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. 2003 Dec 23;100(26):15812-7.
doi: 10.1073/pnas.2634931100. Epub 2003 Dec 10.

Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions

Ping Zhu  1 , Elena Chertova, Julian Bess Jr, Jeffrey D Lifson, Larry O Arthur, Jun Liu, Kenneth A Taylor, Kenneth H Roux
Affiliations

Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions

Ping Zhu et al. Proc Natl Acad Sci U S A. .

Abstract

We used electron tomography to directly visualize trilobed presumptive envelope (env) glycoprotein structures on the surface of negatively stained HIV type 1 (HIV-1) and simian immunodeficiency virus (SIV) virions. Wild-type HIV-1 and SIV virions had an average of 8-10 trimers per virion, consistent with predictions based on biochemical evidence. Mutant SIVs, biochemically demonstrated to contain high levels of the viral env proteins, averaged 70-79 trimers per virion in tomograms. These correlations strongly indicate that the visualized trimers represent env spikes. The env trimers were without obvious geometric distribution pattern or preferred rotational orientation. Combined with biochemical analysis of gag/env ratios in virions, these trimer counts allow calculation of the number of gag molecules per virion, yielding an average value of approximately 1400. Virion and env dimensions were also determined. Image-averaging analysis of SIV env trimers revealed a distinct chirality and strong concordance with recent molecular models. The results directly demonstrate the presence of env trimers on the surface of AIDS virus virions, albeit at numbers much lower than generally appreciated, and have important implications for understanding virion formation, virus interactions with host cells, and virus neutralization.

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Figures

Fig. 2.
Fig. 2.
Representative 3D tomograms from virions with high and low env content. (A) Representative tomogram sections from a SIVmac239 virion with truncated TM glycoprotein and high env content, produced from Sup-T1-CCR5 cells. Trilobed propeller-like presumptive trimer structures are readily apparent (n ≈ 73). (B) Representative tomogram sections from an HIV-1 MN (wild-type) virion from Sup-T1-CCR5 cells (n ≈ 17 trimers). For each tomogram series, the top and bottom sections are in Upper Left and Lower Right, respectively. A representative env spike on the bottom face (black arrow) and projecting from an equatorial section (white arrow) of each virus type is indicated. A cluster of env spikes is located to the right of the bracket (Upper Left) of HIV-1 MN (B). (Bar = 100 nm.)
Fig. 1.
Fig. 1.
HPLC analysis of the SIV and HIV-1 viruses. Details for virus preparations are shown in Table 1. The viruses include two preparations of the same HIV-1 isolate (HIV-1MN) with a full-length TM and low env content, produced from MHC-II-positive (A) and MHC-II-negative (B) cells, one SIV (SIVmac239) with low env content (C), and two SIV preparations with truncated TM glycoproteins and high env content (SIVmac239/tail/CEMX174, D); and SIVmac239/SupT1, E). All show a 1:1 molar ratio of SU/TM. Peaks for viral gag proteins [p1, p6, p7/p8, matrix (MA), capsid (CA)] are labeled. Peaks for gp120SU and gp41/32™ are indicated in red and dark blue, respectively. Peaks containing cellular proteins of interest are indicated as follows: MHC-I, light blue; β2-microglobulin, turquoise; actin (for virus produced from MHC-II cells), lavender; actin and MHC-II (for virus produced from MHC-II+ cells), yellow and green.
Fig. 3.
Fig. 3.
Image-averaged and model-fitted SIV env trimers. Individual trimer images from the top (A) and bottom (B) halves of SIV virions with truncated TM glycoproteins and high env content (SIVmac239/Sup-T1-CCR5 and SIVmac239-tail/CEMx174) were classified, rotated, aligned, and averaged. Trimers were sorted into six categories based on their similarity, aligned to each other by rotation and translation in each category, and then averaged. From top to bottom, the images are from the average of 23, 23, 17, 15, 13, and 8 (A) and 25, 16, 15, 9, 8, and 5 (B) individual trimers, respectively. The glycosylated env trimer molecular model of Kwong et al. (6) was superimposed on the trimer profiles for the corresponding top (as viewed from the outside of the virion) (C) and bottom (as viewed from the inside of the virion) (D) averaged images. The averaged images selected for comparison were both the largest in profile and represented the sets with greatest number of images (i.e., the top image from A and B). (Bars = 10 nm.)
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