The Interaction between HIV-1 Gag and Human Lysyl-tRNA Synthetase during Viral Assembly*

Hassan Javanbakht {ddagger} §, Rabih Halwani {ddagger} §, Shan Cen {ddagger}, Jenan Saadatmand {ddagger} §, Karin Musier-Forsyth ¶, Heinrich Gottlinger || and Lawrence Kleiman {ddagger} § ** {ddagger}{ddagger}

From the Lady Davis Institute for Medical Research and {ddagger}McGill AIDS Center, Jewish General Hospital, §Departments of Medicine and **Microbiology & Immunology, McGill University, Montreal, Quebec H3T 1E2, Canada, Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and ||Department of Cancer, Immunology and AIDS, Dana Farber Cancer Institute, Boston, Massachusetts 02115

Received for publication, February 20, 2003 , and in revised form, April 28, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The life cycle of HIV-11 has been intensely studied (for recent review see Ref. 1). 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 (2). 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 (3, 4). The in vivo interaction of Gag with Gag-Pol has also been well documented (58), 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 (9). 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 (10). 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 (11). 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 (12, 13) 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 (12, 13) indicates that human IleRS, ProRS, and TrpRS are not detected in the virion, whereas other work in one of our laboratories2 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 (13). An HIV-1 population contains, on average, ~20–25 molecules of LysRS/virion (13) similar to the average number of tRNALys molecules/virion (14).

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 (10), the incorporation of LysRS into HIV-1 occurs independently of tRNALys packaging, i.e. it is also packaged efficiently into Gag VLPs (12), which do not selectively package tRNALys (10). 2) Overexpression of LysRS in the cell results in a near doubling of the incorporation of both tRNALys and LysRS into HIV-1 (11). 3) The ability of tRNALys to interact with LysRS is required for the incorporation of tRNALys into the virion (15). 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 (16) and Thermus thermophilus LysRS (17) have been solved. Eukaryotic LysRS is a class II synthetase, forming a closely related subgroup (known as IIb) with aspartyl- and asparginyl-tRNA synthetases (18, 19). The anticodon is a major recognition element for all of the class IIb synthetases including human LysRS (20, 21). 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 (22), 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 (23), and specific residues within the N terminus that function in tRNA binding have been identified recently (24) 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 {beta}-sheet active-site fold and contain three consensus motifs known as motifs 1, 2, and 3 (18). 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.



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FIG. 1.
In vitro interaction between wild-type Gag and wild-type or mutant LysRS. Wild-type and C-terminal-deleted LysRS, which are N-terminal tagged with GST, were expressed in E. coli. The E. coli lysates were adsorbed to glutathione-agarose beads, and after washing, the beads were incubated in binding buffer containing 3 µg purified recombinant HIV-1 Gag. After further washes, beads were resuspended directly in SDS sample buffer, boiled, and subjected to SDS-PAGE. Western blots of the eluted material were probed with either anti-GST (panel B) or anti-Capsid (panel C). Panel A shows the wild-type and mutant LysRS variants tested. The cartoon at the top shows the various LysRS domains and the amino acid positions (numbers) at which they occur. The non-numbered N-terminal squiggle represents glutathione S-transferase. Amino acid sequences deleted are shown graphically as thin lines and are listed to the left of each mutant. N, N-terminal domain; AC, anticodon-binding domain. Motifs 1, 2, and 3 are sequence elements characteristic of class II tRNA synthetases and are associated with functions described under "Experimental Procedures." The right side of panel A 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. These values were obtained from the Western blot data shown in panels B and C using UN-SCAN-IT gelTM automated digitizing system.

 


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—pSVGag-REV response element and CMV-REV were donated by D. Rekosh and M. L. Hammarskjold (both from University of Virginia) (6). Plasmids ZWt, ZWt-p6, and {Delta}Z-Wt-p6 were constructed as previously described (26). 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: {Delta}323–500 (forward primer: 5'-AATCAGTCTAGACAAAATTACCCTATAGTGCAG-3'; reverse primer: 5'-ACTCTGATCACTATCATTGGACCAACAAGGTTTCTGT-3') and {Delta}363–500 (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'); {Delta}1–307 (5'-CTCCGGGAATTCCCGCTTCACAGGAGGTAAAAAATT-3'); {Delta}1–337 (5'-CTC CGGGAATTCCCGGACCAGCGGCTACACTAGAAGA-3'); {Delta}1–378 (5'-CTCCGGGAATTCCCATGCAGAGACGCAATTTTAGGAAC-3'); {Delta}363–500 (5'-AATTATGAATTCCTACAAAACTCTTGCC TTATGGCC-3'); and {Delta}433–500 (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 (22). 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'); {Delta}452–597 (5'-AATTATGAATTCCTACAGGAACTCCCCAACAAGCTTGTCAAGGAG-3'); {Delta}309–597 (5'-AATTATGAATTCCTAACCAACCACAAGCATCTTATGATAGAGTTC-3'; {Delta}267–597 (5'-AATTATGAATTCCTACTATTCAATCTCTAGGAATCCCAG-3'; {Delta}260–597 (5'-AATTATGAATTCCTACTAATCTAAGAAACTTCTTATATA-3'); {Delta}249–597 (5'-AATTATGAATTCTACTACTTAGAGCGGATGATAAATTTCTG-3'); {Delta}207–597 (5'-AATTATGAATTCCTAAGACAGCAGTGTGATTCATACGGAATGAT-3'; and {Delta}1–207 (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: {Delta}506–597 (5'-AATTATGAATTCCTACTACATGGGATCATTCAGGTCAGTAT-3'); {Delta}452–597 (5'-AATTATGAATTCCTACAGGAACTCCCCAACAAGCTTGTCAAGGAG-3'); {Delta}373–597 (5'-AATTATGAATTCCTACTAGTAGGTGACCTTGTAACTGCCTGT-3'); {Delta}309–597 (5'-AATTATGAATTCCTAACCAACCACAAGCATCTTATGATAGAGTTC-3'); {Delta}249–597 (5'-AATTATGAATTCCTACTACTTAGAGCGGATGATAAATTTCTG-3'); and {Delta}207–597 (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 1x 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 {beta}-actin (Sigma), and a monoclonal antibody to C-terminal of HIV-1 capsid, which was used to detect {Delta} 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-{beta}-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 x 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 2x loading buffer (50 mM Tris-HCl, pH 6.8, 100 mM {beta}-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 x 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 2x 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (22). 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-{beta}-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/{beta}-actin are similar for expression of most mutant LysRS constructs but are less than the wild-type LysRS/{beta}-actin ratio, which was set at 1.



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FIG. 2.
The ability of mutant LysRS to be incorporated into Gag VLPs. Plasmids coding for N- and C-terminal LysRS deletion mutants were cotransfected into 293FT cells with a plasmid containing HIV-1 proviral DNA. The LysRS produced contained C-terminal c-Myc sequences. The designation of sequences deleted for each mutant uses amino acid numbers corresponding to those shown at the top of Fig. 1A. The left side of panels A and B show Western blots of lysates of cells cotransfected with HIV-1 proviral DNA and LysRS mutants. The blots are probed, respectively, with anti-LysRS, anti-Myc, and anti-{beta}-actin. Anti-LysRS detects both endogenous and exogenous LysRS, whereas anti-c-Myc detects only exogenous LysRS. The expression of exogenous LysRS listed as the Myc-LysRS/{beta}-actin ratio is shown at the bottom of the blots. The right side of panels A and B show Western blots of lysates of viruses produced from the cells probed, respectively, with anti-LysRS, anti-Myc, and anti-Capsid. The incorporation of exogenous LysRS is listed as the Myc-LysRS/Gag ratio at the bottom of the blots.

 

