2002; 277: 17389-17396Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Therefore, the interaction between Gag and LysRS may be critical for the selective packaging of primer tRNALys3 into the virion and represents a potentially new target for anti-HIV-1 therapy. The sites of interaction between Gag and LysRS are explored in this report using both in vitro and in vivo approaches. As described above, the amino acid sequences within the viral Gag precursor that represent different mature viral proteins have been well delineated. Furthermore, the relatively high sequence conservation among LysRSs and the large amount of structural and biochemical data on aminoacyl-tRNA synthetases has greatly facilitated the design of the truncated LysRS constructs used in these studies. The crystal structures of Escherichia coli LysRS (16Onesti S. Miller A.D. Brick P. Structure. 1995; 3: 163-176Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and Thermus thermophilus LysRS (17Cusack S. Yaremchuk A. Tukalo M. EMBO J. 1996; 15: 6321-6334Crossref PubMed Scopus (150) Google Scholar) have been solved. Eukaryotic LysRS is a class II synthetase, forming a closely related subgroup (known as IIb) with aspartyl- and asparginyl-tRNA synthetases (18Eriani G. Delarue M. Poch O. Gangloff J. Moras D. Nature. 1990; 347: 203-206Crossref PubMed Scopus (1192) Google Scholar, 19Eriani G. Dirheimer G. Gangloff J. Nucleic Acids Res. 1990; 18: 7109-7118Crossref PubMed Scopus (67) Google Scholar). The anticodon is a major recognition element for all of the class IIb synthetases including human LysRS (20Stello T. Hong M. Musier-Forsyth K. Nucleic Acids Res. 1999; 27: 4823-4829Crossref PubMed Scopus (29) Google Scholar, 21Saks M.E. Sampson J.R. Abelson J.N. Science. 1994; 263: 191-197Crossref PubMed Scopus (151) Google Scholar). This subclass is characterized by an N-terminal anticodon-binding domain with a topology known as an oligonucleotide-binding fold, which is positioned downstream of the N-terminal extension found in higher eukaryotes. Although truncation of this extra domain (N-terminal 65 residues) does not significantly affect aminoacylation by human LysRS (22Shiba K. Stello T. Motegi H. Noda T. Musier-Forsyth K. Schimmel P. J. Biol. Chem. 1997; 272: 22809-22816Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), it has been shown that the N-terminally truncated enzyme does display significantly weaker tRNA binding affinity. Thus, hamster LysRS was determined to have 100-fold lower apparent affinity for tRNALys when the N-terminal domain was removed (23Francin M. Kaminska M. Kerjan P. Mirande M. J. Biol. Chem. 2002; 277: 1762-1769Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and specific residues within the N terminus that function in tRNA binding have been identified recently (24Francin M. Mirande M. J. Biol. Chem. 2003; 278: 1472-1479Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) This domain was proposed to provide hamster LysRS with nonspecific tRNA-binding properties. All of the class II synthetases are also characterized by an anti-parallel β-sheet active-site fold and contain three consensus motifs known as motifs 1, 2, and 3 (18Eriani G. Delarue M. Poch O. Gangloff J. Moras D. Nature. 1990; 347: 203-206Crossref PubMed Scopus (1192) Google Scholar). Motif 1 is part of the dimer interface (class II synthetases are dimers or tetramers), whereas motifs 2 and 3 together constitute the aminoacylation active site. The distribution of these different subdomains in LysRS is shown in cartoon form in Fig. 1A. Plasmid Construction—pSVGag-REV response element and CMV-REV were donated by D. Rekosh and M. L. Hammarskjold (both from University of Virginia) (6Smith A.J. Cho M.I. Hammarskjöld M.L. Rekosh D. J. Virol. 