HIV-1 Exploits a Dynamic Multi-aminoacyl-tRNA Synthetase Complex To Enhance Viral Replication

ABSTRACT

A hallmark of retroviruses such as human immunodeficiency virus type 1 (HIV-1) is reverse transcription of genomic RNA to DNA, a process that is primed by cellular tRNAs. HIV-1 recruits human tRNA^Lys3 to serve as the reverse transcription primer via an interaction between lysyl-tRNA synthetase (LysRS) and the HIV-1 Gag polyprotein. LysRS is normally sequestered in a multi-aminoacyl-tRNA synthetase complex (MSC). Previous studies demonstrated that components of the MSC can be mobilized in response to certain cellular stimuli, but how LysRS is redirected from the MSC to viral particles for packaging is unknown. Here, we show that upon HIV-1 infection, a free pool of non-MSC-associated LysRS is observed and partially relocalized to the nucleus. Heat inactivation of HIV-1 blocks nuclear localization of LysRS, but treatment with a reverse transcriptase inhibitor does not, suggesting that the trigger for relocalization occurs prior to reverse transcription. A reduction in HIV-1 infection is observed upon treatment with an inhibitor to mitogen-activated protein kinase that prevents phosphorylation of LysRS on Ser207, release of LysRS from the MSC, and nuclear localization. A phosphomimetic mutant of LysRS (S207D) that lacked the capability to aminoacylate tRNA^Lys3 localized to the nucleus, rescued HIV-1 infectivity, and was packaged into virions. In contrast, a phosphoablative mutant (S207A) remained cytosolic and maintained full aminoacylation activity but failed to rescue infectivity and was not packaged. These findings suggest that HIV-1 takes advantage of the dynamic nature of the MSC to redirect and coopt cellular translation factors to enhance viral replication.

IMPORTANCE Human tRNA^Lys3, the primer for reverse transcription, and LysRS are essential host factors packaged into HIV-1 virions. Previous studies found that tRNA^Lys3 packaging depends on interactions between LysRS and HIV-1 Gag; however, many details regarding the mechanism of tRNA^Lys3 and LysRS packaging remain unknown. LysRS is normally sequestered in a high-molecular-weight multi-aminoacyl-tRNA synthetase complex (MSC), restricting the pool of free LysRS-tRNA^Lys. Mounting evidence suggests that LysRS is released under a variety of stimuli to perform alternative functions within the cell. Here, we show that HIV-1 infection results in a free pool of LysRS that is relocalized to the nucleus of target cells. Blocking this pathway in HIV-1-producing cells resulted in less infectious progeny virions. Understanding the mechanism by which LysRS is recruited into the viral assembly pathway can be exploited for the development of specific and effective therapeutics targeting this nontranslational function.

INTRODUCTION

During assembly of retroviruses, two single-stranded RNA genomes are incorporated into each virion through direct interactions with the retroviral Gag protein (1). The 5′ untranslated region (UTR) of the RNA genome is highly structured and contains several conserved elements critical to the retroviral life cycle, including the primer binding site (PBS) (2). During the process of reverse transcription, a host tRNA that is complementary to the PBS region is unwound and annealed to the genome by Gag (3–7). Reverse transcriptase then initiates cDNA synthesis from the tRNA primer, using the RNA genome as a template to synthesize a double-stranded proviral genome that can be inserted into the host genome (8). In human immunodeficiency virus type 1 (HIV-1) the primer for this process is tRNA^Lys3 (9, 10).

Primer tRNA^Lys3 and the other tRNA^Lys isoacceptor, tRNA^Lys1,2, are selectively packaged into budding virions as an essential step of the HIV-1 life cycle (9). Whereas tRNA^Lys isoacceptors constitute approximately 5 to 6% of total tRNA in cells, 50 to 60% of the low-molecular-weight RNA found in HIV-1 virions is comprised of tRNA^Lys (11). Selective tRNA^Lys packaging is accomplished through an interaction between the HIV-1 Gag protein and human lysyl-tRNA synthetase (LysRS), which is also packaged into HIV-1 virions (12). Aminoacyl-tRNA synthetases such as LysRS are an integral part of the host translation machinery, functioning in the attachment of specific amino acids to cognate tRNA molecules. In mammals, nine tRNA synthetases, including LysRS, and three scaffolding proteins, aminoacyl-tRNA synthetase-interacting proteins (AIMPs), are part of a multi-aminoacyl-tRNA synthetase complex (MSC) (13). It has been shown that nearly all members of the tRNA synthetase family, whether or not they are components of the MSC, carry out alternative nontranslational functions in response to various intra- and extracellular stimuli (14, 15).

Four copies of LysRS are present as dimers within the MSC (16). Each LysRS dimer is tethered to the MSC through interactions with the scaffolding protein p38/AIMP2 (16). LysRS, like other aminoacyl-tRNA synthetases present in the MSC, can be released from the scaffolding protein to carry out alternative functions (17). For example, IgE stimulation of mast cells results in phosphorylation of LysRS on S207. LysRS is then released from the MSC and traffics to the nucleus (18, 19), where it synthesizes a dinucleotide Ap4A, triggering transcriptional activation of several genes (20–22). In another example, following laminin receptor (67LR) stimulation of a variety of human cell lines, LysRS is phosphorylated on residue T52 by mitogen-activated protein kinase (MAPK), released from the MSC, and trafficked to the plasma membrane, where it interacts with 67LR, protecting it from ubiquitin-mediated degradation (23). LysRS can also be released from the MSC and secreted to act as a proinflammatory cytokine (24). In addition to facilitating packaging of the tRNA^Lys primer into HIV-1 virions (25), LysRS enhances the correct placement of tRNA^Lys3 onto the PBS by recognizing a tRNA-like element proximal to the PBS (26, 27). To serve as a reverse transcriptase primer, the tRNA that is annealed to the PBS must be in a nonaminoacylated or “uncharged” state. However, the mechanism to ensure packaging of only uncharged tRNA^Lys3 is unknown.

To begin to elucidate the pathway by which LysRS and uncharged tRNA are diverted from their canonical roles in translation and incorporated into viral particles, we investigated the effect of HIV-1 infection on the MSC and on LysRS localization. We report an unexpected and dramatic cellular response to HIV-1 infection involving LysRS and other components of the MSC. Importantly, these events are directly correlated with the infectivity of progeny HIV-1 virions.

RESULTS

LysRS expression during HIV-1 infection is constant.

To confirm previous observations that LysRS expression is constant during HIV-1 infection (28), we determined that LysRS protein levels do not change in transformed human embryonic kidney cells (HEK293T) and CD4⁺ transformed human T cells (HuT/CCR5) upon single-cycle infection with HIV-1-Luc/vesicular stomatitis virus G (VSV-G) or upon infection with replication-competent NL4-3 virus, respectively (Fig. 1A and B). This suggests either that newly synthesized LysRS is packaged immediately after translation, as previously proposed (28), or alternatively, that LysRS associated with the MSC is released upon HIV-1 infection and packaged into virions.

FIG 1.

LysRS expression and release from the MSC following HIV-1 infection. (A and B) Immunoblots showing the LysRS expression time course over 24 h in HEK293T cells infected with HIV-Luc/VSV-G (A) and HuT CCR5 cells infected with HIV-1_NL4-3 (B). The bar graphs show quantification of LysRS expression relative to GAPDH and normalized to mock samples (uninfected). Data are presented as means ± standard deviations (SD) (n = 3 experiments). (C) Size exclusion chromatography fractionation of mock (top) and HIV-Luc/VSV-G-infected (bottom) HEK293T cells analyzed by immunoblotting. One-milliliter fractions were collected and probed for components of the MSC (LysRS, GluProRS, p38/AIMP2, and LeuRS) and a tRNA synthetase not found in the complex (TrpRS). Fractions 9 to 11 correspond to high-molecular-weight MSC-containing fractions, and fractions 12 to 18 correspond to lower-molecular-weight free proteins. The “ladder” lane inserted between fractions 12 and 13 contains protein molecular weight standards and was used as a marker to distinguish between blots. The band observed in the ladder lane of the infected LysRS row is a cross-reaction between the ladder and secondary antibody.

A free non-MSC-associated pool of LysRS is observed following HIV-1 infection.

Aminoacyl-tRNA synthetases are released from the MSC under specific cellular stresses. To determine whether a free pool of LysRS is available for packaging during HIV-1 infection, cell lysates from HIV-1-Luc/VSV-G-infected and uninfected HEK293T cells were subjected to size exclusion chromatography (SEC) and analyzed by immunoblotting. Fractions were detected with antibodies against various synthetases and a cellular host factor known to be localized within (LysRS, glutamyl-prolyl-tRNA synthetase [GluProRS], leucyl-tRNA synthetase [LeuRS], and p38/AIMP2) and outside (tryptophanyl-tRNA synthetase [TrpRS]) the MSC. Under homeostatic conditions, LysRS, GluProRS, LeuRS, and p38/AIMP2, the main scaffolding protein tethering LysRS to the MSC (29), coeluted in high-molecular-weight fractions, indicating that all detectable LysRS is sequestered in the MSC, as expected (30) (Fig. 1C). In contrast, TrpRS eluted in later fractions, consistent with its presence as a low-molecular-weight “free” protein. Following HIV-1 infection, a significant amount of LysRS shifted to the low-molecular-weight fractions. Furthermore, some of the MSC components (GluProRS and LeuRS) were also present in low-molecular-weight fractions, whereas p38/AIMP2 remained with the higher-molecular-weight complex (Fig. 1C). Thus, a pool of free LysRS is observed upon HIV-1 infection. A previous report indicated that Gag is able to interact with MSC components in cells (31). Interestingly, a minor fraction of HIV-1 Gag coelutes with the high-molecular-weight MSC, suggesting that Gag may interact with the full complex and not just with individual tRNA synthetases (Fig. 1).

LysRS is localized to the nucleus during HIV infection but is retained in the cytoplasm when cells are pretreated with the MEK inhibitor U0126.