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 (12). 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 {Delta}207–597 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 LysRS in Vitro and in Vivo—Fig. 3A shows N- and C-terminal Gag deletion mutants, which were constructed and expressed in E. coli. The E. coli lysates were adsorbed to glutathione-agarose beads and, after washing as described above, incubated with recombinant wild-type His6-LysRS (22). After removing unbound LysRS, the beads were resuspended directly in SDS sample buffer, boiled, and subjected to SDS-PAGE. Western blots were probed with either anti-LysRS or anti-GST. Fig. 3A shows the relative amount of LysRS bound to mutant Gag for each of the constructs tested where the LysRS/Gag ratio for wild-type Gag 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-Capsid. The first lane represents a control experiment performed with GST alone while the other lanes show the wild-type and mutant forms of Gag eluted from the beads. The multiple bands of GST-Gag observed are a common degradation problem seen when Gag is expressed in bacteria (for example, see Ref. 27). We conclude from these results that all of the mutants are expressed. Panel C shows a Western blot of the same samples probed with anti-LysRS. These data show that deletion of the N-terminal 307 amino acids of Gag does not affect its interaction with LysRS, whereas the removal of an additional 30 amino acids ({Delta}1–337) reduced the interaction to 44% of the wild-type binding level. Deletion of the next 41 amino acids ({Delta}1–378) abolished Gag-LysRS binding. C-terminal deletions that included the p6 and NC sequences did not affect the Gag/LysRS interaction. Therefore, these results indicate that the C-terminal third of the capsid region in Gag is important for the Gag/LysRS interaction.



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FIG. 3.
In vitro interaction between wild-type LysRS and wild-type or mutant Gag. Wild-type and N- or C-terminal-deleted Gag, which was N-terminally tagged with GST, were expressed in E. coli. The E. coli lysates were adsorbed to glutathione-agarose beads, and after washing, the beads were incubated in binding buffer containing 4 µg of purified recombinant His6-LysRS. After further washes, beads were resuspended directly in SDS sample buffer, boiled, and subjected to SDS-PAGE. Western blots of the eluted material were probed with either anti-GST (panel B) or anti-LysRS (panel C). Panel A shows the wild-type and mutant Gag variants tested. The cartoon at the top shows the various Gag domains and the amino acid positions (numbers) at which they occur. The non-numbered N-terminal squiggle represents glutathione S-transferase. Amino acid sequences deleted are shown graphically as thin lines and are listed to the left of each mutant. MA, matrix domain; CA, capsid domain; p6, p6 domain. The right side of panel A lists the relative amount of wild-type LysRS bound to wild-type or mutant Gag where the LysRS/Gag ratio for wild-type Gag is given a value of 1.00. These values were obtained from the Western blot data shown in panels B and C.

 

We next examined the ability of LysRS to be packaged into Gag VLPs composed of mutant Gag species (Fig. 4A). Cells were transfected with the plasmids coding for the following Gag constructs: wild-type Gag; ZWt-p6 in which the NC sequence had been replaced with a yeast leucine zipper domain (Z) to allow for protein/protein interactions; ZWt in which both NC and p6 sequences were removed and NC was replaced with Z; {Delta}ZWt-p6, a Gag construct that contains only the signal for myristoylation in the matrix protein; the C-terminal third domain of capsid; the p2 domain; the Z sequence replacing the NC domain; and the p6 domain. It has previously been shown that all of these mutants can efficiently form Gag VLPs (26). 293FT cells were transfected with the plasmids coding for wild-type or mutant Gag constructs, and the ability of the Gag VLPs to package LysRS was assessed by Western blots of VLP lysates probed with anti-Capsid (Fig. 4B, left) or anti-LysRS (Fig. 4B, right). The Western blot data shown in panel 4C indicate that all of the mutants retain the ability to package LysRS. The results indicate that the C-terminal third of capsid and/or the p2 domain may be involved in binding LysRS, which is supported by the in vitro binding data obtained (Fig. 3). However, since the in vitro interactions shown in Fig. 3C indicate that the Gag deletion mutant {Delta}363–500, which lacks p2, still binds to LysRS, p2 is clearly not involved in binding LysRS.



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FIG. 4.
The ability of endogenous LysRS to be incorporated into wild-type or mutant Gag VLPs. Plasmids coding for wild-type or mutant Gag were transfected into 293FT cells. The Gag deletion mutants are shown in panel A, and their construction and has been described previously (26). Wt-Gag, wild-type Gag; ZWt, a construct in which the NC, p1, and p6 domains have been deleted, and NC has been replaced with a yeast leucine zipper domain (Z); ZWt-p6, a construct in which the p6 domain has been added back to ZWt; {Delta}ZWt-p6, a construct in which the deletion includes all but the first eight amino acids of matrix (MA), approximately two-thirds of capsid as well as replacement of the entire NC domain with the Z domain. Western blots of lysates of Gag VLPs produced from cells transfected with the different Gag plasmids and probed with either anti-Capsid or anti-LysRS are shown in panels B and C, respectively. Although monoclonal antibodies that are specifically reactive with HIV-1 capsid (Zepto Metrocs, Inc) were used for the upper gel in panel B, a different monoclonal antibody to C-terminal of HIV-1 capsid was required to detect {Delta}ZWt-p6 (National Institutes of Health AIDS Research and Reference Reagent Program).

