Matrix mediates the functional link between human immunodeficiency virus type 1 RNA nuclear export elements and the assembly competency of Gag in murine cells

Abstract

Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the cis-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4xCTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE- but not 4xCTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.

FIG. 1.

4×CTE-dependent RNA nuclear export and deletion of the MA globular head both increase VLP production in murine cells. (A) Depiction of cytomegalovirus major immediate-early-driven surrogate gRNA transcripts carrying either RRE or 4×CTE nuclear export elements and encoding either wild-type (WT) Gag (GP) or Gag carrying a deletion of the MA globular-head domain (Δ8-126). (B) 3T3 (lanes 1 to 4) or HeLa (lanes 5 to 8) cells were cotransfected with 1 μg or 250 ng (25%), respectively, Gag expression plasmids encoding the indicated transcripts plus plasmids encoding either Rev or luciferase (Luc) as indicated. Cell lysates and VLPs were collected 48 h posttransfection, resolved by SDS-PAGE, and immunoblotted for Gag and HSP90 (loading control).

FIG. 2.

The 4×CTE enhancement of assembly in murine cells requires an intact MA globular-head domain. (A) 3T3 cells were transfected with Gag expression plasmids or dilutions thereof, as indicated. Lysates and VLPs were harvested for immunoblot analysis as described for Fig. 1B. p24^Gag released into the supernatant was quantified by ELISA (bars); the value above each bar represents the change for values normalized to the GP-RRE plus Rev sample (gray bar; lane 8). HSP90 served as a loading control for cell lysates. (B) Comparison of assembly of Δ8-126 Gag mutants encoded by transcripts undergoing either RRE/Rev- or 4×CTE-dependent nuclear export. (C) An experiment similar to that presented in panel B for the Δ8-126 mutant carried out in culture medium containing 1 μM saquinavir to prevent Gag processing. Protein levels were measured by quantitative immunoblotting. A release factor (bars) representing assembly efficiency was calculated as the ratio of Gag detected in VLPs to Gag detected in cell lysates. For comparison, all values were normalized to the Δ8-126-RRE plus Rev condition (gray bar; lane 6). The change is indicated above each bar. (D) Comparison of assembly efficiencies for the Δ8-126 mutant Gag and wild-type Gag encoded by transcripts undergoing 4×CTE-dependent nuclear export.

FIG. 3.

RRE/Rev-derived Gag is intrinsically defective for assembly in murine cells independent of the expression level. (A) 3T3 cells were metabolically radiolabeled with [³⁵S]Met/Cys for 10 min 24 h posttransfection with the indicated amounts of protease-minus Gag (GP-R57G) expression plasmids. Gag was immunoprecipitated from cell lysates using an anti-p24^Gag polyclonal serum and resolved by SDS-PAGE. Radiolabeled protein was quantified by phosphorimager, and the values were normalized to the GP-RRE plus Rev sample (lane 6). (B) Analysis of virus assembly efficiency by quantitative immunoblotting (as described for Fig. 2C) for 3T3 cells transfected in a fashion identical to that for panel A. HSP90 served as a loading control for cell lysates. The change in the release factor is indicated above each bar. (C) Visual comparison of intracellular Gag (GP-R57G) levels for 3T3 cell populations transfected in a fashion identical to that for panels A and B. Cells were seeded on glass coverslips and fixed 48 h posttransfection. Gag was detected by indirect immunofluorescence using an anti-p24^Gag serum and visualized by confocal microscopy. Identical laser settings were used for each acquisition. Representative images are presented. Bars, 10 μm. (D) Comparison of intracellular Gag levels for transfected 3T3 cell populations analyzed by flow cytometry. Cells were transfected in a fashion identical to that for panels A to C prior to being trypsinized, fixed, permeabilized, and stained with a monoclonal anti-p24^Gag serum conjugated to fluorophore RD1 at 48 h posttransfection. The percentage of cells expressing detectable levels of Gag (white bars) or the mean fluorescence intensity for each Gag-positive population (gray bars) for each twofold dilution of 4×CTE plasmid was normalized to the RRE plus Rev condition (bars on right). The mean values for three separate transfections are presented. The error bars represent ±1 standard deviation.

FIG. 4.

The 4×CTE affects assembly independently of NC-dependent Gag multimerization. (A) Depiction of protease-minus Gag (GP-R57G) and the Z_WT-p6 mutant carrying a leucine zipper replacement of NC. (B) A twofold 4×CTE plasmid dilution series followed by quantitative immunoblotting similar to that described for Fig. 2C comparing RRE- and 4×CTE-dependent transcripts for the Z_WT-p6 mutant. The calculated release factors were normalized to the GP-R57G-RRE plus Rev sample (lanes 6 and 7; gray bars). The change is indicated above each bar.

FIG. 5.

Single MA amino acid changes implicated in myristoyl switch regulation improve RRE/Rev-derived Gag assembly efficiency in murine cells. (A) Depiction of N-terminal MA single-amino-acid mutations generated for this analysis. Basic residues are indicated by “+.” (B) 3T3 cells were transfected with the indicated MA mutant and cultured in the presence of saquinavir, and the cells and supernatants were processed for quantitative assembly assay as described for Fig. 2C. Release factors were normalized to the wild-type (WT)-RRE plus Rev sample. The change in the release factor is indicated above each bar. (C) A twofold 4×CTE plasmid dilution series comparing the assembly efficiency of Gag (L21S) mutants derived from 4×CTE-dependent transcripts (lanes 1 to 5) to that derived from RRE/Rev-dependent transcripts (lane 6). As for Fig. 2A, VLP production was assessed both by immunoblotting and p24^Gag ELISA. The change in p24^Gag release is indicated above each bar.

FIG. 6.

MA mutants rescue Gag membrane targeting and particle production. (A) 3T3 cells transfected with Gag or the indicated Gag mutant were fixed 24 h posttransfection prior to indirect immunofluorescence detection of Gag using an anti-p17^Gag polyclonal antiserum. The images were collected using a laser scanning confocal microscope and represent a single optical slice near the cell-coverslip junction. Bars, 10 μm. (B) For the experiment in panel A, coverslips were assessed blindly and >100 transfected cells were scored for the detectable presence of Gag-positive surface punctae, consistent with VLP production. The bars represent average values from duplicate samples.