A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41 - PubMed (original) (raw)

A mutation in the human immunodeficiency virus type 1 Gag protein destabilizes the interaction of the envelope protein subunits gp120 and gp41

Melody R Davis et al. J Virol. 2006 Mar.

Abstract

The Gag protein of human immunodeficiency virus type 1 (HIV-1) associates with the envelope protein complex during virus assembly. The available evidence indicates that this interaction involves recognition of the gp41 cytoplasmic tail (CT) by the matrix protein (MA) region of Pr55(Gag). Here we show that substitution of Asp for Leu at position 49 (L49D) in MA results in a specific reduction in particle-associated gp120 without affecting the levels of gp41. Mutant virions were markedly reduced in single-cycle infectivity despite a relatively modest defect in fusion with target cells. Studies with HIV-1 particles containing decreased levels of envelope proteins suggested that the L49D mutation also inhibits a postentry step in infection. Truncation of the gp41 tail, or pseudotyping by vesicular stomatitis virus glycoprotein, restored both the fusion and infectivity of L49D mutant virions to wild-type levels. Truncation of gp41 also resulted in equivalent levels of gp120 on particles with and without the MA mutation and enhanced the replication of the L49D mutant virus in T cells. The impaired fusion and infectivity of L49D mutant particles were also complemented by a single point mutation in the gp41 CT that disrupted the tyrosine-containing endocytic motif. Our results suggest that an altered interaction between the MA domain of Gag and the gp41 cytoplasmic tail leads to dissociation of gp120 from gp41 during HIV-1 particle assembly, thus resulting in impaired fusion and infectivity.

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Figures

FIG. 1.

FIG. 1.

L49D virus particles exhibit efficient assembly but are poorly infectious. (A) Transient transfection of 293T cells with proviral DNAs resulted in comparable levels of particle production for wild-type (WT; NL4-3) and MA mutant (L49D) particles. Particle yield was determined by p24 ELISA. The data represent the average of at least three independent transfections, with error bars corresponding to 1 standard deviation. (B) Single-cycle infectivity was assayed in P4R5 target cells which contain the lacZ gene under transcriptional control of the HIV-1 promoter. Cells were inoculated with 293T-derived viruses, and infected cells were identified microscopically after staining with X-Gal. Infectivity is presented as infectious units (i.u.)/ng of p24. This figure represents the average of at least three independent assays. *, P < 0.001 from two-tailed Student's t test.

FIG. 2.

FIG. 2.

L49D mutant HIV-1 particles are moderately impaired for fusion. (A) HIV-1 particles were tested for fusion with P4R5 cells. Ratios represent the average of triplicate wells ± standard deviation and are representative of at least three independent assays. WT, wild type; ΔE, Env defective. (B) Virus particles were pelleted through 20% sucrose and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following protein transfer to nitrocellulose, immunoblots were probed for p24/CA, gp41/TM, gp120/SU, p17/MA, and β-lactamase. (C) Band intensities were quantified by LI-COR Odyssey software. (D) ELISA quantitation of gp120 on virus particles. Viruses were pelleted and then dissolved in lysis buffer, and gp120 and p24 were quantified by ELISA. Shown is the mean ratio of gp120 to p24 from three independent assays, with error bars representing 1 standard deviation.

FIG. 3.

FIG. 3.

Envelope titration reveals a second block to L49D infectivity. (A) Infectivity assays. 293T cells were transfected with various ratios of wild-type and Env-defective (ΔE) proviruses with a fixed quantity of the BlaM-Vpr reporter construct. The resulting particles were then assayed for infectivity. Shown are the mean values for triplicate determinations, with error bars representing 1 standard deviation. i.u., infectious units. (B) Assays of virus-cell fusion. Shown are the fusion signals resulting from 100-ng p24 inocula. The data shown are the mean values from triplicate determinations. Results are representative of three independent experiments.

FIG. 4.

FIG. 4.

Truncation of the gp41 CT complements the L49D mutant. (A) Single-cycle infectivity of mutant virus particles lacking the gp41 cytoplasmic tail. Shown are the mean values of three independent experiments, with error bars representing 1 standard deviation. I.U., infectious units. (B) Assays of virus-cell fusion. Shown are the mean values of quadruplicate assays, with error bars representing 1 standard deviation. ΔE, Env defective. (C) Immunoblots of pelleted virus particles for CA, gp41, gp120, MA, and BlaM. (D) Quantification of the protein signals from the immunoblots shown in panel C using the LI-COR Odyssey imaging system and software.

FIG. 5.

FIG. 5.

Pseudotyping by VSV-G suppresses the phenotype of the HIV-1 L49D mutation. (A) Infectivity of pseudotyped HIV-1 particles. Shown are mean values from triplicate determinations, with error bars representing 1 standard deviation. i.u., infectious units. (B) Assays of virus fusion with P4R5 cells. Shown are mean values of triplicate determinations, with error bars representing one standard deviation. Results are representative of at least three independent experiments. ΔE, Env defective. (C) Immunoblots of pelleted virus particles for VSV-G, BlaM, CA, and MA. (D) Quantification of the bands shown in panel C using the Odyssey imaging system.

FIG. 6.

FIG. 6.

HIV-1 Env protein inhibits the infectivity of L49D mutant particles when cell entry is mediated through a heterologous viral Env protein. Dual-enveloped wild-type and L49D mutant particles were generated by cotransfection of cells with Env+ or Env-defective (ΔE) proviral clones with the indicated quantities of plasmid pCEET encoding a CT-truncated ecotropic MLV Env protein. Virus particles were collected and assayed for infection on CD4-negative reporter cells expressing the ecotropic MLV receptor. Shown are the mean infectivity values of triplicate infections, with error bars representing 1 standard deviation. Results are representative of two independent experiments. i.u., infectious units.

FIG. 7.

FIG. 7.

Assays of gp120 shedding and loss of infectivity during incubation of freshly-harvested HIV-1 particles. (A) Viruses were harvested from transfected 293T cells following a 1-h culture period and were then incubated at 37°C for the indicated time periods. Virus stocks were assayed for particle-associated gp120 and p24 by ELISA. Shown is the ratio of gp120 to p24 for each virus sample. (B) Infectivity of incubated HIV-1 particles. Virus stocks were assayed for infectivity with P4R5 cells. Shown are the mean values from triplicate determinations. The results in this figure are representative of two independent experiments, the results of which agreed well. i.u., infectious units.

FIG. 8.

FIG. 8.

Truncation of the gp41 CT enhances the replication of L49D mutant particles. (A) MT-4 cells were inoculated at low multiplicity with the indicated HIV-1 viruses (1 ng p24). Duplicate cultures were evaluated for virus replication every 1 to 2 days by p24 ELISA. The values shown are the averages of duplicate wells from two separate cultures. (B) Regrowth of viruses from cultures shown in panel A. Normalized quantities (1 ng p24) of viruses collected from the cultures shown in panel A were reinoculated onto fresh cultures of MT-4 cells. Duplicate cultures were analyzed, and samples were withdrawn daily. The data shown are the mean values from duplicate wells from two independent assays. •, wild type; ○, CT144; ▪, L49D; □, L49D.CT144.

FIG. 9.

FIG. 9.

Mutation of the gp41 endocytic motif restores L49D fusion and infectivity. (A) Infectivity of HIV-1 particles was determined by titration on P4R5 cells. Values are the means of triplicate determinations, with error bars representing 1 standard deviation. The results are representative of three independent experiments. i.u., infectious units; WT, wild type. (B) Fusion of BlaM-Vpr reporter viruses with P4R5 target cells. The results shown are representative of two independent experiments. (C) Immunoblot analysis of pelleted particles normalized by p24 ELISA. Blots were probed with antibodies specific for BlaM, gp120/SU, p24/CA, p17/MA, and gp41/TM.

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