Amphotropic murine leukaemia virus envelope protein is associated with cholesterol-rich microdomains

Abstract

Background: Cholesterol-rich microdomains like lipid rafts were recently identified as regions within the plasma membrane, which play an important role in the assembly and budding of different viruses, e.g., measles virus and human immunodeficiency virus. For these viruses association of newly synthesized viral proteins with lipid rafts has been shown.

Results: Here we provide evidence for the association of the envelope protein (Env) of the 4070A isolate of amphotropic murine leukaemia virus (A-MLV) with lipid rafts. Using density gradient centrifugation and immunocytochemical analyses, we show that Env co-localizes with cholesterol, ganglioside GM1 and caveolin-1 in these specific regions of the plasma membrane.

Conclusions: These results show that a large amount of A-MLV Env is associated with lipid rafts and suggest that cholesterol-rich microdomains are used as portals for the exit of A-MLV.

Figure 1

A-MLV envelope protein associates with detergent resistant microdomains (DRMs). A) 293T cells producing A-MLV were treated with TX-100 at 4°C and loaded on a discontinuous sucrose gradient. Western blot analyses were performed. Fraction 1 corresponds to the top and fraction 6 corresponds to the bottom of the tube. Fractions 1 to 4 contain the DRMs, fractions 5 to 6 the non-DRM membrane fractions. A-MLV Env is found predominantly in the DRM fractions 2, 3 and 4. EGFP, which is localized in the cytoplasm, remains in the soluble fractions 5 and 6. B) NIH3T3 cells releasing A-MLV were treated with TX-100 at 4°C and loaded on a discontinuous sucrose gradient. Dot blot analyses were performed. Fraction 1 corresponds to the top and fraction 6 corresponds to the bottom of the tube, respectively. B is the background of the dot blot. Fractions were processed in parallel for immunological detection of cav-1 and A-MLV Env. C) Quantification of the dot blot shown in B) using image analysing software. The amounts shown are determined as percentages of the total of all dots; DRM (fractions 1 to 3), non-DRM (fractions 4 to 6). D) Detergent soluble supernatant (non-DRM) and insoluble pellet (DRM) of A-MLV producing NIH3T3 cells treated with TX-100 at 4°C or 37°C were investigated for the amount of envelope protein using dot blot analysis. The results of two independent experiments are shown. The amounts shown are determined as percentages of the total of all dots.

Figure 2

Immunocytochemical investigations of the association of proteins with DRMs. A) NIH3T3 cells producing A-MLV were treated with PBS, TX-100 or MBCD as indicated and subsequently subjected to TX-100 extraction and stained for cav-1, GM1, CD71 and A-MLV Env as indicated. B) Background of the secondary antibody used for cav-1 staining. C) Background of the secondary antibody used for A-MLV Env staining. D) NIH3T3 cells (Env negative) stained for A-MLV Env, negative control (see text for details). Photographs were taken using an oil immersion objective, original magnification 1000×.

Figure 3

A-MLV Env co-localization with cholesterol, GM1 and cav-1. A) A-MLV Env co-localization with cholesterol. NIH3T3 cells producing wild-type A-MLV were treated with filipin for cholesterol detection (left column) and with an A-MLV Env specific antibody (second column) after fixation and treatment with PBS (top) or TX-100 at 4°C (bottom). Co-localization result in pink spots (merged images, third column). The column on the right shows the result of the co-localization finder plugin of the ImageJ program [30] merged with the original A-MLV Env staining. Turquoise colour indicates co-localization of A-MLV Env with cholesterol. B) A-MLV Env and cav-1 co-localization monitored by fluorescence microscopy. Immunofluorescent detection of cav-1 (left) and the A-MLV Env (middle) after treatment with TX-100 at 4°C in NIH3T3 cells producing A-MLV. Co-localization result in yellow spots (right). C) A-MLV Env (left) and GM1 (middle) were detected by immunofluorescence in A-MLV producing NIH3T3 cells after PBS (top) or TX-100 treatment at 4°C (bottom). Co-localization result in yellow spots (right). All photographs were taken using a fluorescence microscope and oil immersion objective, original magnification 1000×.