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Ubiquitination of HIV-1 and MuLV Gag

2000; Elsevier BV; Volume: 278; Issue: 1 Linguagem: Inglês

10.1006/viro.2000.0648

1096-0341

David E. Ott, Lori V. Coren, Elena Chertova, Tracy D. Gagliardi, Ulrich S. Schubert,

Viral-associated cancers and disorders

Our previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6Gag and MuLV p12Gag, that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1NL4-3 p6Gag and both lysines in Moloney MuLV p12Gag. HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6Gag, K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6Gag or MuLV p12Gag is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6Gag does not affect the short-term release of virus from the cell, the maturation of Pr55Gag, or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55Gag can be monoubiquitinated, suggesting that p6Gag is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6Gag, showing that the ubiquitin inside the virus is not initially brought in as a p6Gag conjugate. Although our results establish that monoubiquitination of p6Gag and p12Gag is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding.