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-transcr ibed viral DNA to host DNA. We have probed the organization of functio ns within RSV integrase by constructing mutant derivatives and assayin g 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) Zn2+-binding motif, are greatly im paired in their ability to carry out two reactions characteristic of i ntegrase proteins: specific cleavage of the viral DNA termini and DNA strand transfer. Deletion mutants lacking the carboxyl-terminal 69 ami no acids are also unable to carry out these reactions. However, all de letion mutants that retain the central domain are capable of carrying out disintegration, an in vitro reversal of the normal DNA strand tran sfer reaction, indicating that the catalytic center probably lies with in this central region. Another conserved motif, D-X((39-58))-D-X((35) )-E, is found in this central domain. These findings,vith RSV integras e closely parallel previous findings with human immunodeficiency virus integrase, indicating that a modular catalytic domain is a general fe ature of this family of proteins. Surprisingly, and unlike results obt ained so far with human immunodeficiency virus integrase, efficient st rand transfer activity can be restored to a mutant RSV integrase lacki ng the amino-terminal HHCC domain by fusion to various short peptides. Furthermore, these fusion proteins retain the substrate specificity o f RSV integrase. These data support a model in which the integrase act ivities required for strand transfer in vitro, including substrate rec ognition, multimerization, and catalysis, all lie primarily outside th e aminoterminal HHCC domain.