Rous sarcoma virus integrase protein: mapping functions for catalysis and substrate binding.
Rous sarcoma virus (RSV), like all retroviruses, encodes an integrase protein that is responsible for covalently joining the reverse-transcribed viral DNA to host DNA. We have probed the organization of functions within RSV integrase by constructing mutant derivatives and assaying their activities in vitro. We find that deletion derivatives lacking the amino-terminal 53 amino acids, which contain the conserved H-X(3-7)-H-X(23-32)-C-X(2)-C (HHCC) Zn(2+)-binding motif, are greatly impaired in their ability to carry out two reactions characteristic of integrase proteins: specific cleavage of the viral DNA termini and DNA strand transfer. Deletion mutants lacking the carboxyl-terminal 69 amino acids are also unable to carry out these reactions. However, all deletion mutants that retain the central domain are capable of carrying out disintegration, an in vitro reversal of the normal DNA strand transfer reaction, indicating that the catalytic center probably lies within this central region. Another conserved motif, D-X(39-58)-D-X(35)-E, is found in this central domain. These findings with RSV integrase closely parallel previous findings with human immunodeficiency virus integrase, indicating that a modular catalytic domain is a general feature of this family of proteins. Surprisingly, and unlike results obtained so far with human immunodeficiency virus integrase, efficient strand transfer activity can be restored to a mutant RSV integrase lacking the amino-terminal HHCC domain by fusion to various short peptides. Furthermore, these fusion proteins retain the substrate specificity of RSV integrase. These data support a model in which the integrase activities required for strand transfer in vitro, including substrate recognition, multimerization, and catalysis, all lie primarily outside the amino-terminal HHCC domain.
Bushman
F D
FD
Infectious Disease Laboratory, Salk Institute, La Jolla, California 92037.
Wang
B
B
D000595:Amino Acid Sequence; D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D006678:HIV / Q000201:enzymology; D019426:Integrases; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D017384:Sequence Deletion; D013379:Substrate Specificity; D016662:Virus Integration
D004279:DNA, Viral; D011994:Recombinant Proteins; D004254:DNA Nucleotidyltransferases; D019426:Integrases