The fourth central polypurine tract guides the synthesis of prototype foamy virus plus-strand DNA.

Foamy virus (FV) is a nonpathogenic retrovirus that has the potential to serve as a gene therapy vector. In retroviral replication, the central polypurine tract (cPPT) is used as a primer to synthesize plus-strand DNA. The cPPT is subsequently degraded to produce a single-stranded gap in the double-stranded viral DNA molecule. In the prototype foamy virus (PFV), four cPPT-like motifs have been previously identified, in which there is a gap with uncertain terminals. In this study, we determined the length of the PFV gap varying from 144 to 731 bp. The 3' terminus of the cleavage sites is located between 6272 bp and 6274 bp from the first base of PFV genome, while the 5' terminus is located within a 465 bp range. The start and terminal nucleotides of the gap are located on either side of the fourth cPPT element. Deletion, mutation, and replacement of the fourth cPPT with the Human immunodeficiency virus 1 (HIV-1) cPPT resulted in a significant reduction in modified PFV virions, indicating that the fourth cPPT ought to be the primer that guides the synthesis of PFV plus-strand DNA. These results improve the theoretical basis for understanding FVs replication and will help construct new FV vectors with simple genome sequences containing only the necessary cis elements.