HIV-1 assembly and budding are promising targets for drug design. Gag, the core viral structural protein, requires the aid of host machinery during assembly and egress. Ubiquitination of Gag is proposed to be involved, but its role remains obscure. Here we study ubiquitin derived from one of four human loci, Uba80. Uba80 encodes two proteins, a mono-ubiquitin and Rps27a. We show that ubiquitin, expressed alone or in the context of full-length Uba80 conjugates to many proteins in the viral particle and that the majority of ubiquitin is neither conjugated to Gag nor free, likely linked to host factors. We demonstrate that the C-terminally encoded ubiquitin “extension protein” Rps27a, a small subunit ribosomal protein, interacts with Gag and is packaged into virus particles. In localization studies, overexpression of Uba80 redistributes Gag to the nucleolus. These data suggest that Gag may interact with both Ubiquitin conjugated proteins and Rps27a during assembly.