Ott DE, Coren LV, Chertova EN, Gagliardi TD, Schubert U; HIV DRP Symposium Understanding Antiviral Drug Resistance.

Program Abstr HIV DRP Symp Underst Antivir Drug Resist 1st 2000 Chantilly Va. 2000 Dec 3-6; 1: Abstract no. 18.

AIDS Vaccine Program, SAIC Frederick, National Cancer Institute at Frederick, Frederick, MD;

Our previous studies of HIV-1 and MuLV found that mature Gag products, HIV-1 p6(Gag) and MuLV p12(Gag), were conjugated to a single ubiquitin molecule. Additional experiments have shown that blocking 26S proteasome function with inhibitors sharply reduces HIV-1 Gag processing and budding (presented at this meeting in the two Schubert et al. abstracts). To study the importance of the ubiquitination of p6Gag and p12(Gag), a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1NL4- 3 p6(Gag) and both lysines in Moloney MuLV p12(Gag). HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6Gag, K27 or K33, could be mono-ubiquitinated. However, infectivity assays showed that ubiquitination of HIV-1 p6(Gag) or MuLV p12(Gag) is not required for viral replication in vitro. Pulse-chase radio-labeling of HIV-1-producing cells revealed that ubiquitination of p6(Gag) does not affect short-term virus release from the cell, Pr55(Gag) maturation, or the sensitivity of these processes to proteasome inhibitors. Therefore, the proteasome inhibitor effect is not mediated by the lysines within p6(Gag). Experiments with protease-deficient HIV-1 showed that Pr55(Gag) 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 that free ubiquitin was incorporated into the virions in the absence of the lysines in p6(Gag), showing that the ubiquitin inside the virus is not initially brought in as a p6Gag conjugate. While our results establish that ubiquitination of p6(Gag) and p12(Gag) 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. Our data show that the ubiquitination of p6(Gag) is not responsible for the negative effect of proteasome inhibitors on viral processing and budding. This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-56000. Ulrich Schubert was supported by grant Schu11/2-1 and a Heisenberg grant from the Deutsche Forschungsgemeinschaft.

Publication Types:

• Meeting Abstracts

Keywords:

• HIV-1
• In Vitro
• Lysine
• Ubiquitin
• Virion
• reverse transcriptase, Human immunodeficiency virus 1

Other ID:

• GWAIDS0012012

UI: 102249510

From Meeting Abstracts