1990; 64: 2743-2750Crossref PubMed Google Scholar). Plasmids ZWt, ZWt-p6, and ΔZ-Wt-p6 were constructed as previously described (26Accola M.A. Strack B. Gottlinger H.G. J. Virol. 2000; 74: 5395-5402Crossref PubMed Scopus (253) Google Scholar). Gag deletion mutants for expression in 293FT cells were constructed by PCR amplification of the pSVGag-RRE cDNA and digested with SalI and SpeI whose sites were placed in each of the PCR primers. These fragments were cloned into SpeI-SalI sites of pSVGag-RRE. The following primers were used to construct these Gag mutants: Δ323–500 (forward primer: 5′-AATCAGTCTAGACAAAATTACCCTATAGTGCAG-3′; reverse primer: 5′-ACTCTGATCACTATCATTGGACCAACAAGGTTTCTGT-3′) and Δ363–500 (forward primer: 5′-AATCAGTCTAGACAAAATTACCCTATAGTGCAG-3′; reverse primer: 5′-ACTCTGATCAATCACAAAACTCTTGCCTTATGGCC-3′). These plasmids express truncated Gag when co-transfected with CMV-REV in 293FT cells. All of the wild-type and mutant GST-Gag plasmids were constructed using PCR. The pSVGag-RRE cDNA was PCR-amplified and digested with EcoRI whose sites were introduced in each of the PCR primers. These fragments were cloned into the EcoRI site of pGEX4t2 (Amersham Biosciences). The following primers were used to construct wild-type and mutant GST-Gag: wild type (forward primer: 5′-AATTATGAATTCCTATTATTGTGACGAGGGGTCGTTGCC-3′; reverse primer: 5′-AATTATGAATTCCTATTATTGTGACGAGGGGTCGTTGCC-3′); Δ1–307 (5′-CTCCGGGAATTCCCGCTTCACAGGAGGTAAAAAATT-3′); Δ1–337 (5′-CTC CGGGAATTCCCGGACCAGCGGCTACACTAGAAGA-3′); Δ1–378 (5′-CTCCGGGAATTCCCATGCAGAGACGCAATTTTAGGAAC-3′); Δ363–500 (5′-AATTATGAATTCCTACAAAACTCTTGCC TTATGGCC-3′); and Δ433–500 (5′-AATTATGAATTCCTACCCTAAAAAATTAGCCTGTCTC-3′). These plasmids express wild-type and truncated Gag in BL21 E. coli cells. Plasmid pM368 contains cDNA encoding full-length (1–597 amino acids) human LysRS as previously described (22Shiba K. Stello T. Motegi H. Noda T. Musier-Forsyth K. Schimmel P. J. Biol. Chem. 1997; 272: 22809-22816Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To construct wild-type and mutant LysRS, this cDNA was PCR-amplified and digested with EcoRI, whose sites were placed in each of the PCR primers. These fragments were cloned into the EcoRI site of pcDNA1.0 c-Myc (Invitrogen). We used the following primers: wild-type LysRS (forward primer: 5′-CTCCGGGAATTCTAGCGGCCGTGCAGGCGGCCGAGGTG-3′; reverse primer: AATTATGAATTCCTAGACAGAAGTGCCAACTGTTGTGCT-3′); Δ452–597 (5′-AATTATGAATTCCTACAGGAACTCCCCAACAAGCTTGTCAAGGAG-3′); Δ309–597 (5′-AATTATGAATTCCTAACCAACCACAAGCATCTTATGATAGAGTTC-3′; Δ267–597 (5′-AATTATGAATTCCTACTATTCAATCTCTAGGAATCCCAG-3′; Δ260–597 (5′-AATTATGAATTCCTACTAATCTAAGAAACTTCTTATATA-3′); Δ249–597 (5′-AATTATGAATTCTACTACTTAGAGCGGATGATAAATTTCTG-3′); Δ207–597 (5′-AATTATGAATTCCTAAGACAGCAGTGTGATTCATACGGAATGAT-3′; and Δ1–207 (5′-CTCCGGGAATTCTCCCTGTTTGCATATGTTACCTCATCTTCA-3′. The resulting constructs express c-Myc-tagged wild-type and mutant LysRS proteins once transfected into 293FT cells. All of the wild-type and mutant GST-LysRS plasmids were constructed using PCR. The cDNA was PCR-amplified and digested with EcoRI whose sites were introduced in each of the PCR primers. These fragments were cloned into an EcoRI site of pGEX4t2 (Amersham Biosciences). The following primers were used to construct wild-type and mutant GST-LysRS (wild-type GST-LysRS): forward primer, 5′-CTCCGGGAATTCT AGCGGCCGTGCAGGCGGCCGAGGTG-3′, and reverse primer, 5′-AATTATGAATTCCTAGACAGAAGTGCCAACTGTTGTGCT-3′. Using the same forward primer, the following reverse primers were used for C-terminal deletions: Δ506–597 (5′-AATTATGAATTCCTACTACATGGGATCATTCAGGTCAGTAT-3′); Δ452–597 (5′-AATTATGAATTCCTACAGGAACTCCCCAACAAGCTTGTCAAGGAG-3′); Δ373–597 (5′-AATTATGAATTCCTACTAGTAGGTGACCTTGTAACTGCCTGT-3′); Δ309–597 (5′-AATTATGAATTCCTAACCAACCACAAGCATCTTATGATAGAGTTC-3′); Δ249–597 (5′-AATTATGAATTCCTACTACTTAGAGCGGATGATAAATTTCTG-3′); and Δ207–597 (5′-AATTATGAATTCCTAAGACAGCAGTGTGATCTCATACGGAATGAT-3′). The resulting constructs express wild-type and mutant GST-LysRS proteins in BL21 E. coli cells. Production of Wild-type and Mutant HIV-1 Virus—293FT cells (Invitrogen) are a clonal derivative of the human kidney 293T cell line. They were transfected with wild-type or mutant Gag and LysRS constructs using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Cell culture supernatant was collected 63 h post-transfection. Gag VLPs were pelleted from culture medium by centrifugation in a Beckman 45 Ti rotor at 35,000 rpm for 1 h. The pellet was then purified by centrifugation in a Beckman SW41 rotor at 26,500 rpm for 1 h through 15% sucrose onto a 65% sucrose cushion. The band of purified VLP was removed and pelleted in 1× TNE in a Beckman 45 Ti rotor at 40,000 rpm for 1 h. Protein Analysis—Viral and cellular proteins were extracted with radioimmunoprecipitation assay buffer (10 mm Tris, pH 7.4, 100 mm NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 2 mg of aprotinin/ml, 2 mg of leupeptin/ml, 1 mg of pepstatin A/ml, 100 mg of phenylmethylsulfonyl fluoride/ml). The viral and cell lysates were analyzed by SDS-PAGE (10% acrylamide) followed by blotting onto nitrocellulose membranes (Amersham Biosciences). Detection of protein by Western blotting utilized monoclonal antibodies that are specifically reactive with HIV-1 capsid (Zepto Metrocs, Inc), a polyclonal antibody for human LysRS (Pocono Rabbit Farm and Laboratory, Inc.), a monoclonal antibody for c-Myc (Invitrogen), a monoclonal antibody for GST (Amersham Biosciences), a monoclonal antibody to β-actin (Sigma), and a monoclonal antibody to C-terminal of HIV-1 capsid, which was used to detect Δ ZWt-p6 (National Institutes of Health AIDS Research and Reference Reagent Program). Detection of HIV proteins was performed by enhanced chemiluminescence (PerkinElmer Life Sciences Products) using the following secondary antibodies obtained from Amersham Biosciences: anti-mouse (for capsid and c-Myc), anti-rabbit (for LysRS), anti-goat (for GST), and anti human (for C-terminal capsid). Bacterial Expression and in Vitro Binding Assay—GST-Gag, GST-LysRS, and GST control proteins were expressed in E. coli BL21 (Invitrogen). The recombinant proteins were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside at 30 °C for 3 h. Bacteria were pelleted, washed in STE (0.1 m, NaCl, 10 mm Tris-Cl, pH 8.0, 1 mm EDTA, pH 8.0) buffer, and resuspended in TK buffer (20 mm Tris-HCl, pH 7.5, 100 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 5 mm dithiothreitol, 0.5% Nonidet P-40, 0.5 mm phenylmethylsulfonyl fluoride, and 5% glycerol). The suspended bacteria were sonicated for 30 s on ice. Insoluble materials were centrifuged at 13,000 × g for 10 min. The supernatant was used for GST-pulled down experiments. 20 μl of a 50% (v/v) slurry of glutathione-agarose beads (Sigma) were prepared as described according to the manufacturer's instructions (Amersham Biosciences). The supernatants from wild-type and mutant GST-Gag and GST-LysRS were added to 20 μl of a 50% (v/v) slurry of glutathione-agarose beads at 4 °C for 1 h. Beads were washed twice with TK buffer plus 500 mm NaCl and once with TK buffer alone. Beads containing recombinant proteins were resuspended into 150-μl reaction volume with TK buffer. 3 μg of purified Gag (National Institutes of Health AIDS Research and Reference Reagent Program) or 4 μg of purified His6-LysRS were added to each reaction. Reactions were incubated overnight at 4 °C. Beads were washed three times with TK buffer and resuspended with 40 μl of 2× loading buffer (50 mm Tris-HCl, pH 6.8, 100 mm β-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol), boiled for 5 min, and pelleted. Supernatant (30 μl) was subjected to Western blot analysis for detecting the bound protein, and 5 μl of supernatant was used to detect GST fusion protein. Immunoprecipitation of LysRS/Gag—293FT cells were removed from the plate and washed with phosphate-buffered saline 63 h post-transfection. 