Effects of varying the spacing within the D,D-35-E motif in the catalytic region of retroviral integrase.
The catalytic domain of all retroviral integrases contains an Asp,Asp-35-Glu (D,D-35-E) motif with precisely 35 amino acids between the second aspartate and the glutamate. We have now made several mutations designed to alter the length or flexibility of a mobile loop within this 35-amino-acid spacer region in full-length Rous sarcoma virus integrase. Surprisingly, most of the mutants had enzymatic activity, including ones that shortened or lengthened the loop by up to 6 amino acids. Several size mutants exhibited the two biologically relevant activities of integrase in reactions with Mn(2+), although they were inactive with Mg(2+). No viruses containing integrase with an altered length, however, replicated in cell culture, and these viruses were blocked at the integration step. Thus, the conserved 35-amino-acid spacing is not absolutely required for enzymatic activity, but the correlation between infectivity and Mg(2+)-dependent activity supports magnesium as the metal cofactor used by integrase in vivo.
Konsavage
Wesley M
WM
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, Hershey, Pennsylvania 17033, USA.
Sudol
Malgorzata
M
Katzman
Michael
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D002645:Chickens; D004279:DNA, Viral / Q000235:genetics; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011784:Quail; D052736:Rous sarcoma virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D019427:HIV Integrase; D019426:Integrases; D008274:Magnesium; C514801:p31 integrase protein, Human immunodeficiency virus 1