HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking

Abstract

Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC) assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV) post-transcriptional regulatory element (PRE) mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA) with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

Figure 1. Distinct intracellular distribution and assembly dynamics of Rev-dependent and PRE-dependent HIV-1 Gag.

293T cells were transfected with Rev-dependent (A) and PRE-dependent (B) HIV-1 Gag-GFP and monitored with time-lapse confocal microscopy. Images of different Z sections were overlaid to produce single extended-focus image for each time point. Arrays of individual frames from two time-lapse videos covering about 12 hr compare the distribution and assembly dynamics of Rev-dependent and PRE-dependent HIV-1 Gag.

Figure 2. Deficient assembly and budding of PRE-dependent HIV-1 Gag.

(A) Comparison of mean fluorescence intensity of 293T cells transfected with Rev-dependent (black diamond) or PRE-dependent HIV-1 Gag-GFP (gray square). Ceslls were monitored with time-lapse confocal microscopy as described in Figure 1. 30 randomly selected cells for each were tracked immediately after GFP fluorescence appeared and mean fluorescence intensity was measured. Average mean fluorescence intensity was plot over time. (B) Comparison of mean fluorescence intensity of 293T cells transfected with Rev-dependent (black diamond) or PRE-dependent HIV-1 Gag-GFP (gray square) when Gag-GFP puncta appear. All the 30 Rev-dependent HIV-1 Gag-GFP transfected cells tracked in (A) and 7 of the 30 PRE-dependent HIV-1 Gag-GFP transfected cells tracked in (A) displayed Gag-GFP puncta. (C) Budding of Rev-dependent and PRE-dependent wild type and myristoylation mutant HIV-1 Gag-HA. Rev-dependent and PRE-dependent Gag constructs were transfected into 293T cells. At 24 h post transfection, VLPs (upper panel) were analyzed by immunoblotting using HA antibody and cell lysates (lower panel) were analyzed by immunoblotting using HA and actin antibody. Data are representative of three independent experiments.

Figure 3. Co-assembly of Rev-dependent HIV-1 Gag and PRE-dependent HIV-1 Gag.

(A) Schematic diagram of plasmids expressing Rev-dependent and PRE-dependent HIV-1 Gag-BiFC constructs. (B) Demonstration of co-assembly of Rev-dependent and PRE-dependent HIV-1 Gag by BiFC. HeLa cells grown on glass coverslips were transfected with plasmids expressing the indicated HIV-1 Gag-BiFC pair. At 8 h post transfection, cells were fixed and imaged. Bar: 10 µm.

Figure 4. Coexpression of membrane targeting and assembly competent Rev-dependent HIV-1 Gag rescues PRE-dependent HIV-1 Gag budding.

(A) Schematic diagram of plasmids expressing HA tagged Rev-dependent HIV-1 Gag mutants. (B) Budding of PRE-dependent HIV-1 Gag-GFP upon co-expression with PRE-dependent or Rev-dependent HIV-1 Gag-HA. PRE-dependent HIV-1 Gag-GFP was co-transfected at an equal molar ratio into 293T cells with empty vector, PRE-dependent HIV-1 Gag-HA, or the indicated Rev-dependent HIV-1 Gag-HA constructs described in A. At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using GFP and HA antibody. Data are representative of three independent experiments.

Figure 5. Substitution of HIV-1 matrix with other membrane targeting domains rescued PRE-dependent HIV-1 Gag budding in human cells.

(A) Schematic diagram of plasmids expressing HA tagged PRE-dependent HIV-1 Gag mutants (upper panel) and EIAV Gag mutants (lower panel). (B) Budding of PRE-dependent HIV-1 and EIAV Gag mutants. 293T cells were transfected with the indicated PRE-dependent HIV-1 and EIAV Gag mutants described in (A). At 24 h post transfection, VLPs (upper panel) and cell lysates (lower panel) were analyzed by immunoblotting using HA antibody. Data are representative of three independent experiments.

Figure 6. PRE-dependent HIV-1 Gag failed to associate with lipid raft in human cells.

(A) Both Rev-dependent and PRE-dependent HIV-1 Gag are membrane-associated in human cells. Postnuclear supernatants derived from 293T cells expressing Rev-dependent or PRE-dependent HIV-1 Gag, Rev-dependent or PRE-dependent G2A mutant were subjected to equilibrium flotation centrifugation. Pr55^Gag, TfR and actin were detected by Western blotting. Membrane- and non-membrane-associated fractions were shown. (B) Rev-dependent HIV-1 Gag but not PRE-dependent HIV-1 Gag is associated with Triton X-100 insoluble lipid rafts in human cells. Postnuclear supernatants derived from 293T cells expressing Rev-dependent or PRE-dependent HIV-1 Gag were treated with 0.5% Triton X-100 on ice for 30 min prior to membrane flotation analysis. Pr55^Gag, TfR and actin were detected by Western blotting. Raft and non-raft fractions are indicated. (C) Quantitation of Rev-dependent (left panel) and PRE-dependent (right panel) HIV-1 Gag in each fraction with (open circles) or without (closed squares) cold Triton extraction.