Simian immunodeficiency virus in which nef and U3 sequences do not overlapreplicates efficiently in vitro and in vivo in rhesus macaques

The nef genes of human immunodeficiency virus and simian immunodeficiency v irus (SIV) overlap about 80% of the U3 region of the 3' long terminal repea t (LTR) and contain several essential cis-acting elements (here referred to as the TPI region): a T-rich region, the polypurine tract, and attachment (att) sequences required for integration. We inactivated the TPI region in the nef reading frame of the pathogenic SIVmac239 clone (239wt) by 13 silen t point mutations. To restore viral infectivity, intact cis-regulatory elem ents were inserted just downstream of the mutated nef gene. The resulting S IV genome contains U3 regions that are 384 by shorter than the 517-by 239wt U3 region. Overall, elimination of the duplicated Nef coding sequences tru ncates the proviral genome by 350 bp. Nonetheless, it contains all known co ding sequences and cis-acting elements. The TPI mutant virus expressed func tional Nef and replicated like 239wt in all cell culture assays and in vivo in rhesus macaques. Notably, these SIVmac constructs allow us to study Nef function in the context of replication-competent viruses without the restr ictions of overlapping LTR sequences and important cis-acting elements. The genomes of all known primate lentiviruses contain a large overlap between nef and the U3 region. We demonstrate that this conserved genomic organizat ion is not obligatory for efficient viral replication and pathogenicity.