The role of Pr55(gag) in the annealing of tRNA3Lys to human immunodeficiency virus type 1 genomic RNA

Abstract

During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55(gag) or Pr160(gag-pol). In vivo placement of tRNA3Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55(gag) or mutant Pr160(gag-pol), and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55(gag) inhibit tRNA3Lys placement. The NC mutations in Pr55(gag) reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3Lys even in the presence of wild-type Pr55(gag). This dominant phenotype may indicate that the mutant Pr55(gag) is disrupting an ordered Pr55(gag) structure responsible for the annealing of tRNA3Lys to genomic RNA.

FIG. 1

Inhibition of genomic placement of tRNA₃^Lys by NC mutants in Pr55^gag or Pr160^gag-pol. Placement was measured by the ability of tRNA₃^Lys to be extended six bases in an in vitro reverse transcription reaction with HIV-1 RT and total viral RNA as the source of primer tRNA-template. The transfection of COS7 cells, the isolation of HIV-1 and total viral RNA, and the conditions of reverse transcription were as previously described (5). The sequence of the first six deoxynucleoside triphosphates incorporated is CTGCTA. In the presence of dCTP, dTTP, [α-³²P]dGTP, and ddATP instead of dATP, extension will therefore terminate after six bases. After incubation, the reaction products were precipitated with 2-propanol and resuspended in loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) and the samples were electrophoresed for 16 h at 4°C, with 11% polyacrylamide–7 M urea. COS cells were either transfected with wt HIV proviral DNA or cotransfected with the following plasmids. Wild type is the HIV-1 BH10 proviral clone; BH10.FS is frameshift positive, i.e., it produces Pr160^gag-pol, but not Pr55^gag (2); SVC21.Dr2 is the HIV-1 proviral DNA containing a 2-amino-acid insertion in the connection domain and results in the lack of Pr160^gag-pol incorporation into the virus (12); wt-Gag and wt-Gag-Pol contain wt sequences of Pr55^gag and Pr160^gag-pol, respectively, inserted into pBABY vectors (14) and were donated by D. Rekosh, University of Virginia. They contain the RNA recognition element but lack most of the HIV-1 sequences upstream and downstream of the sequences coding for Gag and Gag-Pol, and the mutant genomic RNA is not packaged. S3-Gag, S3-Gag-Pol, R7-Gag, and R7-Gag-Pol represent the wt-Gag and wt-Gag-Pol sequences which have been modified with the S3(S32-34) and R7R10K11 S NC mutations, respectively. Genomic placement of tRNA₃^Lys was measured in the wt and mutant virus as described in the text. Lanes 5 to 12 in panel A and lanes 5 to 9 in panel B contain the same amount of genomic RNA found in lane 4 of both panels, i.e., 5 × 10⁸ copies. Lanes 1 to 3 in both panels represent fractional amounts of this genomic RNA copy number. Lanes 11 and 8 in panels A and B, respectively, represent the use in the RT reaction of purified human placental tRNA₃^Lys heat annealed to synthetic genomic RNA, as previously described (7), and used as a size marker. Lanes 12 and 9 in panels A and B, respectively, represent virus whose genome lacks a PBS.

FIG. 2

Cytoplasmic expression of Gag and Gag-Pol precursor proteins. Shown is Western blot analysis of wt and mutant viral proteins. COS7 cells were transfected, or cotransfected, with the following plasmids: BH10 (wt HIV-1) (lane 1), R7-Gag and R7-Gag-Pol (lane 2), and S3-Gag and S3-Gag-Pol (lane 3). At 63 h posttransfection, cells were washed twice with ice-cold phosphate-buffered saline, scraped off the tissue culture dishes, and lysed with radioimmunoprecipitation assay buffer (10 mM Tris [pH 7.4], 100 mM NaCl, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 2 mg of aprotinin per ml, 2 mg of leupeptin per ml, 1 mg of pepstatin A per ml, 100 mg of phenylmethylsulfonyl fluoride per ml). Aliquots of total cell lysates were resolved by 10% (wt/vol) sodium dodecyl sulfate–one-dimensional polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. Protein detection utilized monoclonal antibodies reactive to HIV-1 p24 and was performed by enhanced chemiluminescence with a goat anti-mouse cross conjugated to horseradish peroxidase as a secondary antibody.