Mutation of critical serine residues in HIV-1 matrix result in an envelope incorporation defect which can be rescued by truncation of the gp41 cytoplasmic tail

Abstract

The human immunodeficiency virus type 1 (HIV-1) matrix (MA) domain is involved in both early and late events of the viral life cycle. Simultaneous mutation of critical serine residues in MA has been shown previously to dramatically reduce phosphorylation of MA. However, the role of phosphorylation in viral replication remains unclear. Viruses harboring serine to alanine substitutions at positions 9, 67, 72, and 77 are severely impaired in their ability to infect target cells. In addition, the serine mutant viruses are defective in their ability to fuse with target cell membranes. Interestingly, both the fusion defect and the infectivity defect can be rescued by truncation of the long cytoplasmic tail of gp41 envelope protein (gp41CT). Sucrose density gradient analysis also reveals that these mutant viruses have reduced levels of gp120 envelope protein incorporated into the virions as compared to wild type virus. Truncation of the gp41CT rescues the envelope incorporation defect. Here we propose a model in which mutation of specific serine residues prevents MA interaction with lipid rafts during HIV-1 assembly and thereby impairs recruitment of envelope to the sites of viral budding.

Fig. 1. HIV-1 Matrix Serine Mutations

A) The four critical serine residues at positions 9, 67, 72, and 77 are shown schematically in bold lettering in relation to other known functional domains in MA, as well as the other serine residues in MA. The N-terminal myristylation site (N-Myr), nuclear export signal (NES), and nuclear localization sequences (NLS) are denoted in addition to the labeled functional domains. B) Molecular model of HIV-1 MA illustrating the locations of the four critical serine residues. The orientation of the MA molecule is such that the membrane-binding face of MA is in the foreground, with the myristyl group protruding towards the viewer. The molecular model of MA was generated using coordinates from Hill et al (1996) and PyMol software (DeLano Scientific LLC.).

Fig. 2. MA Ser mutant viruses are defective in virus-cell fusion not cell-cell fusion

A) Virus-dependent cell fusion assay was performed with WT, and mutant 4S, 9S, and L12E virus particles added to a 1:1 mixture of U87.CD4.CCR5.GFP cells expressing T7 polymerase or carrying the β-galactosidase gene under the regulation of the T7 promoter. The background level of mismatched Env/coreceptor was subtracted. B) Envelope-dependent cell-cell fusion assay. Proviral clones dEnv, WT, 4S, or 9S were expressed in BSC40 cells with the β-galactosidase gene under the regulation of the T7 promoter. These cells were mixed with U87.CD4.CCR5.GFP cells expressing T7 polymerase, and fusion measured by β-galactosidase activity. Bars represent standard deviations.

Fig. 3. Viral particle morphology is unaffected by serine mutations

Transmission electron microscopic analysis of A) WT, B) 4S, and C) 9S virus particles expressed from transfected 293T cells. The bar represents 164 nm. Sizes of WT, 4S, and 9S particles were measured as 131 + 5.0 nm, 144 + 17 nm, and 134 + 19 nm, respectively. Arrows indicate representative mature virions in each field.

Fig 4. MLV Envelope overcomes the fusion defect of MA 4S Ser mutant

Virus was produced in 293T transfected with each proviral clone with or without the addition of the MLV envelope expression plasmid. The resultant virus was titered on JC53 cells, and mean levels, and standard deviations are shown for 6 replicates of the experiment.

Fig. 5. C-terminal TM deletion overcomes the fusion defect of MA Ser mutants

Virus from WT, 4S, 9S, and L12E clones with or without a gp41 CTD was examined for A) infection of MAGI-R5 cells B) virus-dependent fusion activity, and C) long-term JC53 cell assays.

Fig 6. C-terminal TM deletion overcomes replication defect of MA Ser mutant over multiple replication cycles

Virus from WT, 4S, 9S, WT CTD and 9S CTD was used to infect JC53 cells, which were split 1:2.5 on days 5 and 9. Cells were lysed at each time point and luciferase assays performed.

Fig. 7. Levels of envelope incorporated into Ser mutant virions are reduced

Metabolically-labeled virus from WT, 4S, L12E, and dEnv clones was pelleted through a sucrose cushion, resuspended and centrifuged through 20-60% sucrose density gradients. A) Amount of labeled protein in each fraction was measured using a scintillation counter. Densities of peak fractions were consistently ~1.13-1.14g/ml, suggesting that they contain mature viral particles. B) Immunoprecipitation of peak fractions with AIDS pt sera. C) Densitometry was used to quantify gp120 and p24 bands for each immunoprecipitated fraction in panel B. Levels of envelope incorporation, relative to WT, were measured by calculating the ratio of gp120 to p24 in each peak fraction. The experiments shown are representative figures and reflect the data from at least three independent experiments. The differences in gp120/p24 ratios between WT and 4S were highly significant (2 tailed t test p<.001).

Fig. 8. Rescue of envelope incorporation defect by truncation of gp41CT

Virus from 4S and 4S CTD clones were analyzed by sucrose density gradients. A) Immunoprecipitation of peak fractions with AIDS pt sera is shown. B) Levels of envelope incorporated into mutant virions, as measured by gp120:p24 ratios, are also shown.

Fig. 9. Association of Pr55^Gag with detergent resistant membranes

Lysates from 293T cells transfected with proviral DNA were incubated on ice either in the A) absence or B) and C) presence of cold 0.25% Triton X-100 and subsequently subjected to equilibrium flotation centrifugation. Ten fractions were collected from the top of the gradient, precipitated and resolved by SDS PAGE. Gag in each fraction was detected by western blotting with polyclonal anti-p24 antibody. Detergent resistant membrane (DRM) fractions and detergent soluble (DS) fractions are shown. The experiments shown are representative figures and reflect the data from two independent experiments.

Fig 10. Quantitative analysis of Gas association with detergent resistant membranes

Gag p24 antigen ELISA was used for quantification of sucrose gradient fractions, using lysates from cells transfected with WT, 4S, or 9S virus with or without an MLV pseudotype, as well as WT-CTD and 4S-CTD. Each experiment was repeated on at least two independent occasions with similar results, and are not based on the same gradients shown in Fig 9