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J. Biol. Chem., Vol. 278, Issue 30, 27644-27651, July 25, 2003
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The Interaction between HIV-1 Gag and Human Lysyl-tRNA Synthetase during Viral Assembly*
** 
From the
Lady Davis Institute for Medical Research and
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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (5–8), 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 
-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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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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
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: 323–500 (forward primer:
5'-AATCAGTCTAGACAAAATTACCCTATAGTGCAG-3'; reverse primer:
5'-ACTCTGATCACTATCATTGGACCAACAAGGTTTCTGT-3') and
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'); 1–307
(5'-CTCCGGGAATTCCCGCTTCACAGGAGGTAAAAAATT-3'); 1–337
(5'-CTC CGGGAATTCCCGGACCAGCGGCTACACTAGAAGA-3'); 1–378
(5'-CTCCGGGAATTCCCATGCAGAGACGCAATTTTAGGAAC-3');
363–500 (5'-AATTATGAATTCCTACAAAACTCTTGCC
TTATGGCC-3'); and 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'); 452–597
(5'-AATTATGAATTCCTACAGGAACTCCCCAACAAGCTTGTCAAGGAG-3');
309–597
(5'-AATTATGAATTCCTAACCAACCACAAGCATCTTATGATAGAGTTC-3';
267–597
(5'-AATTATGAATTCCTACTATTCAATCTCTAGGAATCCCAG-3';
260–597
(5'-AATTATGAATTCCTACTAATCTAAGAAACTTCTTATATA-3');
249–597
(5'-AATTATGAATTCTACTACTTAGAGCGGATGATAAATTTCTG-3');
207–597
(5'-AATTATGAATTCCTAAGACAGCAGTGTGATTCATACGGAATGAT-3'; and
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: 506–597
(5'-AATTATGAATTCCTACTACATGGGATCATTCAGGTCAGTAT-3');
452–597
(5'-AATTATGAATTCCTACAGGAACTCCCCAACAAGCTTGTCAAGGAG-3');
373–597
(5'-AATTATGAATTCCTACTAGTAGGTGACCTTGTAACTGCCTGT-3');
309–597
(5'-AATTATGAATTCCTAACCAACCACAAGCATCTTATGATAGAGTTC-3');
249–597
(5'-AATTATGAATTCCTACTACTTAGAGCGGATGATAAATTTCTG-3'); and
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 -actin
(Sigma), and a monoclonal antibody to C-terminal of HIV-1 capsid, which was
used to detect 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--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 -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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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--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/-actin are similar for expression of most mutant LysRS constructs
but are less than the wild-type LysRS/-actin ratio, which was set at
1.
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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
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
(1–337) reduced the interaction to 44% of the wild-type binding
level. Deletion of the next 41 amino acids (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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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; 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 363–500, which lacks p2, still binds to
LysRS, p2 is clearly not involved in binding LysRS.
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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 -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 363–500
(Fig. 3A) interact
with cellular LysRS, but 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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 309–597 interacts with Gag while the LysRS
deletion mutant 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,
32–34),
the p2 spacer region (26,
35,
36), nucleocapsid
(27,
37–39),
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.
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.
2 R. Halwani and L. Kleiman, unpublished work.
|
ACKNOWLEDGMENTS |
|---|
We thank Sandy Fraiberg for assistance in preparation of the paper.
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
- Swanstrom, R., and Wills, J. W. (1997) in Retroviruses (Coffin, J., Hughes, S., and Varmus, H., eds), pp. 263–334, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
- Mak, J., and Kleiman, L. (1997) J. Virol. 71, 8087–8095[Medline] [Order article via Infotrieve]
- Berkowitz, R., Fisher, J., and Goff, S. P. (1996) in Morphogenesis and Maturation of Retroviruses. (Krausslich, H. G., ed), Vol. 214, pp. 177–218, Springer-Verlag New York Inc., New York
- Geigenmüller, U., and Linial, M. L. (1996) J. Virol. 70, 667–671[Abstract]
- Park, J., and Morrow, C. D. (1992) J.
Virol. 66,
6304–6313
[Abstract/Free Full Text] - Smith, A. J., Cho, M. I., Hammarskjöld, M. L., and Rekosh, D.
(1990) J. Virol.
64,
2743–2750
[Abstract/Free Full Text] - Smith, A. J., Srivivasakumar, N., Hammarskjöld, M.-L., and
Rekosh, D. (1993) J. Virol.
67,
2266–2275
[Abstract/Free Full Text] - Srinivasakumar, N., Hammarskjöld, M.-L., and Rekosh, D. (1995) J. Virol. 69, 6106–6114[Abstract]
- Jiang, M., Mak, J., Ladha, A., Cohen, E., Klein, M., Rovinski, B.,
and Kleiman, L. (1993) J. Virol.
67,
3246–3253
[Abstract/Free Full Text] - Mak, J., Jiang, M., Wainberg, M. A., Hammarskjold, M.-L., Rekosh,
D., and Kleiman, L. (1994) J. Virol.
68,
2065–2072
[Abstract/Free Full Text] - Gabor, J., Cen, S., Javanbakht, H., Niu, M., and Kleiman, L.
