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 (E. 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: CA₁₄₆ (intact domain), CA₁₅₁ (truncated domain), and CA₁₄₆-M185A (M185A, CA dimer interface mutant). After confirming that LysU and CA₁₄₆ are dimeric whilst CA₁₅₁ and M185A remain monomeric, we use glutathione S-transferase (GST) pull-down 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 CA₁₄₆ or >500 μM for interactions between LysU and CA₁₅₁ or LysU and M185A. The reason for these binding affinity differences may be that interactions between LysU and CA₁₄₆ take place through dimer–dimer interactions resulting in a α₂β₂ heterotetramer. LysU/CA-C protein interactions are weaker than those reported between hLysRS and the Gag, CA or CA₁₄₆ 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.