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. 2003 Dec;77(24):13389-95.
doi: 10.1128/jvi.77.24.13389-13395.2003.

Alpha interferon inhibits human T-cell leukemia virus type 1 assembly by preventing Gag interaction with rafts

Xuan Feng  1 , Nancy Vander Heyden, Lee Ratner
Affiliations

Alpha interferon inhibits human T-cell leukemia virus type 1 assembly by preventing Gag interaction with rafts

Xuan Feng et al. J Virol. 2003 Dec.

Abstract

Alpha-2a interferon (IFN-alpha2a) has beneficial clinical effects on human T-cell leukemia virus type 1 (HTLV-1) infection, but its antiviral mechanism of action is unknown. Antiviral effects of IFN-alpha2a were studied in 293T cells expressing HTLV-1 proviral DNA and in HTLV-1-infected cells (HOS/PL, MT2, and HUT102). In 293T cells, an 50% inhibitory concentration of 10 U of IFN-alpha2a/ml was determined by p19 antigen ELISA. Analysis of IFN-treated cells demonstrated no defect in viral protein synthesis but did show a decrease in the level of released virus, as determined by immunoblot assays. Electron microscopy studies of IFN-treated cells revealed neither a defect in the site of virus budding nor tethering of virus particles at the plasma membrane, thus arguing against an effect on virus release. Cell fractionation studies and confocal microscopy showed no effect of IFN on Gag association with membranes. However, the level of Gag association with lipid rafts was decreased, suggesting a role of IFN in inhibiting HTLV-1 assembly.

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Figures

FIG. 1.
FIG. 1.
Viral protein synthesis during IFN treatment. 293T cells were pretreated with various amounts of IFN-α2a (0, 5, 10 50, 100, and 500 U/ml) for 16 h and were then transfected with proviral DNA, pACH, in the presence of IFN-α2a for 72 h. The same amount of protein from cell lysates and supernatants was loaded in SDS-10% polyacrylamide gel electrophoresis. Anti-HTLV-1 human MAb against p19Gag and polyclonal antibody against cellular actin were used to react with blots. IFN reduced virion p19Gag in cell supernatants in a dose-dependent manner (A) and had no effect on the viral structural protein Gag, and cellular actin in cell lysates (B). IFN significantly reduced p19Gag production in supernatants, based on quantitation of virion production with p19 ELISA. Representative data from four independent experiments are indicated in panel C. The bar labeled “con” indicated mock-transfected cells.
FIG. 2.
FIG. 2.
Electron microscopic analysis of viral budding. 293T cells were transfected with ACH in the absence or presence of 100 U of IFN/ml for 3 days. Cells were fixed, and viral particles were visualized by electron microscopy. Virus particles are seen budding through the plasma membrane of ACH-expressing cells in the absence of IFN (A) or the presence of IFN treatment (B to D). No virus particles were detected when cells treated with 50 μM proteasome inhibitor lactacystin for 2.5 h prior to harvest. Slightly curved and electron-dense thickenings are seen in panels E to G. Magnification, ×50,000.
FIG. 3.
FIG. 3.
Confocal and immunoblot analysis of Gag membrane localization after IFN treatment. ACH-transfected HeLa cells were treated with IFN for 2 days and then were fixed, permeabilized, and stained with anti-p19Gag. There was a discrete, punctate pattern of Gag protein at the plasma membrane in untreated cells (A), as well as in IFN-treated cells (B). Postnuclear supernatants derived from 293T cells expressing ACH with or without IFN treatment were subjected to equilibrium flotation centrifugation. p19Gag was detected by immunoblot in membrane fractions 1 to 4 and nonmembrane fractions 8 to 10 (C).
FIG. 4.
FIG. 4.
Inhibitory effect of IFN on Gag-raft association. IFN increases the detergent solubility of Gag (A). ACH-transfected 293T cells in the absence (upper panel) or presence (lower panel) of IFN were treated with or without 0.5% Triton X-100 (Tx100) on ice for 20 min before fixation. Gag membrane localization was detected by confocal analysis by using anti-p19Gag. In the presence of Triton X-100, the punctate fluorescence pattern was largely preserved in DRMs without IFN. However, it was largely lost in DRMs from cells treated with 100 U of IFN/ml. IFN blocks Gag-raft colocalization (B). ACH-expressing cells were stained with anti-p19Gag MAb (green) and anti-GM1 (red). p19Gag immunofluorescence colocalized with raft-associated marker GM1 in the absence of IFN (the upper panel), whereas only a small portion of p19Gag was colocalized with GM1 following IFN treatment (the lower panel).
FIG. 5.
FIG. 5.
IFN blocks Gag-raft association. Postnuclear supernatants derived from ACH-transfected 293T cells were treated with or without 0.25% Triton X-100 (Tx100) on ice and were subjected to equilibrium flotation centrifugation. In the absence of IFN, no changes in level of Gag-raft association were detected (A), whereas IFN significantly reduced the level of Gag-raft association (B). Raft-associated proteins caveolin-1 and flotilin-1 were used to determine membrane and DRMs, and TfR was used as a detergent-sensitive (DS), nonraft marker (C).
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