When released from the MSC, LysRS has been shown to traffic to discrete locations within the cell to carry out alternative functions. We therefore examined the cellular localization of LysRS following HIV-1 infection of HEK293T cells. Cells were infected with HIV-1-Luc/VSV-G-pseudotyped virus, as verified by the high degree of luciferase activity in infected lysates (Fig. 2A). Fractionation of cell lysates showed that LysRS is exclusively cytoplasmic in uninfected cells, as expected (Fig. 2B) (22, 32). Surprisingly, following HIV-1 infection, a significant amount of LysRS was detected in the nuclear fraction (Fig. 2B). To visualize the nuclear trafficking more directly, uninfected HEK293T cells or cells infected with green fluorescent protein (GFP) reporter HIV-1/VSV-G-pseudotyped virus were followed by fluorescence microscopy (Fig. 2C, mock and HIV-1 infection, respectively). Consistent with the fractionation studies, nuclear localization of LysRS was observed only after HIV-1 infection, as indicated by the overlap in 4′,6-diamidino-2-phenylindole (DAPI) and LysRS signals. To probe the timing of nuclear localization, cells were next treated with the reverse transcription inhibitor azidothymidine (AZT), which indeed blocked HIV-1 infection, as indicated by reduced GFP levels; however, LysRS nuclear localization was still observed (Fig. 2C). Interestingly, heat-inactivated HIV-1 did not trigger nuclear localization of LysRS. Quantification of LysRS/nuclear colocalization in HIV-1-infected cells indicated a statistically significant overlap in the signals, with a Pearson correlation coefficient of 0.592 ± 0.128, compared to 0.098 ± 0.081 for uninfected cells (Fig. 2D). The overlap observed with heat-inactivated virus was similar to that with mock infection, whereas AZT treatment resulted in an overlap of 0.561 ± 0.130. These results suggest that LysRS release from the MSC and nuclear localization occur prior to HIV-1 reverse transcription and that events following endocytosis-mediated viral entry are required for the cellular response.

FIG 2.

Relocalization of LysRS following HIV-1 infection. (A) Normalized expression of luciferase in HIV-Luc/VSV-G-infected cells with and without U0126 treatment upon infection with HIV-Luc/VSV-G to determine HIV-1 infection. (B) Immunoblots of nuclear/cytoplasmic fractions isolated from uninfected (mock) and HIV-Luc/VSV-G-infected HEK293T cells in the presence and absence of U0126 treatment. GAPDH and lamin A/C were used as markers for cytoplasmic and nuclear fractions, respectively. (C) Immunofluorescence deconvolution microscopy of HEK923T cells in the absence of HIV-1 infection (mock) or upon HIV-1 GFP/VSV-G infection (HIV). The effect of HIV-1 infection following pretreatment with AZT, U0126, or DMSO is also shown, as well as the effect of heat-inactivated HIV-1. DAPI (blue) corresponds to nuclear staining, Alexa Fluor 586 (yellow) represents LysRS staining, and GFP corresponds to HIV-1 GFP (located in the env coding region of the NL4-3 genome) infection (green). Scale bars indicate 15 μm. (D) Calculated Pearson's coefficient of correlation for each condition. Bar represents mean values (n = 50 cells counted per treatment; 3 independent experiments). ***, P < 0.001; ns, not significant (as determined by one-way ANOVA with Tukey's correction for multiple comparisons).

In previous work investigating the effects of mast cell stimulation by IgE, LysRS was shown to be phosphorylated on Ser207, released from the MSC, and localized to the nucleus (22). LysRS release from the MSC and nuclear localization were blocked by treatment with a MAPK/extracellular signal-regulated kinases (MEK) inhibitor, U0126 (22). To determine whether a similar pathway may also be triggered by HIV-1 infection, HEK293T cells were treated with U0126 prior to HIV-1 infection. Interestingly, U0126 treatment blocked LysRS nuclear localization, suggesting a role for LysRS phosphorylation in the response to HIV-1 infection (Fig. 2C and D).

Treatment of HIV-1-producing cells with U0126 reduces infectivity of progeny virions.

To examine the effect of U0126 and inhibition of LysRS nuclear localization on progeny virions, we transfected U0126-treated HEK293T producer cells with a proviral pNL4-3 plasmid, collected the virion-containing supernatants, quantified p24 levels, and measured the infectivity of virions in transformed human osteosarcoma (GHOST) cells. Enzyme-linked immunosorbent assay (ELISA) results showed similar levels of soluble p24 produced from HEK293T cells, regardless of U0126 treatment (Fig. 3B), whereas subsequent infection of GHOST cells showed that a significant decrease in viral infectivity with virus was produced in the presence of U0126 (Fig. 3A). Next, we collected virions from Hut/CCR5 cells infected with NL4-3 or NLAD8 virus, in the absence or presence of U0126, and found that, consistent with the results with HEK293T cells, there was no significant effect on the levels of p24 produced (Fig. 3B). However, the infectivity of virions produced in the presence of U0126 was significantly reduced by 3-fold or 7-fold (Fig. 3A) when normalized to produced virions. These data suggest that U0126 treatment of HIV-1-producing cells reduces viral infectivity.

FIG 3.

MEK inhibitor U0126 reduces infectivity of progeny virions. (A) Relative infectivities of progeny virions collected from untreated cells or cells pretreated with U0126 and infected with HIV-1_NL4-3 or HIV-1_NLAD8 (HuT/CCR5) or transfected with proviral pNL4-3 (HEK293T) by GHOST cell infection. The data in panel A are normalized to the p24 ELISA results shown in panel B. *, P < 0.05; **, P < 0.01; ***, P < 0.001 as determined by Student's t test. Data are presented as means ± SD (n = 3 experiments).

KD of endogenous LysRS and rescue with an S207D LysRS mutant restores infectivity of progeny virions.

