Phosphorylation and proteolytic cleavage of Gag proteins in budded simian immunodeficiency virus

The lentiviral Gag polyprotein (Pr55^Gag) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55^Gag is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55^Gag and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55^Gag, several CA-containing cleavage intermediates of Pr55^Gag, and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55^Gag can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55^Gag and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55^Gag and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.