SEQUENCE COMPARISON AND MUTATIONAL ANALYSIS OF ELEMENTS THAT MAY BE INVOLVED IN THE REGULATION OF DNA-SYNTHESIS IN HIV-1

The large number of sequenced clones of HIV-1 and related viruses made it possible to indicate conserved elements with potential regulatory or structural functions. Such analysis was combined with directed muta genesis in order to investigate the importance of elements that may in fluence the initiation of plus-strand DNA synthesis. The main site for plus-strand initiation is a polypurine tract near the 3' end of the v iral RNA (the 3' PPT). An exact copy of this PPT is located in the mid dle of the genome (the internal PPT). Upstream from the internal PPT t here is an inverted repeat. Mutants designed to upset the internal PPT (i.e., purine to pyrimidine changes), as well as mutants designed to abolish the potential stem-loop formation (changes around the internal PPT or in the upstream inverted repeat) both resulted in viruses with a reduced ability to replicate. Upsetting the stem-loop formation was , however, less harmful than changing the polypurine nature of the PPT . Changing a conserved T on the 3' side of the PPT to a C did not affe ct the phenotype.