Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting

We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5 * end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis-acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5 * splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quan-titatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were con-structed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies. The results confirmed the presence of several previously known functional features in this region of the HIV genome and pro-vided evidence for several novel features, including newly identified cis-acting sequences that appeared to con-tribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication. The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly