Equine infectious anemia virus utilizes host vesicular protein sorting machinery during particle release

Abstract

A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.

FIG. 1.

EIAV Gag VLP production and cellular localization. (A and B) Cell lysates and culture supernatants from transfected 293T cells were subjected to SDS-PAGE and Western blot analysis with anti-EIAV Gag (A) and anti-GFP (B) primary antibodies. Arrows denote Gag products. The asterisk denotes higher-molecular-mass species that frequently cross-react with antibody. Molecular masses are shown to the left of the blots, and lane numbers are shown below. (C) Confocal microscopy of Gag-YFP fusion-transfected HeLa cells. Both panels represent single optical sections. The membrane-localized fluorescence signal was similarly seen in unfixed cells.

FIG. 2.

Effect of dnVPS4 K173A on EIAV Gag budding. (A) 293T cells were transfected with wt or dnVPS4 constructs and subsequently analyzed by SDS-PAGE and immunoblotting with an anti-V5 primary antibody. Equal volumes of cell lysates are loaded in both lanes. (B) 293T cells were transfected with 20 μg of indicated Gag construct and 10 μg of indicated VPS4 construct or empty vector. Cell lysates and culture supernatants were separated by SDS-PAGE and analyzed by immunoblotting with a mouse anti-GFP primary antibody. The arrow denotes Gag fusion proteins. Lane numbers and size markers are shown below and to the left of the blot, respectively. (C) Confocal microscopy of HeLa cells transfected with the indicated Gag and VPS4 constructs. Panels represent a single optical section of cells. Similar staining was visualized in unfixed cells also.

FIG. 3.

TSG101 inhibition does not affect EIAV Gag VLP production. (A) 293T cells were transfected with the TSG101 construct in the presence (lane 1) or absence (lane 2) of siRNA against TSG101. TSG101 expression in transfected cell lysates was assessed by SDS-PAGE and subsequent Western blot analysis with an anti-HA primary antibody. The arrow denotes TSG101 cross-reacting bands. (B) 293T cells were transfected with either HIV-1 Gag (lanes 1 to 4) or EIAV Gag (lanes 5 to 8) in the presence or absence of siRNA against TSG101 (denoted by a plus or minus sign below each lane marker). Cell lysates and culture supernatants were analyzed for HIV-1 and EIAV Gag content by SDS-PAGE and subsequent Western blotting with anti-HIV-1 and anti-GFP antibodies, respectively. Lanes are indicated below the blot.

FIG. 4.

VPS28 fusion rescues budding phenotype in a late-domain mutant and incorporates endogenous TSG101 into particles. 293T cells were transfected with 20 μg of the indicated constructs and separately Western blotted with an anti-EIAV Gag (A) or anti-TSG101 (B) antibody. The arrow denotes EIAV fusion protein (A) or endogenous TSG101 (B) with lane numbers noted below. The bracket marked with an asterisk denotes higher-molecular-mass species that cross-react with the anti-EIAV Gag antibody.