Cytosolic N-myristoyl Gag polyprotein is conjugated with a single molecule of ubiquitin. Conjugation is typically to one of two lysine residues in the p6 domain of Gag but can be to lysine residues in the MA, CA, NC, and SP2 domains of the protein. The specific host cell E2 and E3 proteins that mediate Gag ubiquitination have not been identified. The same studies that first identified the p6 ubiquitination sites in Gag also called the biological significance of Gag ubiquitination into question by demonstrating that Gag proteins in which the p6 ubiquitination sites had been removed by mutagenesis could still assemble efficiently into infectious viral particles (Ott et al. 1998, 2000). Later work, however, identified additional ubiquitination sites throughout the C-terminal region of the Gag polyprotein, and when all of these sites are removed by mutagenesis, both viral assembly involving the mutant Gag polyprotein and infectivity of the resulting viral particles are sharply reduced (Gottwein et al. 2006).