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Journal of Virology, March 2000, p. 2760-2769, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Functional Characterization of the Human Immunodeficiency Virus Type 1 Genome by Genetic Footprinting

Louise Chang Laurent,1,2 Mari N. Olsen,1 Rachel Adams Crowley,1 Harri Savilahti,3 and Patrick O. Brown1,*

Howard Hughes Medical Institute, Stanford University Medical Center, Palo Alto, California 943051; Department of Biochemistry, University of California at San Francisco, San Francisco, California 941432; and Institute of Biotechnology, Viikki Biocenter, University of Helsinki, FIN-00014 Helsinki, Finland3

Received 6 August 1999/Accepted 8 December 1999

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 quantitatively 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 constructed 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 provided evidence for several novel features, including newly identified cis-acting sequences that appeared to contribute 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.

* Corresponding author. Mailing address: Howard Hughes Medical Institute, Stanford University Medical Center, Beckman Center B251, Palo Alto, CA 94305. Phone: (650) 725-7567. Fax: (650) 723-1399. E-mail: pbrown{at}cmgm.stanford.edu.

Journal of Virology, March 2000, p. 2760-2769, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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