 

To determine which domain of Gag was responsible for interaction with LysRS in the cytoplasm of 293FT cells, the latter was transfected with plasmids coding for wild-type Gag and some of the mutant Gag constructs. The interaction was tested by immunoprecipitation with anti-LysRS, and the results are shown in the Western blots presented in Fig. 5. Fig. 5A shows the total expression of the Gag constructs in the cytoplasm using {beta}-actin as a reference, whereas Fig. 5B shows the ability of anti-LysRS to immunoprecipitate the Gag species. wild type Gag, ZWt (Fig. 4A), and {Delta}363–500 (Fig. 3A) interact with cellular LysRS, but {Delta}323–500 does not. With reference to Fig. 3A, these data support an interaction between amino acids 323 and 363 of capsid and LysRS and shows that the p2 region is not critical for Gag-LysRS association, which is consistent with in vitro binding data (Fig. 3C).



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FIG. 5.
Interaction of endogenous LysRS with wild-type or mutant Gag in the cytoplasm of cells transfected with plasmids coding for Gag variants. Interaction was measured by the ability to immunoprecipitate both Gag and LysRS with anti-LysRS. A, Western blot of lysates of transfected cells probed with anti-Capsid (top) or anti-{beta}-actin (bottom), respectively. The Gag/{beta}-actin ratio obtained from quantitation of the gel data is listed below the blots. B, Western blot of anti-LysRS immunoprecipitates from lysates of Gag VLPs probed with anti-Capsid.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Terminal deletion mutations in both HIV-1 Gag and human LysRS have been used to map interacting sites both in vitro and in vivo. In vitro, wild-type and terminally deleted forms of GST-LysRS or GST-Gag were bound to glutathione-agarose and their capability to bind to wild-type Gag or LysRS, respectively, was monitored (Figs. 1 and 3). In vivo, 293FT cells were transfected with plasmids coding for deletion mutants of either LysRS (Fig. 2, A and B) or Gag (Figs. 4 and 5) and the interaction between these proteins was monitored either by measuring the ability of LysRS to be incorporated into extracellular Gag VLPs or by coimmunoprecipitation using anti-LysRS antibodies. Importantly, the same sequences were determined to be essential for Gag/LysRS binding by all of the methods of analysis used.

The importance of amino acids 308–362 at the C terminus of capsid for interaction with LysRS in vitro was suggested by the deletion analysis shown in Fig. 3. N-terminal deletions in Gag up to and including amino acid 307 and C-terminal deletions in Gag extending to amino acid 363 do not affect the in vitro interaction between Gag and wild-type LysRS. This conclusion is supported by in vivo work showing that removal of all of the matrix (with the exception of the first eight N-terminal amino acids in order to maintain the myristoylation site), the N-terminal two-thirds of capsid, all of p6, and replacement of NC sequences with the yeast leucine zipper domain (Z) still allows for the formation of Gag VLPs containing LysRS (Fig. 4). The coimmunoprecipitation data shown in Fig. 5 further help to narrow down the critical region for LysRS interaction to amino acids 323–362. Based upon both the three-dimensional structure of the C-terminal part of the HIV-1 capsid region (28) and in vitro analysis of mutations in this region (27, 28), the sequences in Gag that we have determined are critical for interaction with LysRS are part of the capsid dimer interface. The biological significance of this dimer interface in HIV-1 assembly is not clear, because this interaction is much weaker than the NC/NC interactions, which facilitated by NC/RNA interaction are probably the driving force in Gag oligomerization (27, 28). Nevertheless, this region, which can facilitate homodimerization between capsid molecules, is also important for interaction with LysRS, possibly through the formation of a heterodimer.

