INTRODUCTION

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During human immunodeficiency virus type 1 (HIV-1) assembly, the major cellular tRNA^Lys isoacceptors, tRNA and tRNA, are selectively packaged into the virus (13), and tRNA is used as the primer for the reverse transcriptase-catalyzed synthesis of minus-strand DNA (17). The selective packaging of tRNA^Lys into HIV-1 occurs independently of both genomic RNA packaging (13) and the processing of the viral precursor proteins Pr55^gag and Pr160^gag-pol (18) but does depend on the participation of both of these unprocessed proteins. While Pr55^gag alone is sufficient to form viral particles and binds to both viral genomic RNA (1) and Pr160^gag-pol (21, 25), it is not known if a specific binding of Pr55^gag to tRNA^Lys contributes to tRNA^Lys selective packaging. Evidence for an interaction between Pr55^gag and tRNA comes not from tRNA packaging studies but from tRNA placement studies, which indicate that this protein, and not Pr160^gag-pol, plays a major role in annealing tRNA onto the primer binding site in vitro (9) or in vivo (3).

In considering the interactions involved between viral proteins and tRNA^Lys during packaging, it must be taken into account that tRNAs have been reported to be channeled from one component of the translational machinery to the next and thus may never be free of this synthetic machinery (26). Such components could involve ribosomes, elongation factors, and aminoacyl-tRNA synthetases. Although it has been shown that elongation factor 1-alpha is packaged into HIV-1 via an interaction with Pr55^gag (5), it is not clear how this protein, which binds to all aminoacylated tRNAs, would confer the ability to selectively package tRNA^Lys into the virion. Another tRNA-binding protein in the cytoplasm which is more specific for tRNA^Lys is lysyl-tRNA synthetase (LysRS). This enzyme is an attractive candidate for interacting specifically with viral proteins and may play a role in the transport of the three tRNA^Lys isoacceptors into the virions. In this work, we will show that during viral assembly, LysRS is in fact nonrandomly packaged into HIV-1 through interaction with Pr55^gag and that a truncated LysRS species associated with selective tRNA^Lys packaging is found within the virion.

	MATERIALS AND METHODS

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Plasmid construction. SVC21.BH10 is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA and was a gift from E. Cohen, University of Montreal. pSVGAG-RRE-R and pSVFS5TprotD25G, which code for Gag and unprocessed Gag-Pol, respectively, have been described previously (24, 25). Viral production from either of these two plasmids, which contain the Rev response element (RRE), requires cotransfection with a Rev protein expression vector, such as pCMV-REV. Thus, cotransfection of pSVGAG-RRE-R with pCMV-REV is required to produce virus-like particles containing the unprocessed Pr55^gag precursor protein. In this study, pSVSF5TprotD25G was cotransfected with SVC21P31L, a plasmid coding for HIV-1 proteins including Gag and Rev, but not for stable Gag-Pol. The construction of the mutants SVC21 Dr2 and SVC21 P31L has been described previously (12, 19).

Cell lines. COS7 cells were maintained in Dulbecco modified Eagle medium with 10% fetal bovine serum and antibiotic. H9, PLB, CEMss, and U937 cell lines were grown in RPMI 1640 with 10% fetal bovine serum and antibiotic.

Production of wild-type and mutant HIV-1 virus. Transfection of COS7 cells with the above plasmids by the calcium phosphate method was done as previously described (18). Viruses were isolated from COS7 cell culture medium at 63 h posttransfection or from the cell culture medium of infected cell lines. The virus-containing medium was first centrifuged in a Beckman GS-6R rotor at 3,000 rpm for 30 min, and the supernatant was then filtered through a 0.2-µm-pore-size filter. The viruses in the filtrate were then pelleted by centrifugation in a Beckman Ti45 rotor at 35,000 rpm for 1 h. The viral pellet was then purified by centrifugation with a Beckman SW41 rotor at 26,500 rpm for 1 h through 15% sucrose onto a 65% sucrose cushion.

