Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Growing evidence suggests HIV protease functions beyond cleaving viral polyproteins for viral assembly. Though many substrates have been proposed it remains an open question which host proteins are relevant, endogenous substrates of HIV protease. We have established unbiased, comprehensive proteomic and structural pipelines tailored to identify proteolytic targets and have uncovered novel HIV PR host substrates with known roles in nucleic acid regulation, including eIF3d, a subunit of eukaryotic translation initiation factor 3 (1). This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection. We have initiated a study of the structural, biochemical and molecular mechanisms of these new types of PR-substrate interactions. This resulted in the creation of a “host substrate envelope” with the active site of HIV protease that differs from the viral substrate envelope defined to date. Altogether this work implicates host nucleic acid binding complex proteolysis in productive HIV infection. Uncovering a novel set of proteolytic modifications at the host-virus interface will lead to promising therapeutic host-viral targets for future studies that are less prone to resistance through viral mutation.