To further explore the role of LysRS phosphorylation and nuclear localization in HIV-1 infectivity, we generated a stable HEK293T cell line containing a doxycycline-inducible LysRS-specific short hairpin RNA (shRNA) (LysRS_KD), as well as a nonsilencing (NS) shRNA control cell line (LysRS_NS) (Fig. 4B). The U0126 studies described above suggested that LysRS is posttranslationally modified on S207 following HIV-1 infection. To further explore the role of this residue in HIV-1 infectivity, we generated plasmids encoding codon-optimized full-length (FL) LysRS, as well as the following LysRS mutants: phosphomimetic S207D, phosphoablative S207A, and N-terminally truncated ΔN65 LysRS lacking the putative nuclear localization signal (28) (Fig. 4A). Treatment of knockdown (KD) cells with doxycycline resulted in efficient LysRS knockdown (93% after 72 h) (data not shown). LysRS_KD cells were transfected with pNL4-3, and the virions collected showed a 10-fold reduction in infectivity (Fig. 4C). In contrast, progeny virions produced from LysRS_NS cells maintained comparable levels of infectivity when small interfering RNA (siRNA) expression was induced with doxycycline (Fig. 4D). KD cells were transfected with the FL or mutant LysRS-expressing plasmids, and the impact on progeny virus infectivity was determined. Overexpression of FL and S207D LysRS rescued infectivity to wild-type (WT) levels, whereas overexpression of S207A LysRS and ΔN65 LysRS failed to rescue infectivity (Fig. 4C). These data support the importance of Ser207 phosphorylation and nuclear localization for progeny virion infectivity. A previous study showed that ΔN65 LysRS failed to traffic to the nucleus (28), and in contrast to FL LysRS, overexpression of this variant failed to enhance tRNA^Lys packaging into HIV-1 virions in the presence of endogenous LysRS (25). Nuclear-cytoplasmic fractionation of cell lysates from KD cells transfected with proviral pNL3-4 HIV and either FL, S207D, or S207A LysRS confirmed that both FL LysRS and S207D LysRS trafficked to the nucleus, whereas S207A LysRS did not (Fig. 4E). These experiments suggest that release of LysRS from the MSC and nuclear localization are likely important for virion infectivity.

FIG 4.

Effect of shRNA knockdown of LysRS on HIV-1 infectivity and rescue with LysRS variants. (A) Domain structure of LysRS showing the three Flag-tagged LysRS mutant constructs S207A, S207D, and ΔN65. The 3 conserved class II aminoacyl-tRNA synthetase consensus motifs in the aminoacylation domain are indicated by the hatched bars. (B) Immunoblots showing the expression of LysRS during LysRS-targeted knockdown (KD) and nonsilencing (NS) shRNA induction with 1.5 μg/ml doxycycline (Doxy) over the course of 72 h. GAPDH was used as a loading control. (C) HIV-1 p24 ELISA results (top panel) and infectivity (middle panel) of progeny virions collected from uninduced cells (−), with and without U0126, and KD (+) cells in the absence of added plasmid (WT) or in the presence of expression plasmids encoding S207A, S207D, FL, or ΔN65 LysRS. HEK293T cells were transfected with proviral pNL4-3 HIV-1 normalized to the amount of p24. *, P < 0.05; ***, P < 0.001; ns, not significant (as determined by one-way ANOVA with Tukey's correction for multiple comparisons). Data are presented as means ± SD (n = 3 experiments). Bottom panel, representative immunoblot from producer cells; Flag is used as a marker for exogenous LysRS expression, GAPDH as a loading control, and p55 as an indicator of infection. (D) p24 ELISA results (top panel) and infectivity (middle panel) of progeny virions collected from the NS cell line compared to virions collected from doxycycline-induced cells transfected with proviral pNL4-3 HIV normalized to amount of p24. Differences are not significant (ns) as determined by one-way ANOVA with Tukey's correction for multiple comparisons. Data are presented as means ± SD (n = 3 experiments). Bottom panel, representative immunoblot from NS shRNA producer cells. (E) Nuclear-cytoplasmic fractionations of HEK293T KD cells transfected with mutant LysRS constructs and infected with HIV-Luc/VSV-G. GAPDH and lamin A/C were used as markers for cytoplasmic and nuclear fractions, respectively.

S207D LysRS is packaged into infectious HIV-1 virions, while S207A LysRS is excluded.

To determine the capability of mutant LysRS to be packaged into HIV-1 particles, virions were collected from doxycycline-treated KD cells expressing FL, S027D, S207A, and ΔN65 LysRS. Virions collected from cells expressing either FL, S207D, or ΔN65 LysRS contained Flag-tagged exogenous LysRS (Fig. 5). In contrast, exogenous LysRS was not observed in virions produced from cells expressing S207A LysRS, suggesting that the S207A phosphoablative mutant lacks the ability to be packaged into HIV-1 virions. Moreover, when cells were treated with the U0126 MEK inhibitor, FL LysRS was excluded from virions (Fig. 5), supporting the importance of S207 phosphorylation for LysRS packaging into virions.

FIG 5.

Effect of S207 mutation and N-terminal truncation on LysRS packaging into HIV-1 particles. Flag-tagged LysRS mutants S207D, S207A, and ΔN65 were transfected into doxycycline-treated KD cells. Representative immunoblots of producer cells (top) and virions (bottom) probed using anti-LysRS or anti-p24 antibodies are shown. GAPDH was used as a loading control.

