The factors that determine the path taken by a given RNA molecule are not known. N terminus to LexA. HIV-1 MA coding sequences were amplified by PCR from the proviral clone NL4-3 (1) by using primers 1 and 2. The product of the PCR was digested with restriction enzymes mutations were introduced into pBS-GagX. In this plasmid, the sequence (nucleotides 789 to 2292, according to reference 62) is flanked by an sequence in pBS-GagX Sirt4 contains multiple conservative mutations that act at the RNA level to render expression Rev independent (70). Mutations in MA (AAA, dB5, or MA) or NC (M1-2/BR) coding sequences were introduced into the sequence by replacing an expression plasmids. Rev-independent coding sequences containing the MA mutation (AAA), Verubulin hydrochloride NC mutation (M1-2/BR), or a combination of both mutations (AAA M1-2/BR) were released from the specific pBS-GagX plasmids described above by digestion with coding sequences is driven by an SR promoter (77). Construction of EF1 bacterial expression plasmids. All of the EF1-encoding fragments were cloned into the vector pSE420 (Invitrogen) for bacterial expression. To produce an N-terminal hemagglutinin (HA)-tagged version of EF1, a JG4-5 two-hybrid clone containing the full-length EF1 cDNA plus 30 nucleotides of the 5 untranslated region and around 500 nucleotides of the 3 untranslated region (for a total of 2 kb) was digested with BL21LysS (Novagen) was used for bacterial protein expression. Bacterial lysates were prepared essentially as described before (53), with binding buffer used as resuspension buffer. The lysate was brought to a final glycerol concentration of 20% and frozen in liquid nitrogen. GST fusion proteins to be used in the in vitro translation assay were purified from the bacterial lysate on glutathione affinity columns, eluted with 5 mM glutathione, in 50 mM Tris-Cl (pH 8.0), and frozen in liquid nitrogen with a final glycerol concentration of 20%. The purity and quantity of the proteins were checked by Coomassie gel staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Verubulin hydrochloride by Bradford assay (Bio-Rad). In vitro binding experiments. GST fusion proteins were immobilized onto glutathione-agarose beads Verubulin hydrochloride (Sigma) in binding buffer (20 l, 50% slurry) for 30 min and washed three times in binding buffer. Beads were then incubated with crude lysates expressing native or recombinant EF1 for 1 h at 4C in a final volume of 200 l and then washed three times in binding buffer. Bound proteins were boiled and subjected to SDS-PAGE and Western blotting. In some in vitro binding experiments, the effects of RNase A on the EF1CHIV-1 Gag interaction was examined. For these experiments, lysates expressing the two proteins were diluted in buffer containing 50 mM Tris-Cl (pH 8.0), 1 mM EDTA, and RNase A (Sigma), ranging from 0 to 50 g per 10 mg of total bacterial lysate. The lysates were incubated for 1 h at 37C and then mixed together and assayed as described above. Antibodies and Western blot analysis. Murine monoclonal anti-EF1 antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, N.Y.). Murine monoclonal anti-HIV-1 CA and rabbit polyclonal anti-HIV-1 gp120 antibodies were purchased from Intracel (Cambridge, Mass.). Goat polyclonal anti-M-MuLV CA antibody was a gift from Stephen Goff (Columbia University, New York, N.Y.). Murine monoclonal anti-HA antibody was purchased from Berkeley Antibody Company (Berkeley, Calif.). Western blot analysis was performed essentially as described previously (53). Immunoprecipitation. Human 293T fibroblasts were maintained in complete Dulbeccos modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum. Rev-independent Gag polyproteins were expressed by calcium phosphate transfection (19) of 293T cells by using the SR expression plasmids described above. Cells were lysed 72 h posttransfection in binding buffer, and the lysate was cleared by centrifugation at 14,000 rpm in an Eppendorf microcentrifuge for 20 min at 4C. The lysate was preincubated with 100 l of protein A-Sepharose beads (Sigma; 10% slurry) for 1 h at 4C. Supernatant was removed from the beads and incubated with rabbit polyclonal anti-HIV-1 CA antibody (obtained from Louis Henderson, Frederick Cancer Research and Development Center, Frederick, Mass.) for 2 h at 4C. Protein A-Sepharose beads (100 l) were then added for 1.