Cellular distribution of LysRS in 293FTcells lysed hypotonically. Uninfected or protease-negative HIV-1-transfected 293FT cells (cells transfected with BH10.P− DNA) were lysed at 4°C by Dounce homogenization in hypotonic TE buffer and, after clarification by centrifugation at 1,500 × g, were centrifuged at 100,000 × g for 1 h at 4°C. (A and B) For the detection of LysRS in these fractions, the pellet (P100) was suspended in 1.0 ml of TE buffer, equal to the S100 volume. Western blots of the S100 and P100 from uninfected cells (A) or HIV-1-transfected cells (B) were probed with anti-LysRS. (C and D) Sucrose flotation analysis. The P100 fractions from uninfected cells (C) and HIV-1-transfected cells (D) were resolved by discontinuous sucrose gradient centrifugation into membrane-bound and membrane-free proteins (flotation analysis). Aliquots of each gradient fraction were analyzed by Western blots probed with anti-LysRS. I, interface between 10 and 65% sucrose layers, where membrane-bound protein localizes; B, bottom fractions of gradient, where membrane-free protein remains during centrifugation. Fraction numbers increase from top to bottom of gradient. (E and F) Association of LysRS with membrane. Each fraction from sucrose gradients of uninfected cells was immunoprecipitated with anti-LysRS, and Western blots of the immunoprecipitates were probed with antibodies to the membrane markers CD45 (E) or Na⁺/K⁺ ATPase (F). (G and H). Cellular distribution of p38 in uninfected 293FT cells. (G) Western blots of the S100 and P100 probed with anti-p38. (H) The P100 fraction from uninfected cells was resolved by discontinuous sucrose gradient centrifugation into membrane-bound and membrane-free proteins. Aliquots of each gradient fraction were analyzed by Western blots probed with anti-p38.

Cellular distribution of LysRS in 293FT cells lysed with Triton X-100. Uninfected and protease-negative HIV-1-transfected 293FT cells were lysed at 4°C in Triton X-100 buffer, and the postnuclear supernatants were resolved into S100 and P100. (A, and B) Western blots of the S100 and P100 from uninfected cells (A) or HIV-1-transfected cells (B) were probed with anti-LysRS. (C) Western blots of the S100 and P100 from uninfected cells were probed with anti-p38. (D and E) The P100 fractions from uninfected cells (D) and HIV-1-transfected cells (E) were resolved on iodixanol gradients, and aliquots of each gradient fraction were analyzed by Western blots probed with anti-LysRS. Denser fractions found at the bottom of the gradient are represented by larger fraction numbers. (F and G) Each iodixanol gradient fraction from HIV-1-transfected cells was immunoprecipitated with either anti-IN (F) or anti-LysRS (G), and the immunoprecipitates were analyzed by Western blots probed with anti-reverse transcriptase and anti-CA. (H and I) 293FT cells were transfected with the plasmid hGag, which expresses only Gag. Cells were lysed at 4°C in Triton X-100 buffer, and the postnuclear supernatants were resolved into S100 and P100. The P100 was resolved on an iodixanol gradient, and gradient fractions were immunoprecipitated with either anti-CA (H) or anti-LysRS (I). Western blots of the immunoprecipitates were probed with anti-CA.

The selective interaction of LysRS with Gag and its incorporation into HIV-1. (A) Detection of aaRSs and non-aaRSs in HIV-1. Viruses produced from 293FT cells transfected with protease-negative HIV-1 proviral DNA were pelleted from cell culture medium and purified by centrifugation through 15% sucrose onto a 65% sucrose cushion. Purified viruses were either left untreated or treated with the protease subtilisin (subt.) before viral lysis. Cell and viral lysates were analyzed by Western blots. Blots were probed with antibodies to the proteins shown and were analyzed by enhanced chemiluminescence by using anti-mouse or anti-rabbit as a secondary antibody. (B) Detection of aaRSs and non-aaRSs in a cytoplasmic Gag immunoprecipitate (IP). 293FT cells transfected with protease-negative HIV-1 proviral DNA were lysed in TNT buffer 64 h posttransfection. After removal of nuclei and broken cells at 1,500 × g, Gag was immunoprecipitated from the clarified lysate with anti-CA. After heating the immunoprecipitate to release the precipitated proteins, the precipitate was analyzed by Western blots, which were probed with antibodies to the proteins shown and were analyzed by enhanced chemiluminescence by using anti-rabbit as a secondary antibody.

Interaction of wild-type or mutant LysRS with p38. Plasmids coding for wild-type or C-terminal deletion mutant LysRS tagged at the N terminus with Myc were transfected into 293FT cells. (A) Wild-type and mutant LysRS variants tested. The cartoon at the top shows the various LysRS domains and the amino acid positions (numbers) at which they occur. The unnumbered N-terminal squiggle represents Myc. Deleted amino acid sequences are shown graphically as thin lines and are listed to the left of each mutant. The N-terminal domain (N), the anticodon binding domain (AC) and motifs 1, 2, and 3 (M1, M2, and M3) are sequence elements characteristic of class II tRNA synthetases and are associated with tRNA binding (N and AC), LysRS dimerization (M1), and aminoacylation (M2 and M3). (B and C) Western blots of lysates of cells transfected with plasmids coding for the different LysRS species. Blots were probed with either anti-Myc (B) or anti-p38 (C). (D) Cell lysates were treated with anti-p38, and the p38 immunoprecipitate was analyzed by Western blots probed with anti-Myc.

Distribution of wild-type and mutant LysRS between nuclei and cytoplasm. 293FT cells were either nontransfected (−) or transfected with a plasmid coding for full-length LysRS (pLysRS.CF) or a truncated LysRS missing the N-terminal 65 amino acids (pLysRS.CT). The exogenous LysRS species contain C-terminal V5. Cells were lysed in PBS buffer containing 0.1% Nonidet P-40 and 0.1% Triton X-100 as described in Materials and Methods. Nuclei were pelleted from the total cell lysate by centrifugation at 1,000 × g for 10 min and purified as described in Materials and Methods, and the nuclear extract was prepared by sonication. Total cell lysate (T), nuclear extract (N), and the postnuclear supernatant (C) were analyzed by Western blotting. (A) The distribution of endogenous LysRS in nontransfected cells and of LysRS.CF and LysRS.CT in transfected cells. Endogenous LysRS was detected with anti-LysRS, while LysRS.CF and LysRS.CT were detected with anti-V5. (B) A Western blot similar to that shown in panel A but probed with anti-tubulin. (C) A Western blot similar to that shown in panel A but probed with anti-YYI, a nuclear transcription factor.

Ability of wild-type and mutant LysRS to bind to cell membrane. Uninfected 293FT cells were transfected with plasmids coding for either wild type LysRS, N-terminal deletion mutant LysRS (Δ1-207), or C-terminal deletion mutant LysRS (Δ260-597 or Δ207-597). All LysRS species contain an N-terminal Myc tag. Cells were lysed hypotonically, and the cytoplasmic expression of these species, as analyzed in Western blots of uninfected cell lysate probed with anti-Myc, is shown (A). The P100 from each postnuclear supernatant was isolated and resolved by sucrose flotation. The membrane-associated fraction (fraction 2) from each gradient was immunoprecipitated with anti-Myc and resolved by Western blots probed with anti-Myc (B).

Kinetics of interaction between newly synthesized LysRS and Gag. 293FT cells were either cotransfected with hGag and LysRS cDNA (A) or transfected with hGag alone (B). Cells were metabolically labeled 64 h posttransfection with [³⁵S]methionine/cysteine for 10 min and then chased for various lengths of time in medium containing 100 μM cysteine and methionine as described in Materials and Methods. Aliquots of cells obtained at various times were lysed in TNT buffer and centrifuged at 1,500 × g to produce the S1 supernatant. Gag/LysRS complexes were then immunoprecipitated from this supernatant and analyzed by one-dimensional SDS-PAGE and autoradiography. (A) One-dimensional SDS-PAGE and autoradiography of immunoprecipitates from the S1 supernatants of cells cotransfected with hGag and LysRS. Lanes 1 to 4 show anti-CA immunoprecipitates from cells lysed at 0 to 60 min postlabeling. Lane 5 shows an anti-V5 immunoprecipitate from cells lysed 30 min postlabeling. The ratios of the signals for LysRS/Gag are shown below the lanes. (B) One-dimensional SDS-PAGE and autoradiography of immunoprecipitates from the S1 supernatants of cells transfected with hGag alone. Lanes 1 to 4 show anti-CA immunoprecipitates from cells lysed at 0 to 60 min postlabeling. Lane 5 shows an anti-LysRS immunoprecipitate from cells lysed 30 min postlabeling.