S207D binds but does not aminoacylate tRNA^Lys3.

To determine the effect of S207 mutation on the canonical function of LysRS, in vitro aminoacylation assays were performed. Phosphoablative S207A LysRS aminoacylated in vitro-transcribed tRNA^Lys3 at a rate comparable to that for WT LysRS. However, aminoacylation by the phosphomimetic mutant, S207D, was undetectable (Fig. 6A). Intriguingly, electrophoretic mobility shift assays (EMSAs) showed that both the S207A and S207D mutants retain their ability to bind tRNA^Lys3, with apparent binding affinities of 1.3 ± 0.62 μM and 0.47 ± 0.19 μM, respectively (Fig. 6B). These affinities are similar to that measured for WT LysRS, 0.93 ± 0.32 μM.

FIG 6.

Effect of S207 mutation on LysRS aminoacylation and tRNA binding. (A) In vitro aminoacylation of tRNA^Lys3 by WT, S207A, and S207D LysRS. Conditions are described in Materials and Methods. The results are the averages and SD from 3 trials. (B) Binding of WT, S207A, and S207D LysRS to tRNA^Lys3 was measured by EMSAs performed as described in Materials and Methods. Results are the averages and SD from three trials.

DISCUSSION

HIV-1 requires that its reverse transcription primer, human tRNA^Lys3, be packaged into progeny virions in order to ensure productive infection of target cells (9, 33, 34). This is facilitated by the interaction of LysRS with HIV-1 Gag (12, 25, 28, 35–37). Here, we present evidence that HIV-1 infection triggers release of LysRS, as well as other tRNA synthetases, from the MSC. Surprisingly, the released LysRS traffics to the nucleus, a response that appears to require a specific phosphorylation event on S207, as previously identified in IgE-stimulated mast cells (22). In the latter case, nuclear LysRS was shown to synthesize Ap4A and activate genes controlled by microphthalmia-associated transcription factor. While we do not yet know the precise role for LysRS nuclear localization in HIV-1 infectivity, blocking this pathway by addition of a MEK inhibitor or specific mutation of Ser207 to Ala reduces infectivity of progeny virions. In contrast, the S207D phosphomimetic mutant restored progeny virion infectivity from producer cells lacking the majority of endogenous LysRS. Importantly, S207D LysRS is packaged into HIV-1 particles, whereas S207A is excluded (Fig. 5), and treatment with U0126 prevents FL LysRS packaging. Thus, while these data do not prove that S207 phosphorylation controls LysRS packaging into HIV-1 particles, taken together, these data suggest that the restoration of infectivity may be correlated with phosphorylation and packaging capability. However, further experiments should be conducted to directly determine the phosphorylation state of cellular and viral LysRS. Previous studies suggested that U0126 negatively affects X4- but not R5-tropic HIV-1 infection in resting peripheral blood CD4⁺ T lymphocytes (38). Therefore, it is possible that we are observing multiple effects upon treatment with the MEK inhibitor on the production of progeny virions from NL4-3-infected HuT/CCR5 cells.

The finding that ΔN65 LysRS is unable to rescue progeny virion infectivity despite the fact that it is packaged is in agreement with previous studies reporting that (i) expression of this truncation mutant did not enhance tRNA^Lys incorporation into virions (25, 39) and (ii) this mutant displays a 50-fold reduction in tRNA binding in vitro. Thus, the lack of rescue with ΔN65 LysRS may be due to lack of nuclear trafficking and/or reduced tRNA binding and packaging. Another previous study concluded that viral LysRS was unlikely to originate from the MSC because p38/AIMP2 was not found in virions (28). However, it is now clear that MSC components can be released under a variety of conditions and mobilized to perform other cellular functions (14–16). Our SEC data show that a significant fraction of LysRS, as well as other tRNA synthetases that are exclusively in the high-molecular-weight MSC in uninfected cells, is in the free fractions following HIV-1 infection, while the p38/AIMP2 protein remains bound to the complex. LysRS exists as a dimer of dimers in the MSC and is considered one of the integral structural components of the complex (40). Therefore, release of LysRS may cause other tRNA synthetases to become less stably associated with the MSC, accounting for their release.

The MSC is thought to enhance association of tRNAs with their cognate tRNA synthetases (41), increasing the likelihood that a released pool of LysRS would be bound to tRNA^Lys. The N-terminal domain of p38/AIMP2 forms hydrophobic interactions with the N-terminal anticodon binding domain of one LysRS monomer and the C-terminal aminoacylation domain of the other (16). HIV-1 Gag interacts with LysRS via its motif 1 dimerization interface (35, 42). The S207 residue lies proximal to the LysRS homodimerization interface and the p38/AIMP2-binding interface in the anticodon binding domain of LysRS (16). The phosphomimetic S207D LysRS mutant is unable to bind p38/AIMP2 or any component of the MSC (16), which would increase the pool of free LysRS. Additionally, S207D LysRS has been shown to trigger a more “open” conformation, resulting in abolition of aminoacylation but not tRNA binding capacity in vitro (16, 43). Our data are consistent with these previous findings and also show that S207D LysRS still binds tRNA^Lys3 as well as WT LysRS. In addition, S207A LysRS, which is not packaged, maintains full tRNA binding and aminoacylation capability (Fig. 6). These findings are also consistent with previous reports showing the presence of exclusively uncharged tRNA in virions (44) and the requirement for deacylated tRNA for priming of reverse transcription (45). Since Gag binds the dimerization face of LysRS, the more open conformation of the S207D variant may favor Gag binding and displacement of one LysRS monomer.

