Matrix-induced inhibition of membrane binding contributes to human immunodeficiency virus type 1 particle assembly defects in murine cells - PubMed (original) (raw)
Matrix-induced inhibition of membrane binding contributes to human immunodeficiency virus type 1 particle assembly defects in murine cells
Theodora Hatziioannou et al. J Virol. 2005 Dec.
Abstract
Defective human immunodeficiency virus type 1 (HIV-1) assembly in murine cells is accompanied by poor plasma membrane binding and proteolytic processing of the HIV-1 Gag precursor. Here, we show that such defects are induced by the propensity of the HIV-1 MA globular head to inhibit membrane binding and particle assembly, particularly at the low expression levels observed in murine cells. Simple additions to or deletion of the MA globular head can improve the yield of infectious virions from murine cells by >50-fold. Expression level and autoinhibition can be important confounding variables in studies of HIV-1 assembly and contribute to defects encountered in murine cells.
Figures
FIG. 1.
Simple modifications of HIV-1 MA markedly improve membrane binding in murine cells. (A) Schematic representation of the constructs used in this study. WT is wild-type unmodified Gag with intact MA capsid (CA), nucleocapsid (NC), and p6 domains. S-Gag is appended with a 10-residue peptide MGSSKSKPKD from c-Src, while δGH lacks MA residues 7 to 110. (B) Flotation analysis of Gag membrane interactions. Clarified homogenates of WT-infected HeLa (upper panel) and 3T3/CycT (lower panel) cells were mixed with 80% sucrose and overlaid with 65% and 10% sucrose prior to ultracentrifugation. A Western blot analysis using an α-CA antibody (183-H12-5C) of gradient fractions is shown. Membrane-associated proteins appear in the top three fractions, while cytosolic proteins appear in the bottom three fractions. Note that a longer exposure of the 3T3/CycT-derived blot is shown. (C) Myristoylation of Pr55Gag in both human and murine cells. [3H]myristate-labeled Gag proteins from WT HIV-1-infected HeLa and 3T3/CycT cells were immunoprecipitated by using serum from an HIV-1-infected individual and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography (upper panel). The lower pane shows the same immunoprecipitates analyzed for Pr55Gag content by Western blotting with the 183-H12-5C antibody (lower panels). (D and E) Modifications of Pr55Gag improve membrane binding and processing. Analyses were carried out as described for panel B except that the S-Gag and δGH viruses were used to infect HeLa cells.
FIG. 2.
MA modifications enhance infectious virion release in HOS and 3T3/CycT cells. (A) Western blot analysis of HOS cells (left) and 3T3/CycT cells (right) using antibodies specific for late gene expression (Gag, upper panel) and early gene expression (GFP, center panel). The lower panel shows Western blot analysis of Gag pelleted through a 20% sucrose cushion following filtration (0.2 μm) of culture supernatants. Also shown below the blots are the concentrations of p24 capsid (CA) (pg/ml) measured in the culture supernatants by enzyme-linked immunosorbent assay. (B) Infectious virus yield from transfected HOS and 3T3/CycT cells. The VSV-G-pseudotyped virions were used to inoculate HOS cells, and the GFP-positive target cells were enumerated 48 h later by fluorescence-activated cell sorter analysis, as previously described. Titers are given as infectious units (i.u.) per ml of supernatant. Figures above the bars represent the increase (_n_-fold) in titer over that of the WT construct.
FIG. 3.
Efficiently expressed, codon-optimized Gag-Pol constructs yield HIV-1 virus-like particles in murine cells, but MA-induced assembly defects can be recapitulated by expression at lower levels. (A) Western blot analysis of 3T3/CycT cell lysates (upper and middle panels) and virions or VLPs pelleted through 20% sucrose (lower panels) following transfection with 1 μg pSYNGP (cytomegalovirus promoter) plus 1 μg pCMV/GFP (left lane) or 2 μg of the R7/NL/δEnv/GFP proviral plasmid (right lane). Western blots were probed as described in the legend to Fig. 1. (B) Western blot analysis using an α-capsid (α-CA) antibody (183-H12-5C) of cell lysates (upper panels) and VLPs (lower panels) following transfection of HOS cells (left panels) or 3T3/CycT cells (right panels) with pSYNGP-derived WT, S-Gag, or δGH Gag-Pol expression vector. Each group of three lanes shows cells transfected with decreasing amounts (from left to right, 1 μg, 0.5 μg, and 0.25 μg) of each pSYNGP-derived construct.
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