The final stages of budding and release of a retroviral particle

The final stages of budding and release of a retroviral particle from the cell require the late (L) domain of Gag. is incorporated into HIV-2 virions. Expression of the N-terminal Ubc-conjugation homology domain of Tsg101 inhibits the release of Rabbit Polyclonal to MED24. HIV-2 virus PIK-93 particles. Overexpression of Tsg101 results in an increase in the level of ubiquitination of HIV-2 Gag. Our results provide evidence for recruitment of the ubiquitination machinery of the cell during late stages of the viral life cycle mediated by the viral Gag protein. Retroviral Gag proteins play a crucial role in the virus life cycle mediating virion assembly association with viral genomic RNA and virus release (47). The Gag protein is synthesized as a polyprotein precursor that on activation of the viral protease during the budding process is cleaved to yield the mature virion components: matrix (MA) capsid (CA) nucleocapsid (NC) p6 and two spacer peptides PIK-93 p2 and p1. Gag proteins contain conserved domains required for membrane targeting (M) discussion between Gag polyproteins (I) and past due stages of pathogen particle set up and release through the cell (L). Even though the amino acidity sequences from the L domains aren’t conserved among retroviruses they may be functionally comparable and compatible between different people from the (36). The L site is necessary for the ultimate phases of virion launch through the cell (14 20 54 55 and has been reported to connect to the ubiquitination equipment (37 43 46 52 Further proof for a job of ubiquitin and ubiquitin ligases in retroviral budding offers result from the observation that depletion of intracellular swimming pools of free of charge ubiquitin by treatment of cells with proteasome inhibitors leads to inhibition of retroviral budding (37 43 Ubiquitin can be discovered within retroviral contaminants (1 33 34 40 and a little proportion of human being immunodeficiency pathogen type 1 (HIV-1) p6 and Moloney murine leukemia pathogen (MoMLV) p12 (both of which contain the L domain) present in virions is monoubiquitinated (14 56 A number of cellular proteins interact with Gag. Cyclophilin A a prolyl isomerase interacts with HIV-1 Gag (30) and is incorporated into virions enhancing virion infectivity (12). HO3 a histidyl-tRNA synthetase binds to MA of HIV-1 (26). KIF4 a microtubule-associated motor protein interacts with retroviral Gag proteins including those of HIV-1 (48) and MoMLV (23). The cytoskeletal proteins actin ezrin moesin and cofilin have been found in virions (35). Human translation factor elongation factor 1α (EF1α) interacts with HIV-1 Gag MA and NC domains and is incorporated into virions (5). A novel virion-associated protein called VAN binds to HIV-1 MA (16). The C-terminal fragment of nucleolin interacts with MoMLV Gag inhibiting virion assembly (3). HIV-1 and HIV-2 are both members of the lentivirus subfamily of PIK-93 retroviruses. While they have similar genetic organization there is limited homology at the nucleotide and amino acid levels (17). HIV-2 differs from HIV-1 in the mechanism it uses to select its genomic RNA for PIK-93 encapsidation (21) suggesting that the Gag proteins of HIV-1 and HIV-2 may interact with different cellular proteins. In this report we describe the identification of Tsg101 as an HIV-2 Gag-interacting protein. Tsg101 interacts specifically with HIV-2 Gag in vitro and in vivo. The PTAPP motif in p6 of Gag and the N-terminal Ubc-homology domain of Tsg101 are required for the interaction. Furthermore we demonstrate that Tsg101 is incorporated into HIV-2 particles. Overexpression of full-length Tsg101 causes an increased level of ubiquitination of Gag. Expression of the N-terminal Ubc-conjugation homology PIK-93 domain of Tsg101 (amino acid residues 1 to 167) inhibits the release of HIV-2 particles. MATERIALS AND METHODS Construction of plasmids for use in the yeast two-hybrid system. Yeast expression plasmids pLexA (bait) and pB42AD (prey) carrying and selection markers respectively were obtained from Clontech. Gag coding sequences of HIV-1 HXB2 (11) and HIV-2 ROD (17) were cloned between the Turbo DNA polymerase (Stratagene) PIK-93 with IGAGF (5′-CATAGAATTCATGGGTGCGAGAGCGTCA-3′) and 1GAGR (5′-CATGTAGTCGACTTATTGTGACGAGGGGTC-3′) primers for HIV-1 Gag or 2GAGF (5′-CTATGAATTCATGGGCGCGAGAAACTCC-3′) and 2GAGR (5′-CACGTCGTCGACCTACTGGTCTTTTCCAAA-3′) primers for HIV-2 Gag. The resulting PCR products were digested with gene between and genes was constructed by cloning the Gag open reading frame from pGexGag2 as an gene (nucleotides 2114 to 5067) and therefore expresses uncleaved Gag polyprotein. Tsg101 was.