Our results also support a role in Gag interaction for LysRS amino acid sequences within motif 1, a domain known to be critical for dimerization of class II aminoacyl-tRNA synthetases (16, 17, 30, 31). In particular, the importance of amino acids 208–259 in LysRS was shown by the fact that the N-terminal deletion of residues 1–207 (Fig. 2A) and the C-terminal deletion of residues 260–597 (Fig. 2B) do not affect packaging of LysRS into Gag VLPs, whereas constructs with deletions that include residues 208–259 abolish packaging (Fig. 2, A and B). Although no N-terminal LysRS deletion construct was tested for its interaction with Gag in vitro, the fact that the LysRS deletion mutant {Delta}309–597 interacts with Gag while the LysRS deletion mutant {Delta}249–597 does not (Fig. 1) supports the conclusions derived from in vivo data.

As described under Introduction, eukaryotic LysRS is a class IIb synthetase with a distribution of functional domains shown in cartoon form in Fig. 1A. Thus, the N-terminal deletion of residues 1–207 removes regions that are important for binding to tRNALys, including the N-terminal extension and the anticodon-binding domain (Fig. 1), yet this construct can still interact with Gag (Fig. 2A). The C-terminal deletion of residues 260–597 removes motifs 2 and 3 sequences that constitute the catalytic domain of LysRS. These regions are also dispensable for Gag interaction (Fig. 2B). A multiple sequence alignment of LysRSs from different species showed that the aminoacylation catalytic domain is highly conserved in this enzyme (22). Therefore, our results suggest that the interaction of LysRS with Gag occurs independent of its ability to bind strongly to and aminoacylate tRNALys. This finding supports earlier findings that LysRS is packaged into Gag VLPs or mutant virions independent of tRNALys packaging (12). Thus, we have previously shown that Gag VLPs efficiently package viral genomic RNA (10) and LysRS (12) but do not package tRNALys, which requires the additional presence of Gag-Pol (10). Gag-Pol is presumably required to stabilize the presence of tRNALys in the Gag·Gag-Pol·tRNALys·LysRS-packaging complex.

In contrast to LysRS packaging, which can occur independent of tRNALys packaging, we previously showed that the latter was directly correlated with LysRS interaction (15). In particular, tRNA anticodon mutants that were poorly aminoacylated were also not efficiently packaged. Thus, tRNA packaging appears to depend upon productive interaction with LysRS, whereas LysRS packaging depends only on interaction with Gag.

In conclusion, our data show that residues responsible for the homodimerization of capsid and LysRS are also critical for facilitating the interaction between these two molecules. This observation suggests that the interaction between LysRS and Gag involves heterodimer formation using the same interface used by each molecule for homodimerization. This implies that LysRS may be incorporated into the virion as a monomer. The effect of this interaction upon the multimerization of the Gag molecule would be more difficult to predict since putative regions of interactions between Gag molecules have been identified as occurring at multiple sites in the C-terminal half of Gag and include the C-terminal half of capsid (28, 3234), the p2 spacer region (26, 35, 36), nucleocapsid (27, 3739), and p6 (29). Also, the virus is composed of ~1500 Gag molecules (1), and it is very probable that only a small fraction of these molecules is involved in the interaction with LysRS.

Alternatively, homodimers of LysRS and Gag may be required in the LysRS/Gag interaction to allow sequences elsewhere in these molecules to participate in this interaction. This possibility is less probably since the experiments performed herein, in vitro and in vivo, indicate that sequences deleted both upstream or downstream of the dimerization sites in LysRS and in Gag are not required for the formation of the Gag·LysRS complex.


    FOOTNOTES
 
* This work was supported in part by grants from the Canadian Institutes for Health Research and the Canadian Foundation for AIDS Research. This work was performed by H. Javanbakht in partial fulfillment of the Ph.D. degree at McGill University, Montreal, Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Lady Davis Institute for Medical Research-Jewish General Hospital, 3755 Cote St. Catherine Rd., Montreal, Quebec H3T 1E2, Canada. Tel.: 514-340-8260; Fax: 514-340-7502; E-mail: lawrence.kleiman{at}mcgill.ca.

1 The 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. Back

2 R. Halwani and L. Kleiman, unpublished work. Back


    ACKNOWLEDGMENTS
 
We thank Sandy Fraiberg for assistance in preparation of the paper.



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