293FT cells from 100-mm plates were lysed in 500 μl of TNT buffer. Insoluble material was pelleted at 1800 × g for 30 min. The supernatant was used for immunoprecipitation. Anti-LysRS was first cross-linked to Sepharose beads. 40 μl of antibody and 400 μl of 50% (w/v) protein A-Sepharose (Amersham Biosciences) were incubated together in 10 ml of 0.2 m triethanolamine, pH 9. Dimethyl pimelimidate cross-linker (Pierce) was then added to a final concentration of 20 mm, and the mixture was incubated for 1 h at room temperature. The beads were then washed with 5 ml of 0.2 m triethanolamine, pH 9, and further incubated in 10 ml of 0.2 m triethanolamine for another 2 h at room temperature. Equal amounts of protein (∼200–500 μg as determined by the Bio-Rad assay) were incubated with 30-μl antibody cross-linked to protein A-Sepharose for 1 h at 4 °C. The immunoprecipitate was then washed three times with TNT buffer and twice with phosphate-buffered saline. After the final supernatant was removed, 30 μl of 2× sample buffer (120 mm Tris HCl, pH 6.8, 20% glycerol, 4% SDS, and 0.02% bromphenol blue) was added and the precipitate was then boiled for 5 min to release the precipitated proteins. After microcentrifugation, the resulting supernatant was analyzed using Western blots. Interaction of Mutant and Wild-type LysRS with Wild-type Gag in Vitro— Fig. 1 shows a schematic diagram of the domain architecture of human LysRS. This enzyme contains the three consensus sequences (motifs 1, 2, and 3) common to all of the class II synthetases as well as an N-terminal extension proximal to the anticodon-binding domain (22Shiba K. Stello T. Motegi H. Noda T. Musier-Forsyth K. Schimmel P. J. Biol. Chem. 1997; 272: 22809-22816Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Wild-type LysRS and C-terminally truncated LysRS variants were tagged with GST on the N terminus and expressed in E. coli. The E. coli lysates were adsorbed to glutathione-agarose beads followed by three washing steps with buffer containing 20 mm Tris-HCl, pH 7.5, and 100 mm NaCl. The washed beads were then incubated in binding buffer containing purified recombinant HIV-1 Gag. The mixture was then washed three times in buffer containing 100 mm NaCl and twice in buffer containing 200 mm NaCl. Beads were then resuspended directly in SDS sample buffer, boiled, and subjected to SDS-PAGE. Western blots were probed with either anti-GST or anti-Capsid to detect Gag. Fig. 1A shows the wild-type and mutant LysRS constructs tested and lists the relative amount of Gag bound to mutant LysRS where the Gag/LysRS ratio for wild-type LysRS is given a value of 1.00. The Western blot data supporting these results are shown in panels B and C. Panel B shows a Western blot of the gel probed with anti-LysRS. The first lane represents a control experiment performed with GST alone, whereas the other lanes show the wild-type and mutant forms of LysRS eluted from the beads. In panel C, a Western blot of the same sample was probed with anti-Capsid. These data show that removal of the C-terminal 288 amino acids from LysRS (full-length = 597 amino acids) did not prevent Gag binding but further removal of an additional C-terminal 60 amino acids resulted in severely reduced binding. These data suggest that the sequence between amino acids 249 and 309 in motif I of LysRS is required for binding to Gag in vitro. Incorporation of Mutant and Wild-type c-Myc-tagged LysRS into Gag Viral-like Particles in Vivo—We next tested truncated LysRS variants