Influence of the amino-terminal sequence on the structure and function of HIV integrase

Abstract

Background: Antiretroviral therapy (ART) can mitigate the morbidity and mortality caused by the human immunodeficiency virus (HIV). Successful development of ART can be accelerated by accurate structural and biochemical data on targets and their responses to inhibitors. One important ART target, HIV integrase (IN), has historically been studied in vitro in a modified form adapted to bacterial overexpression, with a methionine or a longer fusion protein sequence at the N-terminus. In contrast, IN present in viral particles is produced by proteolytic cleavage of the Pol polyprotein, which leaves a phenylalanine at the N-terminus (IN 1F). Inspection of available structures suggested that added residues on the N-terminus might disrupt proper protein folding and formation of multimeric complexes. Results: We purified HIV-1 IN 1F 1-212 and solved its structure at 2.4 Å resolution, which showed extension of an N-terminal helix compared to the published structure of IN 1-212. Full-length IN 1F showed increased in vitro catalytic activity in assays of coupled joining of the two viral DNA ends compared to two IN variants containing additional N-terminal residues. IN 1F was also altered in its sensitivity to inhibitors, showing decreased sensitivity to the strandtransfer inhibitor raltegravir and increased sensitivity to allosteric integrase inhibitors. In solution, IN 1F exists as monomers and dimers, in contrast to other IN preparations which exist as higher-order oligomers. Conclusions: The structural, biochemical, and biophysical characterization of IN 1F reveals the conformation of the native HIV-1 IN N-terminus and accompanying unique biochemical and biophysical properties. IN 1F thus represents an improved reagent for use in integration reactions in vitro and the development of antiretroviral agents.

Structure of a HIV-1 IN-Allosteric inhibitor complex at 2.93 Å resolution: Routes to inhibitor optimization

“…IN (NL4-3) constructs were expressed and purified as described previously [10,12,101,102]. Individual IN domains (CCD F185K [11], CTD(220-271), CTD(220-270) L242A [52], and CTD (220-288)) were inserted into an expression vector containing an N-terminal His7-Flag-SUMO tag [103]. Proteins were purified with nickel-nitriloacetic acid resin and subjected to a second nickel-nitriloacetic column after fusion proteins were liberated by cleavage with Ulp1 protease (Thermofisher Scientific).…”

Structure of a HIV-1 IN-Allosteric Inhibitor Complex at 2.93 Å Resolution: Routes to Inhibitor Optimization

“…IN (NL4-3) constructs were expressed and purif ied as described previously (10,12,57,58). Individual IN domains (CCD and CTD) were inserted into an expression vector containing an N-terminal His7-Flag-SUMO tag (59). Proteins were purif ied with nickel-nitriloacetic acid resin and subjected to a second nickel-nitriloacetic column af ter f usion proteins were liberated by cleavage with Ulp1 protease (Thermof isher Scientif ic).…”

HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro

“…We therefore compared the biophysical properties and activity of HIV-1 intasomes assembled with Sso7d-IN and wild-type IN and devised a strategy to obtain cryo-EM structures of wild-type HIV-1 intasomes. Since it has been reported that the amino-terminal sequence of HIV-1 integrase can influence the oligomerization and function of IN, 11 we also modified the expression construct so that the protein has the native phenylalanine at the N-terminus, rather the additional four amino acids (GSHM) resulting from cleavage of the His-tag with thrombin.…”