Context-dependent effects of L domains and ubiquitination on viral budding

Abstract

Many enveloped viruses encode late assembly domains, or L domains, that facilitate virion egress. PTAP-type L domains act by recruiting the ESCRT-I (endosomal sorting complex required for transport I) component Tsg101, and YPXL/LXXLF-type L domains recruit AIP-1/ALIX, both of which are class E vacuolar protein sorting (VPS) factors, normally required for the generation of vesicles within endosomes. The binding cofactors for PPXY-type L domains have not been unambiguously resolved but may include Nedd4-like ubiquitin ligases. Largely because they act as autonomous binding sites for host factors, L domains are generally transferable and active in a context-independent manner. Ebola virus matrix protein (EbVP40) contains two overlapping L-domain motifs within the sequence ILPTAPPEYMEA. Here, we show that both motifs are required for efficient EbVP40 budding. However, upon transplantation into two different retroviral contexts, the relative contributions of the PTAP and PPEY motifs differ markedly. In a murine leukemia virus carrying the EbVP40 sequence, both motifs contributed to overall L domain activity, and budding proceeded in a partly Tsg101-independent manner. Conversely, when transplanted into the context of human immunodeficiency virus type 1 (HIV-1), EbVP40 L-domain activity was entirely due to a PTAP-Tsg101 interaction. In fact, a number of PPXY-type L domains were inactive in the context of HIV-1. Surprisingly, PTAP and YPXL-type L domains that simulated HIV-1 budding reduced the amount of ubiquitin conjugated to Gag, while inactive PPXY-type L domains increased Gag ubiquitination. These observations suggest that active L domains recruit deubiquitinating enzymes as a consequence of class E VPS factor recruitment. Moreover, context-dependent L-domain function may reflect distinct requirements for host functions during the morphogenesis of different viral particles or the underlying presence of additional, as yet undiscovered L domains.

FIG.1.

Tsg101-PTAP interaction is necessary but not sufficient to mediate the late stages of EbVP40 virus-like particle release. (A) Alignment of HIV-1 p6 and EbVP40 L-domain sequences, showing conservation of the PTAP but not PPXY motifs (boxed residues). Residues targeted for mutagenesis that selectively eliminate the PTAP motif (P7L) or the PPXY motif (P11L and Y13A) are underlined. The methionine residue in the EbVP40 sequence represents the amino terminus of the protein. (B) Western blot analysis of cell lysates and extracellular virus-like particles upon expression of Myc epitope-tagged wild-type (WT) and mutant EbVP40 proteins in 293T cells. (C) Subcellular localization and filament formation by wild-type and L-domain mutant forms of a GFP-EbVP40 fusion protein. (D) Yeast two-hybrid analysis of EbVP40-Tsg101 interaction. The chart shows β-galactosidase activity (in optical density [OD] units) in lysates of S. cerevisiae Y190 cells expressing GAL4-Tsg101 and the indicated wild-type or mutant form of VP16-EbVP40 fusion protein.

FIG. 2.

L-domain activity of PTAP and PPXY motifs from the EbVP40 L domain in the context of MLV Gag. (A) Schematic representation of the MLV/hPTAP and MLV/ePTAP proviral constructs. The endogenous PPPY motif in MLV is replaced by residues from the HIV-1 PTAP L domain or the EbVP40 L domain, as indicated. Residues that were mutated in these constructs are underlined. In both cases P7L refers to the first proline residue in the PTAP motif. Coincidentally, this residue is the seventh residue from the N terminus of both HIV-1 p6 and EbVP40. (B) Effect of mutations in the transplanted HIV-1 and EbVP40 L domains on MLV virion formation. Infectious virus production by cells transfected with a GFP-expressing MLV vector, a vesicular stomatitis virus G expression vector, and the indicated wild-type (WT) or mutant MLV/hPTAP or MLV/ePTAP proviral plasmids. Infectious virions were measured by flow cytometry analysis of cells inoculated with culture supernatants and are given in infectious units (i.u.).

FIG. 3.