for their capability to be packaged into Gag VLPs in vivo. 293FT cells were cotransfected with a plasmid coding for wild-type HIV-1 Gag and a plasmid coding for wild-type or N- or C-terminal deleted LysRS tagged with c-Myc. The expression in the cell of the different LysRS species was determined, and the results are shown in the left panels in Fig. 2, A and B. Western blots of cell lysates were probed with anti-LysRS (Fig. 2, A and B, top panels), anti-c-Myc (middle panels), or anti-β-actin (bottom panels). Anti-LysRS detects both endogenous and exogenous LysRS, whereas anti-c-Myc detects only exogenous wild-type and mutant forms of LysRS. The ratios of mutant LysRS/β-actin are similar for expression of most mutant LysRS constructs but are less than the wild-type LysRS/β-actin ratio, which was set at 1. In the cell lysate of both transfected and non-transfected cells, there is also less abundant protein species staining with anti-LysRS that has a lower molecular mass (62–63 kDa) than the endogenous LysRS (68 kDa). The smaller molecular mass species also appears in the viral lysate where its abundance relative to the full-length 68-kDa LysRS band has increased as previously reported (12Cen S. Khorchid A. Javanbakht H. Gabor J. Stello T. Shiba K. Musier-Forsyth K. Kleiman L. J. Virol. 2001; 75: 5043-5048Crossref PubMed Scopus (116) Google Scholar). The source of this species (proteolytic processing of full-length LysRS or translation from an alternatively spliced mRNA) is not yet known. The overexpression of wild-type LysRS from an exogenous plasmid (lane 5 in the upper two panels in Fig. 1A), which results in a LysRS species larger than the endogenous wild-type species because it contains a 20 amino acid N-terminal c-Myc tag, does not seem to increase the relative abundance of the 62–63-kDa LysRS species in the cytoplasm. The incorporation of the LysRS variants into virions is shown on the right side of Fig. 2, A and B, which shows Western blots of viral lysates probed with anti-LysRS (top), anti-c-Myc (middle), and anti-Capsid (bottom). Deletion of the N-terminal 207 amino acids does not affect the ability of LysRS to be incorporated into Gag VLPs, whereas the deletion of the C-terminal amino acids (207–597) inhibits LysRS packaging. The anti-LysRS used in the top panel does show a small amount of Δ207–597 incorporated into the virus, whereas anti-c-Myc (middle panel) detects none of this species in the virion. The ratios listed at the bottom of the panel use the Myc-LysRS/Gag ratio because the anti-Myc is expected to show less variability in detecting the different deleted LysRS species than the anti-LysRS. Further mapping shown in Fig. 2, A and B, reveals that a critical region in LysRS for incorporation lies between amino acids 249 and 309, i.e. C-terminal deletions not including this region do not affect packaging. Finer mapping shown in Fig. 2B shows further that a critical region for LysRS incorporation lies between amino acids 249 and 260, i.e. C-terminal deletions of LysRS up to and including amino acid 260 do not affect LysRS packaging, whereas LysRS with a C-terminal deletion up to and including amino acid 249 is not incorporated into Gag VLPs. Taken together, the results shown in Figs. 1 and 2 show that the in vitro interaction between Gag and LysRS is inhibited when the LysRS C-terminal deletion includes the sequence between amino acids 249 and 309, whereas the packaging of LysRS into Gag VLPs is inhibited when the C-terminal deletion of LysRS includes amino acids 249–260. The similarity of results obtained in vitro and in vivo indicates that the interaction between Gag and LysRS in vivo is likely to be a direct one. Interaction of Mutant and Wild-type Gag with Wild-type
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