Murine leukemia virus glycosylated Gag (gPr80gag) facilitates interferon-sensitive virus release through lipid rafts

Abstract

Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65(gag), the precursor for internal structural (Gag) proteins. Whereas gPr80gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80gag-facilitated release occurs through lipid rafts, because gPr80gag-negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MbetaCD, and there is less Pr65gag associated with detergent-resistant membranes in mutant-infected cells. gPr80gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.

Fig. 1.

Effect of IFN-α on virus release from 43D and 17-5 cells. (A) 43D and 17-5 cells were treated with different concentrations of mouse IFN for 24 h, after which media were replaced and the cells and released viruses were collected 6 h after incubation. The same portions of cells and released viruses were subjected to the Western blots with anti-CA^p30. (B) Virus release efficiency from 43D and 17-5 cells was calculated (means ± SD from three replicate experiments). (C and D) PA317/BAG/ΔMCS and PA317/BAG/p8065-2 cells were analyzed as in A and B.

Fig. 2.

Differential Gag localization in lipid rafts. (A) Lysates (1% Triton X-100) from 43D and 17-5 cells were analyzed by membrane flotation in 5–30% sucrose gradients. Low-density fractions represented detergent-resistant membranes (DRMs)/lipid rafts, whereas high-density fractions represented protein in detergent-soluble membranes (DSMs) or cytosolic proteins. Gradient fractions were analyzed by Western blots for viral CA^p30, Caveolin (lipid rafts marker), and CD-71 (nonlipid rafts marker). The amount of Pr65^gag was quantified by densitometry. The percentage of Pr65^gag in lipid rafts is shown in parentheses. The band migrating more rapidly than Pr65^gag represents a partial proteolytic cleavage product. There are two bands corresponding to gPr80^gag (absent from 17-5 cells) that correspond to different states of glycosylation. (B) 43D cells (a and b) and 17-5 cells (c and d) were incubated with ice-cold PBS (a and c) or 0.5% Triton X-100 (b and d) followed by fixation with paraformaldehyde and then incubation with anti-CA^p30 antibody. The antigens and nuclei were visualized by secondary antibodies conjugated with Alexa 488 and DAPI (to visualize the nuclei). The regions indicated by arrows were enlarged in the insets.

Fig. 3.

MβCD impairment of viral release. (A) 43D and 17-5 cells were treated with different concentrations of MβCD and the amount of virus release after 6 h was assayed as in Fig. 1. (B) Viral release efficiencies from 43D and 17-5 cells were calculated as in Fig. 1 (means of two experiments ± SD). (C) 43D and 17-5 cells were incubated with or without 5 mM MβCD for 12 h. Viruses were harvested and analyzed by density gradient centrifugation, and distribution of CA^p30 was analyzed by Western blots and densitometry. The lower amounts of virus released from the MβCD-treated cells likely reflected cell toxicity after longer incubation in MβCD and serum-free medium.

Fig. 4.

gPr80^gag increases the efficiency of HIV-1 particle release. The HIV-1 Gag-Pol expression vector was transfected with or without p8065-2 into 293T cells. (A) The cells and media were harvested 48 h after transfection, and the same portions of cells and viruses were analyzed by Western blots with anti-HIV CA^p24 and anti-β-Tubulin antibodies. The relative amounts of Gag (B) in cells and viral release efficiencies (C) were quantified (means of two experiments, ±SD).