HIV-1 genomic RNA contains a centrally located PPT (cPPT) within the pol gene that, like 3'PPT, is spared by RNase H during minus-strand DNA synthesis and persists to prime plus-strand DNA synthesis. This ribonucleotide primes the synthesis of a plus-strand DNA extending through the U3 and R regions of the HIV sequence and terminating in the PBS region (the tRNA primer-binding site). This DNA segment is known as plus-strand strong-stop DNA (+sssDNA) (Telesnitsky and Goff 1997; Pullen et al. 1993; Huber and Richardson 1990). cPPT priming is important for efficient viral replication (Alizon et al. 1992; Rausch and Le Grice 2004). Several features of cPPT priming in vivo remain to be clarified.