Molecular and genetic determinants of rous sarcoma virus integrase for concerted DNA integration.
Site-directed mutagenesis of recombinant Rous sarcoma virus (RSV) integrase (IN) allowed us to gain insights into the protein-protein and protein-DNA interactions involved in reconstituted IN-viral DNA complexes capable of efficient concerted DNA integration (termed full-site). At 4 nM IN, wild-type (wt) RSV IN incorporates approximately 30% of the input donor into full-site integration products after 10 min of incubation at 37 degrees C, which is equivalent to isolated retrovirus preintegration complexes for full-site integration activity. DNase I protection analysis demonstrated that wt IN was able to protect the viral DNA ends, mapping approximately 20 bp from the end. We had previously mapped the replication capabilities of several RSV IN mutants (A48P and P115S) which appeared to affect viral DNA integration in vivo. Surprisingly, recombinant RSV A48P IN retained wt IN properties even though the virus carrying this mutation had significantly reduced integrated viral DNA in comparison to wt viral DNA in virus-infected cells. Recombinant RSV P115S IN also displayed all of the properties of wt RSV IN. Upon heating of dimeric P115S IN in solution at 57 degrees C, it became apparent that the mutation in the catalytic core of RSV IN exhibited the same thermolabile properties for 3' OH processing and strand transfer (half-site and full-site integration) activities consistent with the observed temperature-sensitive defect for integration in vivo. The average half-life for inactivation of the three activities were similar, ranging from 1.6 to 1.9 min independent of the IN concentrations in the assay mixtures. Wt IN was stable under the same heat treatment. DNase I protection analysis of several conservative and nonconservative substitutions at W233 (a highly conserved residue of the retrovirus C-terminal domain) suggests that this region is involved in protein-DNA interactions at the viral DNA attachment site. Our data suggest that the use of recombinant RSV IN to investigate efficient full-site integration in vitro with reference to integration in vivo is promising.
St. Louis University Health Sciences Center, Institute for Molecular Virology, Missouri 63110, USA.
Roger
R
Chiu
Duane P
DP
Grandgenett
D000595:Amino Acid Sequence; D001287:Attachment Sites, Microbiological; D001358:Avian Sarcoma Viruses / Q000201:enzymology* / Q000502:physiology; D018983:DNA Footprinting; D004279:DNA, Viral / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000378:metabolism; D019281:Dimerization; D006358:Hot Temperature; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D016662:Virus Integration; D014779:Virus Replication
D004279:DNA, Viral; D019426:Integrases; D003851:Deoxyribonucleases