Western blotting. Sucrose-gradient-purified virions were resuspended in 1× radioimmunoprecipitation assay buffer (RIPA buffer: 10 mM Tris [pH 7.4], 100 mM NaCl, 1% deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40, protease inhibitor cocktail tablets [Boehringer Mannheim]). Western blot analysis was performed using either 300 µg of cellular protein or 10 µg of viral protein, as determined by the Bradford assay (2). The cellular and viral lysates were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) followed by blotting onto nitrocellulose membranes (Gelman Sciences). Detection of protein on the Western blot utilized monoclonal antibodies or antisera specifically reactive with viral p24 and gp120 as well as with different aminoacyl-tRNA synthetases. Mouse anti-p24 and rabbit anti-gp120 antibodies were purchased from Intracel Corp. Rabbit anti-LysRS, anti-ProRS, and anti-IleRS antibodies were isolated following three subcutaneous injections of purified protein with 3- to 4-week intervals between injections (150 to 300 µg of total protein). An N-terminally truncated form of human LysRS (22) and a C-terminally truncated form of human IleRS (23) were used in these preparations. Human ProRS is derived from the C-terminal domain (amino acid residues 926 to 1440) of human glutamyl-prolyl-tRNA synthetase and was purified as described previously (11). Western blots were analyzed by enhanced chemiluminescence (ECL kit; Amersham Life Sciences) using goat anti-mouse or donkey anti-rabbit (Amersham Life Sciences) as a secondary antibody. The sizes of the detected protein bands were estimated using prestained high-molecular-mass protein markers (GIBCO/BRL).

Optiprep gradient. Virions were sometimes purified by replacing centrifugation through sucrose with centrifugation in an Optiprep velocity gradient (60% [wt/vol] iodixanol; Life Technologies). Iodixanol gradients were prepared in phosphate-buffered saline as 11 steps in 1.2% increments ranging from 6 to 18%. Virions were layered onto the top of the gradient and centrifuged for 1.5 h at 26,500 rpm in a Beckman SW41 rotor. Fractions were collected from the top of the gradient. Aliquots were resuspended in phosphate-buffered saline and centrifuged for 1 h at 40,000 rpm in a Beckman Ti50.3 rotor. The resulting pellets were resuspended in RIPA buffer and resolved using SDS-PAGE, followed by either Coomassie blue staining or Western blot analysis.

Subtilisin digestion assay. Subtilisin digestion assays were performed essentially according to the method of Ott et al. (20). The purified virions were mock treated or treated with 1 mg of subtilisin (Boehringer Mannheim)/ml in digestion buffer (10 mM Tris-HCl [pH 8], 1 mM CaCl₂, bovine serum albumin) for 16 h at 37°C. Subtilisin was inactivated by phenylmethylsulfonyl fluoride. Virions were then repelleted, resuspended in 2× loading buffer (120 mM Tris-HCl [pH 6.8], 20% glycerol, 4% SDS, 200 mM dithiothreitol, 002% [wt/vol] bromophenol blue) and subjected to SDS-PAGE, followed by Western blot analysis, using anti-LysRS, anti-p24, and anti-gp120.

Expression and purification of recombinant human lysyl-tRNA synthetase. His₆-tagged full-length human LysRS was overexpressed in Escherichia coli and purified as previously described (22).

	RESULTS

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LysRS is incorporated nonrandomly into HIV-1. Figure 1A shows Western blots of some aminoacyl-tRNA synthetases found in the cytoplasms of COS7 cells transfected with HIV-1 and in the viruses produced. Panel a represents a Western blot of either viral or cytoplasmic proteins probed with an antibody to human LysRS. In both the COS cell cytoplasm and the viruses, LysRS species can be detected in three sizes. The apparent molecular weights (M_rs) of these peptides, determined by SDS-PAGE (Fig. 1 and 3), are 70,000 for the large species, 63,000 for the intermediate species, and 62,000 for the small species. The large species predominate in the cytoplasm, while in the virus, both large and intermediate species are present. The sizes of the LysRS species determined by SDS-PAGE are only approximate, since the calculated size of the human LysRS coded by a full-length LysRS cDNA is 597 amino acids, with an M_r of 68,034 (22).

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Detection of aminoacyl-tRNA synthetases in HIV-1. Virions were pelleted from cell culture medium and purified by centrifugation through 15% sucrose onto a 65% sucrose cushion. (A) Western blots of aminoacyl-tRNA synthetases found in the cytoplasms of HIV-1-transfected COS7 cells and in the viruses produced from these cells. Western blots of cell lysates (lane C) or viral lysates (lane V) were probed with antibody to LysRS (a), IleRS (b), or ProRS (c). Numbers at the left of each panel represent molecular weight markers. GluProRS, glutamyl-prolyl-tRNA synthetase. (B) Resistance of virus-associated proteins to the protease subtilisin. Purified virions were either left untreated (N) or treated (S) with subtilisin, and after subtilisin inactivation, viruses were lysed, and Western blots of viral lysate were probed with antibodies to CAp24 (a), gp120 (b), or LysRS (c). Purified His6-LysRS was left untreated or treated with subtilisin (d). Lane K contains purified, His6-tagged human LysRS, which in panel c has not been exposed to protease.

View larger version (58K): [in this window] [in a new window]   FIG. 2.   Detection of LysRS in viruses purified by centrifugation through Optiprep gradients. Western blots of fractions from Optiprep gradients. (A) Blot probed with anti-CA. Lane V, viral lysate before Optiprep gradient. (B) Blot probed with anti-LysRS. Lane K, purified His6-tagged LysRS. (C) Blot stained with Coomassie blue. Twenty times more viral lysate was used than was used for panels A and B in order to clearly detect protein by the stain. Lane M, marker proteins. (D) Blot of pellet of material from cell culture media of nontransfected COS7 cells, probed with anti-LysRS.

Sizes of LysRS incorporated into HIV-1 produced from transfected COS7 cells and chronically infected cell lines. Although both large and intermediate-size LysRS species are found in HIV-1 produced from COS7 cells, the intermediate-size peptide is the major LysRS found in HIV-1 produced from chronically infected cell lines. This is shown in the Western blots probed with anti-LysRS, shown in Fig. 3. In the cytoplasm of H9 cells, uninfected (Fig. 3, lane 10) or chronically infected with HIV-1 (lane 9), the major LysRS species is the large species, with a small amount of small species also present. Similar results are also found in the cytoplasm of PLB, CEMss, and U937 cells (data not shown). On the other hand, in virions produced from these four chronically infected cell lines, the major LysRS species packaged is the intermediate-size LysRS species.

View larger version (77K): [in this window] [in a new window]   FIG. 3.   Detection of LysRS in cell lysates and lysates of sucrose-purified viruses produced from chronically infected cell lines. Western blots are probed with anti-LysRS. Cell lysates are from uninfected () or infected (+) cells. Numbers at the left represent molecular weight markers. LysRS, purified His6-tagged LysRS.

Relationship between LysRS and tRNA^Lys incorporation in HIV-1. Mutant viruses previously shown to be deficient in tRNA^Lys incorporation (12, 19) were produced by transfecting COS7 cells with wild-type and mutant HIV-1 proviral DNA, and the incorporation of LysRS into the virions was analyzed by Western blots, as shown in Fig. 4A. Lanes 1 and 7 show purified His₆-tagged-LysRS and LysRS found in the COS7 cell cytoplasm, respectively. Lanes 2 and 3 represent protein from wild-type or protease-negative viruses, respectively. Both viruses have been shown to selectively incorporate tRNA^Lys (13, 14), and lanes 2 and 3 show they both contain the large and intermediate-size species of LysRS. Lanes 4 to 6 represent Western blots of protein from the mutant virus-like particles (VLPs) P31L, Dr2, and Pr55^gag, none of which incorporates either Pr160^gag-pol or tRNA^Lys (12, 14, 18, 19). P31L contains a substitution of P for L at position 31 in the nucleocapsid protein (NCp7) in the basic amino acid sequence between the two Cys-His boxes. This mutation causes the rapid degradation of Pr160^gag-pol in the cytoplasm (12). Dr2 is a substitution mutation in the connection domain of reverse transcriptase, in which F₃₈₉ is replaced with F₃₈₉AG, and also causes the rapid degradation of Pr160^gag-pol in the cytoplasm (19). Lane 6 represents protein from Pr55^gag VLPs produced by transfecting COS cells with the vector pSVGAG-RRE, which codes only for Pr55^gag (25). These three different VLPs, which do not selectively package tRNA^Lys, do not contain the intermediate-size LysRS species but do contain the large and small species of LysRS. Thus, the incorporation of LysRS into viral particles appears dependent upon the Pr55^gag protein and is independent of tRNA^Lys or Pr160^gag-pol incorporation. However, the presence of intermediate-size LysRS in viruses appears to be directly correlated with the packaging of tRNA^Lys and Pr160^gag-pol.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Detection of LysRS in lysates of sucrose-purified viruses produced from COS7 cells transfected with wild-type and mutant HIV-1 DNA. (A) Western blot of viral lysates probed with anti-LysRS. LysRS, purified His6-tagged LysRS. wt, wild type. PR(), viral protease-negative. P31L, substitution mutation in the region between the two Cys-His boxes in the nucleocapsid. Dr2, insertion mutation in the connection domain of reverse transcriptase. Gag, Pr55gag particles which do not contain Pr160gag-pol. COS7, cytoplasmic lysate. (B) Western blot of viral lysate probed with anti-LysRS. Lanes: 2, wt: 3, P31L; 4 and 5, viruses from cells cotransfected with P31L DNA and DNA coding for either wild-type Pr160gag-pol (4) or wild-type Pr55gag (5).

	DISCUSSION

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In this work, we have provided evidence for the incorporation of human LysRS into HIV-1. This evidence included detection of LysRS in virions purified by centrifugation using either sucrose or Optiprep gradients. Two other human aminoacyl-tRNA synthetases, ProRS and IleRS, were not detected in virions, though they were readily detected in the cytoplasm of HIV-1-transfected cells. While purified LysRS was susceptible to degradation by the protease subtilisin, LysRS detected in viruses was resistant to subtilisin digestion under reaction conditions in which external envelope protein gp120 was degraded.

We detect LysRS in three sizes, with apparent molecular weights on SDS gels of 70,000 (large species), 63,000 (intermediate species), and 62,000 (small species). The results shown in Fig. 4 indicate that Pr55^gag alone among the viral proteins is sufficient for incorporating LysRS. The Gag VLPs do not incorporate either tRNA^Lys or Pr160^gag-pol, and the intermediate LysRS is replaced with the small species. The three types of Pr55^gag VLPs (Fig. 4A, lanes 4 to 6) do not incorporate either tRNA^Lys or Pr160^gag-pol. The VLPs which contain only Pr55^gag (Fig. 4A, lane 6) are produced by cotransfecting cells with pSVGAG-RRE-R and pCMV-REV. The HIV-1 proviral DNA in the former plasmid not only lacks viral sequences downstream of Gag (except for the RRE), but a simian virus 40 late promoter region has replaced all viral sequences upstream of nucleotide 679 in the viral DNA. The VLPs produced are defective in incorporating the truncated genomic RNA as well as tRNA^Lys and Pr160^gag-pol (18, 24, 25). Pr55^gag may interact with a cytoplasmic tRNA^Lys-LysRS complex and destabilize it, thereby releasing the tRNA^Lys and resulting in the incorporation of LysRS alone into the Gag VLP. The additional presence of Pr160^gag-pol may serve to stabilize the Pr55^gag-tRNA^Lys-LysRS ternary complex, since Pr160^gag-pol interacts with both tRNA^Lys (14) and Pr55^gag (21, 25).

