Directed DNA Shuffling of Retrovirus and Retrotransposon Integrase Protein Domains

Abstract

Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.

GCN2 phosphorylates HIV-1 integrase and decreases HIV-1 replication by limiting viral integration

“…To better understand the role of the IN CCD, a chimeric protein (HPH) was used that harbors the HIV-1 IN NTD (residues 1–56) fused to the PFV IN CCD (residues 116–278 corresponding to HIV-1 IN residues 57–209) 19 and the HIV-1 IN CTD (residues 221–288). In this context, GCN2 was able to phosphorylate the chimeric protein (Fig.…”

“…Vectors for the bacterial expression of eIF2α and exotic INs (Ty3 and chimers) were kind gifts from Dr. Sandmeyer. The Ty3 retrotransposon IN and the chimeric protein HPH (containing a CCD corresponding to PFV IN surrounded by the NTD and CTD of HIV-1 IN) were purified according to previous publication 19 . Cellular eIF2α was expressed and purified as previously reported 20 .…”

Retrotransposon replication in plants

“…Mechanistic studies on retrotransposon INs have advanced farthest with those of yeast Ty1 and Ty3, in keeping with the general advanced state of these systems [79]. Unravelling the target specificity of Ty3 IN for integration at Pol III transcription initiation sites has been of particular interest [80 ] as has been, conversely, the question of substrate specificity for Ty1 IN [81].…”

Among retroviral integrases prototype foamy virus integrase displays unique biochemical activities

“…Studies of PFV intasome activity are routinely performed in the presence of NaCl and MgCl2 or MgSO4 [25,28,30,[33][34][35][36][37][38][39][40]. PFV IN strand transfer showed no difference in the presence of MgSO4 or MgCl2 indicating no significant effects of sulfite or chloride co-ions (Figure 2) [25].…”