Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.Upon entering cells, retroviruses convert their single-stranded RNA genomes to double-stranded DNA. As minus-strand DNA is synthesized, the RNase H activity of reverse transcriptase (RT) degrades the RNA in the RNA/DNA duplex. However, one portion of the RNA, the polypurine tract (PPT), is resistant to this RNase H degradation. The PPT RNA is subsequently used as the primer for plus-strand synthesis (2).The region of the retroviral genome required for plus-strand priming was initially characterized by Sorge and Hughes, who noted that more than 9 but not more than 29 bases upstream of the primer cleavage site are required for avian sarcoma virus replication (30). In a previous study, we established that sequences as far upstream as Ϫ28 (where Ϫ1 refers to the base immediately upstream of the cleavage site) are required for Moloney murine leukemia virus (Mo-MLV) plus-strand priming and that a T-rich stretch in this region is critical (24). Noad et al. have similarly established that T-rich sequences upstream of the PPT are required for plus-strand priming for the pararetrovirus cauliflower mosaic virus (16). Additionally, Ilyinskii et al. have demonstrated that the T stretch upstream of the simian immunodeficiency virus (SIV) PPT is required for SIV replication (11), and a T stretch upstream of the Ty1 PPT is important for plus-strand priming and transposition of that yeast retroelement (33).Several reports have examined roles of sequences within the PPT in plus-strand priming: most using model templates in purified reactions. Rattray and Champoux demonstrated that when PPTs with mutations at position Ϫ1, Ϫ2, Ϫ4, or Ϫ7 were tested, additional cleavage sites appeared, suggesting that the integrity of these positions is necessary for Mo-...