ESCRT-III protein requirements for HIV-1 budding

Abstract

Two early-acting components of the cellular ESCRT pathway, ESCRT-I and ALIX, participate directly in HIV-1 budding. The membrane fission activities of ESCRT-III subunits are also presumably required, but humans express 11 different CHMP/ESCRT-III proteins whose functional contributions are not yet clear. We therefore depleted cells of each of the different CHMP proteins and protein families and examined the effects on HIV-1 budding. Virus release was profoundly inhibited by codepletion of either CHMP2 or CHMP4 family members, resulting in ≥100-fold titer reductions. CHMP2A and CHMP4B proteins bound one another, and this interaction was required for budding. By contrast, virus release was reduced only modestly by depletion of CHMP3 and CHMP1 proteins (2- to 8-fold titer reductions) and was unaffected by depletion of other human ESCRT-III proteins. HIV-1 budding therefore requires only a subset of the known human ESCRT-III proteins, with the CHMP2 and CHMP4 families playing key functional roles.

Figure 1. HIV-1 Release and Infectivity Can Be Modestly Affected by Depletion of Individual ESCRT-III Proteins

Viral titers produced by cells expressing HIV-1 and treated with control siRNAs (panel 1, lanes 1 and 2), with siRNAs that targeted TSG101 (positive control, lane 3) or the designated ESCRT-III proteins (lanes 4-14) (n = 6 titer measurements from two independent experiments ± S.D.). Lower western blots show virion-associated viral Gag proteins released into the supernatant (panel 2, “Virus”, anti-CA and anti-MA), intracellular viral Gag protein levels (panel 3, “Cell”, anti-CA and anti-MA), endogenous cellular ESCRT proteins (panels 4-14), or α-Tubulin (panel 15, loading controls). (see also Figure S1).

Figure 2. CHMP2 and CHMP4 Protein Families Are Required for HIV-1 Release and Infectivity

(A,B) Viral titers, virion-associated Gag proteins, and intracellular protein levels in 293T cells expressing HIV-1 and depleted or co-depleted of the designated (A) CHMP2 or (B) CHMP4 family members. Figure panels are equivalent to those shown in Figure 1 (n = 3 titer measurements ± S.D.). (C,D) Thin-sectioned, transmission electron micrographs of arrested HIV-1 budding particles in cells lacking CHMP2A/B (C) or CHMP4A/B (D). Scale bars are 100 nm. (E) Quantification of arrested budding particles without (grey) or with (black) visible striations within their necks (see arrows in Figure 2C for examples). (see also Figure S2).

Figure 3. Identification and Characterization of CHMP2A:CHMP4B Interactions

(A) Panel showing yeast two-hybrid interactions between the designated ESCRT proteins fused to activation (AD) or DNA binding (DBD) domains (or with empty vector controls). The following interactions were judged to be positive (AD fusions listed first): CHMP1A:CHMP2A_1-146, CHMP1A:CHMP2A_1-146,C4-, CHMP4B:CHMP2A_1-146, CHMP4B:CHMP4B, CHMP4B_C2-:CHMP4B, VPS4A:CHMP2A. VPS4A also interacted very weakly with CHMP4B in this assay. (B) Upper: homology model of CHMP2A showing mutations that blocked binding of CHMP4B (designated CHMP2A_C4-) or VPS4A and B (CHMP2A_VPS4-). Lower: homology model of CHMP4B showing mutations that blocked binding of CHMP2A (CHMP4B_C2-) or ALIX (CHMP4B_ALIX-). Homology models were generated by threading the CHMP2A and CHMP4B sequences onto the known CHMP3 structure (pdb 3FRT) (Bajorek et al., 2009b; Muziol et al., 2006) using SWISS-MODEL (Arnold et al., 2006) and COOT (Emsley and Cowtan, 2004), and displayed using PYMOL (DeLano, 2008). (C,D) GST pull-down analyses demonstrating that recombinant CHMP2A and CHMP4B proteins interact and that the mutations in the CHMP2A_C4- (C) and CHMP4B_C2- (D) constructs inhibit these interactions. (C) SDS-PAGE/Coomassie detection of matrix-bound proteins following incubation of immobilized GST (lanes 1-3, negative controls) or GST-CHMP4B proteins (lanes 4-6) with no added proteins (lanes 1 and 4, negative controls), or with purified wild type CHMP2A (lanes 2 and 5) or CHMP2A_C4- (lanes 3 and 6). Input CHMP2A proteins are shown in lanes 7 and 8 (2% of total). (D) SDS-PAGE/Coomassie detection of matrix-bound proteins following incubation of immobilized GST (lanes 1 and 2, negative controls), wild type GST-CHMP4B (lanes 3 and 4) or GST-CHMP4B_C2- (lanes 5 and 6) with no added proteins (lanes 1, 3 and 5) or with purified wild type CHMP2A (lanes 2, 4, and 6). Input CHMP2A is shown in lane 7 (2% of total). (see also Figure S3).

Figure 4. Point Mutations that Inhibit CHMP2A-CHMP4B Interactions also Block HIV-1 Budding

(A) Viral titers, virion-associated Gag proteins, and intracellular protein levels in 293T cells expressing HIV-1 (lanes 1-5) and treated with a control siRNA (lane 1), or with siRNAs that co-depleted CHMP2A/B (lanes 2-5), together with an empty control vector (lane 2) or with vectors expressing siRNA-resistant wild type CHMP2A (lane 3) CHMP2A_VPS4- (lane 4) or CHMP2A_C4- (lane 5). Figure panels are equivalent to those in Figures 1 and 2 (n = 9 titer measurements from 3 independent experiments ± S.D.). (B) This experiment is equivalent to Figure 4A except that cells were co-depleted of CHMP4A/B (lanes 2-5) and the siRNA-resistant rescue constructs expressed wild type CHMP4B (lane 3), CHMP4B_ALIX- (lane 4) or CHMP4B_C2- (lane 5) (n = 6-12 titer measurements from 2-4 independent experiments ± S.D.). (see also Figure S4).