Altogether, these data support a model in which initial steps of HIV-1 infection trigger the phosphorylation and release of LysRS from the MSC (Fig. 7). Based on our data, we cannot rule out an alternative model wherein infection triggers phosphorylation of newly synthesized LysRS, which prevents association with the MSC (Fig. 7). It has been demonstrated in other retroviruses, such as Rous sarcoma virus, that Gag traffics through the nucleus (46). Although HIV-1 Gag is not confirmed to traffic through the nucleus during assembly, we cannot exclude the possibility that a small fraction of Gag could interact with nuclear LysRS. Our data suggest that HIV-1 Gag exploits the dynamic nature of the MSC to recruit free LysRS-tRNA^Lys to the plasma membrane for incorporation into assembling virions. The virus ensures packaging of a deacylated primer by packaging an isoform of LysRS that is defective in tRNA aminoacylation but still capable of strong tRNA binding. The findings presented here suggest potential new targets for future therapeutic intervention.

FIG 7.

Proposed model of LysRS recruitment to budding virions. (1 and 2) Infection of cells by HIV-1 results in phosphorylation of either MSC-associated or newly synthesized LysRS (1), resulting in LysRS-tRNA^Lys release from the MSC or preventing association with the MSC, respectively (2). (3) Free LysRS-tRNA^Lys translocates to the nucleus and interacts with Gag (likely in the cytoplasm). (4) The tRNA packaging complex containing an uncharged tRNA is brought to sites of viral assembly at the plasma membrane and packaged into virions. The tRNA synthetases present in the MSC are represented by their single-letter amino acid abbreviations, and the associated cellular factors are designated 1 to 3.

MATERIALS AND METHODS

Plasmids, antivirals, and inhibitors.

WT virus strains HIV-1_NL4-3 and HIV-1_NLAD8 were expressed from pNL4-3 (47) (NIH AIDS Reagent Program) and pNLAD8 (Eric Freed, National Cancer Institute) (48). Single-round derivatives HIV-Luc and HIV-GFP were expressed from pNL-Luc R⁺E⁻ (49, 50) and pNL4-3GFPR⁺E⁻, obtained through the NIH AIDS reagent program (cat. no. 111000). VSV-G glycoprotein was expressed using pMD2.G. pCDNA3.FLLysRS, encoding FL C-terminally Flag-tagged human cytoplasmic LysRS codon optimized for expression in mammalian cells, was synthesized by Genewiz. Site-directed ligase-independent mutagenesis (51) was used to make plasmids encoding S207A, S207D, and ΔN65 LysRS mutants (pCDNA3.S207ALysRS, pCDNA3.207DLysRS, and pCDNA3ΔN65.LysRS, respectively). AZT was obtained from the NIH AIDS reagent exchange program. The MEK inhibitor U0126 monoethanolate was obtained from Sigma-Aldrich (U120.1MG).

Cell culture and virus production.

HEK293T cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (complete DMEM), while transformed human T cells (HuT/CCR5) were maintained in Roswell Park Memorial Institute (RPMI) medium supplemented with 1 μg/ml puromycin and 500 μg/ml Geneticin. GHOST X4/R5 cells were maintained in complete DMEM, 1 μg/ml puromycin, 500 μg/ml Geneticin, and 100 μg/ml hygromycin B.

HIV-Luc and HIV-GFP virus stocks were produced by cotransfecting HEK293T cells (3.5 × 10⁶) with 10 μg pNL4-3LucR⁺E⁻ or pNL4-3GFPR⁺E⁻, along with 4 μg pMD2.G, using the calcium phosphate method (52). HIV-1_NL4-3 and HIV-1_NLAD8 stocks were produced by transfecting HEK293T cells (3.5 × 10⁶) with 15 μg of pNL4-3 or pNLAD8, respectively, using the calcium phosphate method (53). Supernatants were collected at 48 h posttransfection, and the virus titer was determined by GHOST cell titration. GHOST cells (4 × 10⁵) were infected with increasing volumes of virus-containing supernatant. Cells were harvested at 48 h postinfection, fixed, and analyzed by flow cytometry as previously described (52).

Plasmids (pTripZ) containing a doxycycline-inducible LysRS-specific (5′-TCTTCATGACAAACAGCTC-3′) or nonsilencing (proprietary) shRNA were obtained from Dharmacon and used to create stable HEK293T LysRS knockdown (KD) and nonsilencing (NS) cell lines by lentivirus transduction according to the manufacturer's protocol. Stable cell lines were grown in complete DMEM and 1 μg/ml puromycin.

Immunoblotting.