EbVP40 L-domain function is partly Tsg101 independent in the context of MLV Gag. (A) 293T cells were transfected with a GFP-expressing MLV vector, a vesicular stomatitis virus G expression plasmid, and the indicated proviral plasmids, and infectious virion production was measured as in Fig. 2. Also included in the transfection mixture were siRNA duplexes directed at firefly luciferase (Luc, as a control) or Tsg101, as indicated. (B) 293T cells were transfected with an HIV-1 Tat-expressing MLV vector, a vesicular stomatitis virus G expression plasmid, and the indicated proviral plasmids, and infectious virion production was measured by activation of β-galactosidase expression following inoculation of HeLa P4/R5 cells. Plasmids expressing dominant negative fragments of Tsg101 that inhibit PTAP-dependent HIV-1 budding were included in some transfections, and the yield of infectious virions is given as a percentage of that obtained in their absence.

FIG.4.

Failure of PPXY-type L domains to support HIV-1 budding. (A) 293T cells were transfected with wild-type (WT) or mutant forms of full-length HIV-1 proviral plasmids containing the natural PTAP motif, a variant EbVP40 PTAPPEY motif, or a PPPY motif from MLV. (B) Western blot analysis with an anti-HIV-1 CA antibody of cell lysates and extracellular virions harvested by ultracentrifugation is shown; the scale on the left shows the migration of molecular size markers (in kilodaltons). (C) 293T cell were transfected with a p6-deleted HIV-1 provirus along with trans-complementing Gagδp6 protein fused to either HIV-1 p6, equine infectious anemia virus p9, Rous sarcoma virus p2, or wild-type or mutant forms of MLV p12, as indicated. Cell lysates and virion particles were analyzed by Western blotting as in panel B.

FIG. 5.

Opposite effects of L domains on MLV and HIV-1 Gag ubiquitination. (A) Wild-type (WT) and mutant (PPPY-deleted [δPY]) forms of MLV Gag fused to GST were expressed in 293T cells along with an HA-tagged form of ubiquitin. The left panels show Western blot analyses of extracellular virus-like particles and cell lysates with an anti-MLV CA antibody. The right two panels show anti-HA and anti-GST Western blot analyses of the MLV Gag-GST fusion proteins purified from cell lysates with glutathione-agarose beads; the positions of Gag GST- and HA-ubiquitin conjugates are indicated. (B) HIV-1 Gagδp6 proteins fused to the indicated viral L domains or PTAP- or PPXY-containing peptide motifs were coexpressed in 293T cells along with an HA-tagged form of ubiquitin. In this case, HIV-1 Gag was immunoprecipitated with serum from an HIV-positive patient, and the immunoprecipitates were analyzed by Western blotting with anti-HIV-1 CA and anti-HA monoclonal antibodies. The band marked with an asterisk in the blots probed with anti-HA is the immunoglobulin G heavy chain. In both A and B, the scales to the side of the blots indicate the positions of molecular size markers (in kilodaltons).

FIG. 6.

Opposite effects of PTAP and PPEY motifs in the EbVP40 L domain on ubiquitination and lack of contribution of the PPEY motif to HIV-1 budding. (A) HIV-1 Gagδp6 proteins fused to the indicated wild-type (WT) and mutant forms of the EbVP40 L domain peptide (ILPTAPPEYMEA; the P7, P11, and Y13 mutated residues are in italics) were coexpressed with HA-tagged ubiquitin in 293T cells. Subsequently, HIV-1 Gag was immunoprecipitated with serum from an HIV-positive patient, and the immunoprecipitates were analyzed by Western blotting with anti-HIV-1 CA and anti-HA monoclonal antibodies, as in Fig. 5B. (B) Ability of the same Gagδp6-EbVP40 L domain fusion proteins to complement PTAP-defective HIV-1 budding. Infectious virion formation by 293T cells cotransfected with plasmids expressing a PTAP-defective HIV-1 provirus and Gagδp6 fusion proteins containing the indicated wild-type and mutant L domain motifs was measured by inoculation of HeLa P4/R5 cells. Gagδp6-p6 and Gagδp6-hPTAP were included as positive controls. (C and D) Equivalent effects of Tsg101 depletion on infectious HIV-1 virion formation mediated by HIV-1 (C) and EbVP40 (D) L-domain motifs.