(2002) J. Virol.
76,
9096–9102
[Abstract/Free Full Text] - Cen, S., Khorchid, A., Javanbakht, H., Gabor, J., Stello, T.,
Shiba, K., Musier-Forsyth, K., and Kleiman, L. (2001)
J. Virol. 75,
5043–5048
[Abstract/Free Full Text] - Cen, S., Javanbakht, H., Kim, S., Shiba, K., Craven, R., Rein, A.,
Ewalt, K., Schimmel, P., Musier-Forsyth, K., and Kleiman, L.
(2002) J. Virol.
76,
13111–13115
[Abstract/Free Full Text] - Huang, Y., Mak, J., Cao, Q., Li, Z., Wainberg, M. A., and Kleiman,
L. (1994) J. Virol.
68,
7676–7683
[Abstract/Free Full Text] - Javanbakht, H., Cen, S., Musier-Forsyth, K., and Kleiman, L.
(2002) J. Biol. Chem.
277,
17389–17396
[Abstract/Free Full Text] - Onesti, S., Miller, A. D., and Brick, P. (1995) Structure 3, 163–176[Medline] [Order article via Infotrieve]
- Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 6321–6334[Medline] [Order article via Infotrieve]
- Eriani, G., Delarue, M., Poch, O., Gangloff, J., and Moras, D. (1990) Nature 347, 203–206[CrossRef][Medline] [Order article via Infotrieve]
- Eriani, G., Dirheimer, G., and Gangloff, J. (1990)
Nucleic Acids Res. 18,
7109–7118
[Abstract/Free Full Text] - Stello, T., Hong, M., and Musier-Forsyth, K. (1999)
Nucleic Acids Res. 27,
4823–4829
[Abstract/Free Full Text] - Saks, M. E., Sampson, J. R., and Abelson, J. N. (1994)
Science 263,
191–197
[Abstract/Free Full Text] - Shiba, K., Stello, T., Motegi, H., Noda, T., Musier-Forsyth, K.,
and Schimmel, P. (1997) J. Biol. Chem.
272,
22809–22816
[Abstract/Free Full Text] - Francin, M., Kaminska, M., Kerjan, P., and Mirande, M.
(2002) J. Biol. Chem.
277,
1762–1769
[Abstract/Free Full Text] - Francin, M., and Mirande, M. (2003) J.
Biol. Chem. 278,
1472–1479
[Abstract/Free Full Text] - Deleted in proof
- Accola, M. A., Strack, B., and Gottlinger, H. G.
(2000) J. Virol.
74,
5395–5402
[Abstract/Free Full Text] - Burniston, M. T., Cimarelli, A., Colgan, J., Curtis, S. P., and
Luban, J. (1999) J. Virol.
73,
8527–8540
[Abstract/Free Full Text] - Gamble, T. R., Yoo, S., Vajdos, F., Von Schwedler, U. K.,
McCutcheon, J., and Sundquist, W. I. (1997)
Science 278,
849–853
[Abstract/Free Full Text] - Garnier, L., Ratner, L., Rovinski, B., Cao, S. X., and Wills, J. W.
(1998) J. Virol.
72,
4667–4677
[Abstract/Free Full Text] - Cavarelli, J., Rees, B., Ruff, M., Thierry, J. C., and Moras, D. (1993) Nature 362, 181–184[CrossRef][Medline] [Order article via Infotrieve]
- Eriani, G., Cavarelli, J., Martin, F., Dirheimer, G., Moras, D.,
and Gangloff, J. (1993) Proc. Natl. Acad. Sci. U. S.
A. 90,
10816–10820
[Abstract/Free Full Text] - Berthet-Colominas, C., Monaco, S., Novelli, A., Sibai, G., Mallet, F., and Cusack, S. (1999) EMBO J. 18, 1124–1136[CrossRef][Medline] [Order article via Infotrieve]
- Li, S., Hill, C. P., Sundquist, W. I., and Finch, J. T. (2000) Nature 407, 409–413[CrossRef][Medline] [Order article via Infotrieve]
- Momany, C., Kovari, L., Prongay, A., Keller, W., Gitti, R., Lee, B., Gorbalenya, A., Tong, L., McClure, J., Ehrlich, L., Summers, M., Carter, C., and Rossmann, M. (1996) Nat. Struct. Biol. 3, 763–770[CrossRef][Medline] [Order article via Infotrieve]
- Kräusslich, H.-G., Fäcke, M., Heuser, A.-M., Konvalinka, J., and Zentgraf, H. (1995) J. Virol. 69, 3407–3419[Abstract]
- Morikawa, Y., Hockley, D. J., Nermut, M. V., and Jones, I. M.
(2000) J. Virol.
74,
16–23
[Abstract/Free Full Text] - Campbell, S., and Vogt, V. M. (1995) J. Virol. 69, 6487–6497[Abstract]
- Craven, R. C., and Parent, L. J. (1996) Curr. Top. Microbiol. Immunol. 214, 65–94[Medline] [Order article via Infotrieve]
- Sandefur, S., Varthakavi, V., and Spearman, P. (1998)
J. Virol. 72,
2723–2732
[Abstract/Free Full Text]
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