Characterization of a replication-defective human immunodeficiency virus type 1 att site mutant that is blocked after the 3' processing step of retroviral integration.
Two activities of retroviral integrase, 3' processing and DNA strand transfer, are required to integrate viral cDNA into a host cell chromosome. Integrase activity has been analyzed in vitro using purified protein and recombinant DNA substrates that model the U3 and U5 ends of viral cDNA or by using viral preintegration complexes (PICs) that form during virus infection. Numerous studies have investigated changes in integrase or viral DNA for effects on both 3' processing and DNA strand transfer activities using purified protein, but similar analyses have not been carried out using PICs. Here, we analyzed PICs from human immunodeficiency virus type 1 (HIV-1) strain 604del, an integration-defective mutant lacking 26 bp of U5, and revE1, a revertant of 604del containing an additional 19-bp deletion, for levels of 3' processing activity that occurred in infected cells and for levels of in vitro DNA strand transfer activity. Whereas revE1 supported one-third to one-half of the level of wild-type DNA strand transfer activity, the level of 604del DNA strand transfer activity was undetectable. Surprisingly, integrase similarly processed the 3' ends of 604del and revE1 in vivo. We therefore conclude that 604del is blocked in its ability to replicate in cells after the 3' processing step of retroviral integration. Whereas Western blotting showed that wild-type, revE1, and 604del PICs contained similar levels of integrase protein, Mu-mediated PCR footprinting revealed only minimal protein-DNA complex formation at the ends of 604del cDNA. We propose that 604del is replication defective because proteins important for DNA strand transfer activity do not stably associate with this cDNA after in vivo 3' processing by integrase.
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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Chen
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Engelman
D015153:Blotting, Western; D002457:Cell Extracts; D002460:Cell Line; D018983:DNA Footprinting; D004279:DNA, Viral / Q000032:analysis; D015694:Gene Products, rev / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D019426:Integrases / Q000378:metabolism; D009154:Mutation; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000235:genetics*; D017384:Sequence Deletion; D016662:Virus Integration / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication; D054321:rev Gene Products, Human Immunodeficiency Virus
D002457:Cell Extracts; D004279:DNA, Viral; D015694:Gene Products, rev; D011991:Receptors, Virus; D054321:rev Gene Products, Human Immunodeficiency Virus; D019426:Integrases