FIG. 1.

Effect of expression of wild-type or N-terminal-truncated LysRS upon the cytoplasmic and viral concentrations of LysRS. HIV-1 was produced and isolated from HIV-1-transfected COS7 cells as previously described (6). Sucrose gradient-purified virions and cells were lysed by suspension in 1× radioimmunoprecipitation assay buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 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 (1). The cellular and viral lysates were resolved by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, which was followed by blotting onto nitrocellulose membranes (Gelman Sciences) and detection by antibody. Detection of protein on the Western blot utilized monoclonal antibodies or antisera that were specifically reactive with viral capsid (mouse antibody; Intracel), β-actin (mouse antibody; Sigma Aldrich), and human LysRS (rabbit antibody [26]). 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, and the results were quantitated using the UN-SCAN-IT gel automated digitizing system. (A through C) Cytoplasmic concentration of LysRS. Western blot analysis of COS7 cell lysates, probed with either anti-LysRS (panel A) or anti-β-actin (panel B). Panel C shows the LysRS/β-actin ratio as determined by quantitative analysis of the bands in panels A and B. Lane K, purified His₆-tagged human LysRS, containing the appended N-terminal MRGSHHHHHHSSGWVD sequence (6). The other lanes represent COS7 cells transfected with the following plasmids: lane 1, BH10.P-; lane 2, BH10.P- and LysRS.F; and lane 3, BH10.P- and Δ1-65 LysRS. BH10.P- is a simian virus 40-based vector that contains full-length wild-type HIV-1 proviral DNA containing an inactive viral protease (20, 23). LysRS.F contains cDNA encoding full-length (1 to 597 amino acids) human LysRS cloned into pcDNA3.1 (Invitrogen) and was constructed through PCR amplification of the cDNA as previously described (14). To produce an N-terminal-truncated Δ1-65 LysRS encoding amino acids 66 to 597, the sense primer was complementary to a sequence downstream of the sequence encoding the N-terminal amino acids. (D through F) Viral concentrations of LysRS. Western blot analysis of viral lysates probed with anti-LysRS (panel D) or anti-CA (panel E). Panel F shows the LysRS/Gag ratio determined from the data in panels D and E. Lanes 1 to 3 represent viruses produced from COS7 cells transfected with the following plasmids: lane 1, BH10.P-; lane 2, BH10.P- and LysRS.F; and lane 3, BH10.P- and Δ1-65 LysRS. Lane K, purified His₆-tagged human LysRS. To purify this protein, the full-length LysRS PCR fragment was cloned into the bacterial expression vector pET-21b(+) (Clontech), which expresses the protein with a C-terminal His₆ tag. The protein was overexpressed in E. coli and purified as previously described (26). The bar graphs in panels C and F represent the means of results of experiments performed at least three times, and the error bars represent standard deviations.