Cells or viruses were lysed in cell lysis buffer (CLB) (Cell Signaling Technology) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Total cellular lysate (20 μg) was loaded onto sodium dodecyl sulfate–12% polyacrylamide gels. Following electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). Antibodies used to probe membranes were obtained from Cell Signaling Technology (mouse anti-lamin A/C), Bethyl (rabbit anti-TrpRS, rabbit anti-GluProRS, rabbit anti-LeuRS, and rabbit anti-LysRS), Sigma-Aldrich (mouse anti-Flag), Thermo Scientific (mouse anti-HIVp24 and rabbit anti-AIMP2), and Bio-Rad (rabbit anti-glyceraldehyde-3-phosphate dehydrogenase [anti-GAPDH]). Rabbit anti-LysRS was a gift from Lawrence Kleiman (Lady Davis Institute for Medical Research). Following incubation with the primary antibody, blots were washed and incubated with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (Promega) or HRP-conjugated goat anti-mouse IgG (Santa Cruz) secondary antibodies. Super-Signal chemiluminescence substrates (Pierce) were used to detect secondary antibodies. Protein bands were visualized on a GE Amersham Imager 600 and quantified using ImageQuant (GE). HIV-1 p24 concentrations were determined using an HIV p24 ELISA kit (ZeptoMetrix).

Immunofluorescence and deconvolution microscopy.

HEK293T cells (50,000) were grown on coverslips prior to treatment with virus or inhibitors or transfection with LysRS plasmids. Approximately 2 h before infection, cells were pretreated with dimethyl sulfoxide (DMSO) (control), 10 μM AZT, or 10 μM U0126. Treatments were continued during HIV-1 infection and supplemented into media postinfection. In the case of heat-inactivated HIV-1, virus was inactivated by heating virions at 56°C for 2 h to denature viral proteins, as previously described (54, 55). Cells were fixed with 4% paraformaldehyde (in 1× phosphate-buffered saline) at 24 h postinfection for 20 min at room temperature, followed by permeabilization with 0.1% Triton X-100 for 5 min at room temperature as previously described (56). Cells were blocked with 5% (wt/vol) bovine serum albumin (in 1× phosphate-buffered saline) for 1 h at room temperature and incubated for 1 h at room temperature with rabbit anti-LysRS (1:500 dilution in 2% FBS in 1× phosphate-buffered saline). Alexa Fluor 568-labeled donkey anti-rabbit antibody (1:500 dilution in 2% FBS in 1× phosphate-buffered saline) was used as the secondary antibody (Life Technologies). DAPI (Molecular Probes) was used to stain nuclei (1:47,000 dilution in 1× phosphate-buffered saline). Deconvolution images were acquired at room temperature using an inverted DeltaVision microscope (GE) with an oil immersion 60×/NA 1.4 objective lens. Subsequent colocalization analysis to determine Pearson's coefficient of correlation and processing of images were performed using the DeltaVision software and ImageJ. Statistical analysis was performed using a one-way analysis of variance (ANOVA) with Tukey's correction for multiple comparisons.

Flow cytometry.

HEK293T cells were harvested at 24 and 48 h postinfection or transfection with HIV-GFP/VSV-G and fixed in a mixture of 2% paraformaldehyde and 1% FBS in phosphate-buffered saline. The percentage of infected cells was determined on a Guava EasyCyte (Millipore) with FlowJo analysis (Tree Star) (52).

SEC.

For size exclusion chromatography (SEC), HEK293T cells (7 × 10⁶) were uninfected or infected with HIV-Luc/VSV-G at a multiplicity of infection (MOI) of 1 and 24 h later were lysed with 1× CLB. Cell extracts were clarified, applied to a Superdex 200 gel filtration column (30 by 1 cm; GE Healthcare) using a BioLogic DuoFlow fast protein liquid chromatography (FPLC) system (Bio-Rad), and eluted at a flow rate of 0.3 ml per min in buffer containing 20 mM HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 0.5% Triton X-100. Blue dextran was used as a molecular weight marker (22). Collected fractions were immunoblotted for LysRS, GluProRS, TrpRS, LeuRS, and AIMP2 using the antibodies described above.

Cytoplasmic-nuclear fractionation and luciferase assay.

HEK293T cells were grown in 6-well plates and either left uninfected (mock) or infected with HIV-1-Luc/VSV-G for 2 h. Some samples were treated with 10 μM U0126 prior to HIV-1 infection. For analysis of LysRS mutants, KD stable cells were grown in 6-well plates for 36 h with 1.5 μg/ml doxycycline. At 36 h, cells were transfected overnight with pcDNA.3 (empty vector), pCDNA3.FLLysRS, pCDNA3.S207ALysRS, or pCDNA3.S207DLysRS. The medium was replaced with complete DMEM containing 1.5 μg/ml doxycycline, and the cells were infected with HIV-Luc/VSV-G for 2 h. At 24 h (untransfected) or 72 h (transfected), four wells were collected using phosphate-buffered saline, combined, and fractionated using an NE-PER cytoplasmic nuclear fractionation kit (Pierce). The contents of two wells were combined and lysed in CLB for total lysate fractions. The remainder of the cells were combined, lysed in luciferase reporter lysis buffer (Promega), and clarified. Luciferase expression of cells was determined using the luciferase assay system (Promega).

HIV-1 infectivity assays.

