Analysis of human immunodeficiency virus type 1 Gag ubiquitination.
Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.
Abteilung Virologie, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.
Eva
E
Gottwein
Hans-Georg
HG
Kräusslich
D020816:Amino Acid Motifs; D002460:Cell Line; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D025801:Ubiquitin / Q000378:metabolism*; D014771:Virion / Q000737:chemistry
D015683:Gene Products, gag; D025801:Ubiquitin; D008239:Lysine