Avian retrovirus DNA internal attachment site requirements for full-site integration in vitro.
Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the cis-acting attachment (att) sites at the viral termini and the viral integrase (IN) in trans. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal att DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal att site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in trans. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in trans between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal att sequences to mediate full-site integration in vitro is similar to the internal att site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.
Chiu
R
R
Institute for Molecular Virology, St. Louis University Health Sciences Center, St. Louis, Missouri 63110, USA.
Grandgenett
D P
DP
D009189:Avian Myeloblastosis Virus / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003101:Collodion; D017124:Conserved Sequence; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D066298:In Vitro Techniques; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D020079:Terminal Repeat Sequences; D016662:Virus Integration
D004279:DNA, Viral; D011991:Receptors, Virus; D003101:Collodion; D019426:Integrases