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

We present a detailed and quantitative analysis of the functional character istics of the 1,000-nucleotide segment at the 5' end of the human immunodef iciency virus type 1 (HIV-1) RNA genome. This segment of the viral genome c ontains several important cis-acting sequences, including the TAR, polyaden ylation, viral att site, minus-strand primer-binding site, and 5' splice do nor sequences, as well as coding sequences for the matrix protein and the N -terminal half of the capsid protein. The genetic footprinting technique wa s used to determine quantitatively the abilities of 134 independent inserti on mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete revers e transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and an alyzed en masse, greatly decreasing the labor typically involved in mutagen esis studies, The results confirmed the presence of several previously know n functional features in this region of the HIV genome and provided evidenc e for several novel features, including nea ly identified cis-acting sequen ces that appeared to contribute to (i) the formation of stable viral transc ripts, (ii) viral RNA packaging, and (iii) an early step in viral replicati on, The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcrip ts. Finally, in contrast to previous reports, the results of this study sug gested that detrimental mutations in the matrix and capsid proteins princip ally interfered with viral assembly.