Destabilization of the LysRS-tRNA^Lys complex by the large number of Pr55^gag molecules in the cell might be expected to inhibit translation. There are a number of possible reasons why this does not happen. Most Pr55^gag molecules may not bind LysRS, either because Pr55^gag molecules without Pr160^gag-pol have a weaker affinity for LysRS or because Pr55^gag only interacts with LysRS as a multimeric Pr55^gag complex. Additionally, the destabilization of tRNA^Lys-LysRS may release free nonacylated tRNA^Lys, a molecule which has been shown in yeast to induce the synthesis of more LysRS (16), which could help maintain the cytoplasmic concentrations of tRNA^Lys- LysRS and lysine-tRNA^Lys required for translation.

We do not yet know if Pr55^gag interacts directly with LysRS. Since the plasmid coding for the Pr55^gag protein, pSVGAG-RRE-R, codes only for this protein (24), Vpr, a viral protein which was shown to interact with human LysRS both in vitro and in the yeast two-hybrid system (27), is not needed for the incorporation of LysRS into the Pr55^gag particles. We have also previously shown that tRNA^Lys is selectively incorporated into HIV-1 that is missing Vpr (14). On the other hand, Pr55^gag might interact indirectly with LysRS via another cellular tRNA-binding protein, such as elongation factor 1-alpha, which has been shown to interact with Pr55^gag and to be incorporated into HIV-1 during assembly (5).

The dominant LysRS form in viruses produced from the human cell lines is the intermediate form (Fig. 3). Since truncation of LysRS to the small species also occurs in Gag VLPs, processing does not depend upon the presence of either Pr160^gag-pol or tRNA^Lys but may be limited by them to production of the intermediate species. The predominance of large LysRS in the cytoplasm and intermediate LysRS in the viruses (particularly in viruses produced from human cell lines) suggests that the intermediate and small LysRS species may be generated by proteolysis of the large species, a phenomenon observed during the in vitro proteolytic cleavage of the N-terminal regions of dimeric yeast (6) or sheep (7) LysRS to truncated homodimers. The detection of LysRS heterodimers in sheep has also been reported (7). However, if a protease is involved, it is not a viral protease, since processing of LysRS occurs both in Gag VLPs and in protease-negative virions. A recent report does indicate that the human cytoplasmic and mitochondrial LysRSs are generated by alternative splicing of the same primary RNA transcript (29). The mitochondrial LysRS contains extra amino acid sequences used for mitochondrial targeting in the N-terminal region, and because it is larger than the cytoplasmic LysRS, it is unlikely to be represented by the intermediate and small species observed in the present studies. Alternate RNA splicing has also been reported for generating human cytoplasmic cysteinyl-tRNA synthetase (15).

Very little processed LysRS is detected in the cytoplasm of chronically infected cells (Fig. 3), and this is the small species. These data appear to support the possibility that the processing of the large LysRS species to the intermediate species occurs during or after viral release from the cell. We cannot exclude the possibilities either that nondetectable amounts of intermediate LysRS in the cytoplasm are selectively packaged into the virus or that the scarcity of the intermediate LysRS species in the cytoplasm is due to the fact that it is selectively packaged into the virus. The presence of both large and intermediate species of LysRS in HIV-1 produced from COS7 cells does indicate that the large species is capable of being packaged into the virion.

It has been shown that removal of N-terminal sequence from yeast AspRS weakens binding of the enzyme to the tRNA^Asp, as shown by an increase in both the K_d for tRNA binding and K_m of the aminoacylation reaction of approximately 2 orders of magnitude (10). On the other hand, human LysRS missing the N-terminal 65 amino acids did not display significantly reduced in vitro aminoacylation kinetics (22), implying a tRNA^Lys binding affinity similar to that for wild-type LysRS. A reduced affinity of the intermediate LysRS for tRNA^Lys might therefore be due to other LysRS sequences missing or to a cellular environment different from that tested in vitro.