Characterization of replication defects induced by mutations in the basic domain and C-terminus of HIV-1 matrix

Abstract

Extensive mutagenesis has defined distinct functional domains in the HIV-1 matrix domain (MA). In an attempt to more clearly define functions of regions of MA which affect viral entry, we analyzed mutations in the N-terminal basic and the C-terminal helical domains. Deletions of 8-10 amino acid residues of the C-terminal fifth helix of MA resulted in viruses that were only mildly defective in infectivity and fusion. The defect exhibited by these mutations could largely be attributed to a reduction in levels of viral envelope incorporated into mature virions. Truncation of the gp41 cytoplasmic tail (gp41CT) could rescue the phenotype of one of these mutants. In contrast, mutations of multiple basic residues in the N-terminus of MA were severely defective in both infectivity and fusion. While these mutations induce severe envelope incorporation defects, they also result in virus crippled at a post-entry step, since truncation of the gp41CT could not rescue the infectivity defect.

Fig 1. N-terminal basic domain mutations and C-terminal deletions in MA

A) Mutations used in this study are shown in relation to known functional domains in MA. The MA18,20,22 mutation is comprised of substitutions K18I, R20G, and R22A. The MA32,33 mutation is comprised of substitutions K32I and H33L. D9 and D10 are 10 and 8 amino acid deletions, respectively, in the C-terminal tail of 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 and C) Molecular modeling of N-terminal basic domain mutations and C-terminal deletions, respectively. The model was generated with PyMol software (DeLano Scientific LLC.) based on coordinates from Hill et al (1996).

Fig 2. Effect of N-terminal and C-terminal mutants on infectivity

Equivalent amounts of virus, based on p24 ELISA, were used to infect MAGI-5 (CD4+, R5+, X4+) cells in triplicate. β-gal activity was measured 40 hrs post transfection by counting blue cells. Studies were performed with viruses with or without C-terminal truncation of gp41 (CTD). Values are reported as a percentage of WT (134) infectivity.

Fig 3. N and C-terminal mutants are defective in fusion

Equivalent amounts of virus, based on p24 ELISA, were 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. Studies were performed with viruses with or without C-terminal truncation of gp41 (CTD). A) Fusion of WT (p134) and the D10 mutant virus. Experiments were done in triplicate and values are reported as relative levels of β-galactosidase (β-gal) activity compared to p134 wild type (134WT). A virus lacking envelope (dEnv) was used as a control. B) Fusion of N-terminal mutants. Error bars are derived from triplicate measurements of a representative experiment of a total of 5 independent experiments.

Fig 4. C-terminal Matrix Mutants Display Mild Defects in Envelope Incorporation

Metabolically-labeled virus produced from 134 (WT), D9 and D10 proviral clones was pelleted through a sucrose cushion, resuspended and centrifuged through 20-60% sucrose density gradients. A) CPM of 1ml sucrose gradient fractions 1-16, collected from top to bottom. The range of peak fraction densities is shown, as measured by refractometry. B) SDS-PAGE analysis of viral proteins immunoprecipitated from peak fractions with pooled AIDS patient sera (numbered above each lane according to fraction number). Bands delineating p17 Matrix, p24 Capsid, and gp120 Env are shown. Sizes of molecular weight markers are shown to the left of the gel. C) gp120:p24 ratios from peak fractions as calculated by densitometry are shown.

Fig 5. N-terminal MA mutants display severe defects in envelope incorporation

Metabolically-labeled virus produced from 134 (WT), MA18,20,22, MA32,33 and envelope deficient (Env-) proviral clones was pelleted through a sucrose cushion, resuspended and centrifuged through 20-60% sucrose density gradients. A) Immunoprecipitation of peak fractions was performed with AIDS patient sera. Lane numbers correspond to gradient fraction numbers B) gp120:p24 ratio calculated by densitometry.