Abstract: The human immunodeficiency virus (HIV) is a retrovirus that is the causative agent of the acquired immunodeficiency syndrome (AIDS), a disease which compromises the immune system. While current therapies inhibit several processes of viral replication, the development of drug resistance remains a risk for the HIV-positive population. Membrane targeting, a process which offers potential as a new therapeutic target, involves the trafficking of Gag polyprotein (Gag) to the plasma membrane via interaction between Gag’s matrix domain (MA) and phosphatidylinositol- 4,5-bisphosphate (PI(4,5)P2). Recent studies have shown that cellular tRNAs interact with MA’s basic patch prior to assembly, preventing interaction with PI(4,5)P2, but the underlying mechanism for the shift from MA-tRNA association to MA-PI(4,5)P2 remains elusive. In order to better understand this mechanism, this work aims to characterize the structure and interactions of the MA-tRNA complex using nuclear magnetic resonance (NMR) spectroscopy, a method of structure determination that requires conditions in which the complex is stable. To identify these conditions, 1D NMR titrations were performed to determine conditions for proper folding of the tRNA, and electric mobility shift assays (EMSAs) were employed to determine the stoichiometric ratio of MA to tRNA necessary for complex formation. Ultimately, better understanding these aspects of HIV biology may aid in the design of novel therapies.