| The life cycle of human immunodeficiency virus type 1 (HIV-1), including assembly and reverse transcription, is mediated by protein-protein and protein-nucleic acid interactions. A host cell tRNALys3 plays an essential role as primer of HIV-1 reverse transcription. This host cell tRNA Lys3 molecule must be packaged into newly forming virions for it to function as the reverse transcription primer, and for the virus to be infectious. The HIV-1 proteins Gag and GagPol, as well as the host cell factor lysyl-tRNA synthetase (LysRS), are required for specific packaging of tRNALys3 into virions. LysRS is thought to be the signal that allows for specific packaging of the tRNALys3 primer. Gag alone is sufficient for packaging LysRS into virus-like particles. Thus, it is believed that specific Gag/LysRS interactions are critical for tRNALys3 packaging. In vitro pull-down assays and in vivo LysRS packaging studies mapped the Gag/LysRS interaction to motif 1 of LysRS and to the C-terminal domain (CTD) of the capsid (CA) domain of Gag.; Further in vitro characterization of the interaction between HIV-1 Gag and human LysRS was accomplished using affinity pull-down assays, fluorescence anisotropy measurements, and gel chromatography. An equilibrium binding constant of 310 +/- 80 nM was measured for the Gag/LysRS interaction, and the CA domain of Gag binds to LysRS with a similar affinity. Point mutations in the dimerization domains of LysRS and Gag, along with gel chromatography studies, support the formation of a Gag/LysRS heterodimer. Nuclear magnetic resonance spectroscopy studies reveal that residues in and proximal to helix 4 of CA-CTD are perturbed upon LysRS binding. Alanine scanning mutagenesis studies targeting residues in the helix 4 region, and peptide binding assays, are consistent with a critical role for helix 4 of CA-CTD in mediating interactions with LysRS.; The HIV-1 nucleocapsid protein (NC) plays an essential role in the HIV-1 life cycle. The large scale solid-phase peptide synthesis of full-length (55 amino acids) and truncated forms of NC were carried out. This synthesis yielded ∼60 mg of full-length protein and enabled numerous studies of NC's nucleic acid chaperone function in HIV-1 that would not have otherwise been possible. |
