Ubiquitination of HIV-1 and MuLV Gag

Our previous biochemical studies of HIV-1 and MuLV virions isolated and ide ntified mature Gag products, HIV-1 p6(Gag) and MuLV p12(Gag), that were con jugated to a single ubiquitin. To study the importance of the monoubiquitin ation of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1(NL4-3) p6( Gag) and both lysines in Moloney MuLV p12(Gag). HPLC and immunoblot analysi s of the HIV-1 mutants demonstrated that either of the lysines in p6(Gag), K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6(Gag) or MuLV p12(Gag) is not required f or viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6(Gag) does not affect the shor t-term release of Virus from the cell, the maturation or Pr55(Gag), Or the sensitivity of these processes to proteasome inhibitors. Experiments with p rotease-deficient HIV-1 showed that Pr55(Gag) can be monoubiquitinated, sug gesting that p6(Gag) is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorp orated into the virions in the absence of the lysines in p6(Gag), showing t hat the ubiquitin inside the virus is not initially brought in as a p6(Gag) conjugate. Although our results establish that monoubiquitination of p6(Ga g) and p12(Gag) is not required for Viral replication in vitro, this modifi cation may be a by-product of interactions between Gag and cellular protein s during assembly and budding, (C) 2000 Academic Press.