MUTATIONAL ANALYSIS OF CIS-ACTING PACKAGING SIGNALS IN HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA

We previously identified blocks of sequence near the 5' end of the hum an immunodeficiency virus (HIV-1) genome which conferred on RNA the ab ility to bind specifically to the HIV-1 Gag polyprotein, Pr55(gag) (J. Luban and S. P. Goff, J. Virol. 65:3203-3212, 1991; R. Berkowitz, J. Luhan, and S. P, Goff, J. Virol. 67:7190-7200, 1993). sere ne report t he use of an RNase protection assay to quantify the effect of deletion of these sequences on RNA packaging into virions. First, we demonstra ted with wild-type HIV-1 sequences that in comparison with spliced vir al RNA, full-length viral genomic RNA is enriched 20-fold in virions. A previously described mutation with deletion of sequences between the major splice donor and the first codon of gag (A. Lever, H. Gottlinge r, W. Haseltine, and J. Sodroski, J. Virol. 63:4085-4087, 1989) disrup ted these ratios such that different HIV-1 RNA forms were packaged in direct proportion to cytoplasmic concentrations. The effect of deletio n mutations preceding and within gag coding sequence on packaging was then tested in competition with RNAs containing wild-type packaging se quences, Using this system we were able to demonstrate significant eff ects on packaging of RNAs with mutations immediately preceding the fir st codon of gag. The greatest reduction in packaging was seen with RNA s lacking the first 40 nucleotides of gag coding sequence, although se quences more 3' had slight additional effects.