Escherichia coli LysU is a potential surrogate for human lysyl tRNA synthetase in interactions with the C-terminal domain of HIV-1 capsid protein

摘要

Human lysyl-tRNA synthetase (hLysRS) is known to interact directly with human immunodeficiency virus type-1 (HIV-1) GagPol polyproteins, and both hLysRS with tRNA~(Lys3) are selectively packaged into emerging HIV-1 viral particles. This packaging process appears to be mediated by contact between the motif 1 helix h7 of hLysRS and the C-terminal dimerization domain of the HIV-1 capsid protein (CA) segment of Gag or GagPol. Given similarities between hLysRS and Escherichia coli (￡. coli) heat shock protein LysU, we investigate if LysU might be an hLysRS surrogate for interactions with Gag or GagPol proteins. We report on a series of studies involving three CA C-domains: CA_(146) (intact domain), CA_(151) (truncated domain), and CA_(146)-M185A (M185A, CA dimer interface mutant). After confirming that LysU and CA_(146) are dimeric whilst CA_(151) and M185A remain monomeric, we use glutathione S-transferase (GST) pulldown assays to demonstrate the existence of specific interactions between LysU and all three CA-C domains. By means of 1H-NMR titration experiments, we estimate K_d values of 50 μM for the interaction between LysU and CA_(146)or >500μM for interactions between LysU and CA_(151) or LysU and M185A. The reason for these binding affinity differences may be that interactions between LysU and CA_(146) take place through dimer-dimer interactions resulting in a α_2β_2 heterotetramer. LysU/CA-C protein interactions are weaker than those reported between hLysRS and the Gag, CA or CA_(146) proteins, and hLysRS/Gag binding interactions have also been suggested to involve only αβ heterodimer formation. Nevertheless, we propose that LysU could act as a surrogate for hLysRS with respect to Gag and GagPol polyprotein interactions although arguably not sufficiently for LysU to act as an inhibitor of the HIV-1 life cycle without further adaptation or mutation. Potentially, LysU and/or LysU mutants could represent a new class of anti-HIV-1 therapeutic agent.