Engineered Hyperactive Integrase for Concerted HIV-1 DNA Integration

Abstract

The DNA cutting and joining reactions of HIV-1 integration are catalyzed by integrase (IN), a viral protein that functions as a tetramer bridging the two viral DNA ends (intasome). Two major obstacles for biochemical and structural studies of HIV-1 intasomes are 1) the low efficiency of assembly with oligonucleotide DNA substrates, and 2) the non-specific aggregation of both intasomes and free IN in the reaction mixture. By fusing IN with a small non-specific DNA binding protein, Sulfolobus solfataricus chromosomal protein Sso7d (PDB: 1BNZ), we have engineered a highly soluble and hyperactive IN. Unlike wild-type IN, it efficiently catalyzes intasome assembly and concerted integration with oligonucleotide DNA substrates. The fusion IN protein also functions to integrate viral reverse transcripts during HIV-infection. The hyperactive HIV-1 IN may assist in facilitating future biochemical and structural studies of HIV-1 intasomes. Understanding the mechanistic basis of the Sso7d-IN fusion protein could provide insight into the factors that have hindered biophysical studies of wild-type HIV-1 IN and intasomes.