To determine the effect of the MEK inhibitor U0126 on progeny virion infectivity, HuT/CCR5 cells (1 × 10⁶) with and without U0126 were pretreated and infected with NL4-3 or NLAD8 virus. The medium was harvested at 24 h postinfection, filtered through a 0.45-μm syringe filter, and concentrated by ultracentrifugation into a 25% sucrose cushion. Virus was resuspended overnight. The HIV p24 content was determined via ELISA, and infectivity was determined by GHOST cell titration (52). HEK293T cells (400,000) were pretreated with U0126 and transfected with pNL4-3. The supernatant was collected after 48 h and filtered, and the titer was analyzed as described above. To determine the role of S207 phosphorylation in HIV-1 infection, stable cell lines were grown in complete DMEM supplemented with 1.5 μg/ml doxycycline for 36 h and then transfected with pNL4-3 and pCDNA3.FLLysRS, pCDNA3.S207ALysRS, pCDNA3.S207DLysRS, or pCDNA3.ΔN65LysRS. Doxycycline treatment was maintained for the entirety of the experiment. Virus-containing medium was collected after 72 h and processed in the same manner as for HEK293T cells. Cells were also collected, lysed, and immunoblotted for LysRS expression. Statistical analysis was performed using a one-way ANOVA with Tukey's correction for multiple comparisons.

LysRS packaging into HIV-1 virions.

To determine the effect of phosphomimetic and phosphoablative mutants and U0126 treatment on LysRS packaging into HIV-1 particles, LysRS KD cells were grown in complete DMEM supplemented with 1.5 μg/ml doxycycline for 36 h, followed by transfection with pNL4-3 and pCDNA3.FLLysRS, pCDNA3.S207ALysRS, pCDNA3.S207DLysRS, or pCDNA3.ΔN65LysRS. To test the effects of MEK inhibition on FL LysRS packaging, 10 μM U0126 was added to cells at 2 h prior to transfection with pCDNA3.FLLysRS and pNL4-3. Doxycycline and U0126 treatment was maintained for the entirety of the experiment. Virus-containing medium was collected after 72 h, filtered through a 0.45-μm syringe filter, and concentrated by ultracentrifugation through a 25% sucrose cushion. Virus was resuspended for 4 h at 37°C in 20 mM Tris (pH 8.0), 1 mM CaCl₂, and 250 μg/ml subtilisin to digest free proteins in the supernatant. Reactions were quenched by treatment with of 1 mM phenylmethylsulfonyl fluoride for 20 min at room temperature (57). The samples were diluted to 5 ml with buffer containing 10 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM NaCl, loaded onto a 5-ml 25% sucrose cushion, and concentrated by ultracentrifugation. Virions were lysed in 1× CLB overnight at 4°C and analyzed by immunoblotting using the LysRS antibody described above. Cells were also collected, lysed, and analyzed by immunoblotting for FL and mutant LysRS expression.

Preparation of WT, S207A, and S207D human LysRS.

S207A and S207D mutations were incorporated into plasmid pM368 encoding full-length human LysRS (58) using site-directed ligase-independent mutagenesis (51). Human LysRS protein was overexpressed and purified from Escherichia coli as previously described (58) with some modification. Briefly, expression vectors were transformed into BL21-CodonPlus (DE3)-RIL cells, and cultures were grown to an optical density at 600 nm (OD₆₀₀) of 0.6 to 0.8. WT and S207D LysRS protein expression was induced by the addition of 0.1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) and incubation at room temperature overnight. S207A LysRS was induced by the addition of 1 mM IPTG followed by incubation at 37°C for 4 h. Cells were pelleted and lysed, and LysRS protein was purified using His-select nickel affinity columns (Sigma-Aldrich). Proteins were further purified by SEC on a Superdex 200 increase 10/300 GL (GE Healthcare Life Sciences). LysRS concentrations were determined by Bradford assay, and enzymes were stored at −20°C in 40 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM dithiothreitol (DTT), and 40% glycerol.

Human tRNA^Lys3 preparation.

Human tRNA^Lys3 was in vitro transcribed using T7 RNA polymerase (59) and gel purified using denaturing 10% polyacrylamide gel electrophoresis. The excised full-length tRNA^Lys3 band was eluted, and the supernatant was butanol extracted and ethanol precipitated to recover the RNA. Purified tRNA^Lys3 was labeled at the 5′ end with [γ-³²P]ATP (PerkinElmer) using standard protocols.

Aminoacylation assays.

Aminoacylation assays were performed in 100 μg/ml bovine serum albumin (BSA), 20 mM KCl, 20 mM β-mercaptoethanol, 10 mM MgCl₂, 50 mM HEPES, 4 mM ATP, 20 μM lysine, 2.5 μM [³H]lysine (PerkinElmer), and 0.5 μM human tRNA^Lys3. Specific activity readings were taken in triplicate to determine ³H counts per minute per picomole of lysine. Experiments were initiated by addition of 5 nM WT, S207D, or S207A LysRS. Samples were taken every 20 s and quenched on a filter disk (Whatman 3MM) presoaked with 5% trichloroacetic acid (TCA). Filter disks were placed in excess 5% TCA, washed 3 times, and dried prior to counting in a liquid scintillation counter. All assays were performed in triplicate.

EMSAs.

Electrophoretic mobility shift assays (EMSAs) were performed by incubating WT or mutant LysRS (0 to 10 μM) with 40 nM ³²P-labeled tRNA^Lys3 at room temperature for 30 min in 20 mM HEPES (pH 7.4), 60 mM NaCl, 10 mM KCl, 1 mM MgCl₂, 0.4 mM DTT, and 8% glycerol. Samples were electrophoresed on native (89 mM Tris, 45 mM borate, 1 mM MgCl₂) 10% polyacrylamide gels and analyzed by phosphorimaging on a GE Typhoon FLA 9500 imager. Bands were quantified using ImageJ software (60), and dissociation constants were calculated by fitting data to the Hill equation.