Late domain-dependent inhibition of equine infectious anemia virus budding

Abstract

The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5') inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3') potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5', TSG-3', and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5' correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3', whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.

FIG. 1.

The virus budding defect induced by deletion of the YPDL motif in EIAV p9 is reversed by introducing the PPPY or PTAP motif. (A) The schematic organization of the WT and mutant EIAV Gag chimeras used in this study. The location of the L domain of EIAV (YPDL) is shown. The open box within p9 represents the YPDL deletion (ΔYPDL). The EIAV/WT p9 sequence YPDL is replaced by SRSA (EIAV/ΔYPDL), ASAPPPPYVG (EAIV/PPPY), or RPEPTAPP (EIAV/PTAP). CA, capsid protein; NC, nucleocapsid protein. (B) 293T cells were transfected with vectors expressing EIAV/WT Gag, the ΔYPDL deletion mutant, or the PPPY and PTAP chimeras. Transfected cells were metabolically labeled overnight with [³⁵S]Met-[³⁵S]Cys; cell and viral lysates were radioimmunoprecipitated with horse anti-EIAV antiserum (Materials and Methods). The position of the EIAV Gag precursor (Pr55^Gag) is indicated on the left, and the position of molecular mass markers is shown on the right. (C) Virus release efficiency, determined by phosphorimager analysis and calculated as the amount of VLP-associated Gag divided by the total (cell plus VLP) Gag. The data are averages from at least three independent experiments plus or minus the standard error.

FIG. 2.

TSG-5′ inhibits EIAV/PTAP, but not EIAV/WT or EIAV/PPPY, VLP release. 293T cells were transfected with vectors expressing EIAV/WT, EIAV/PPPY, or EIAV/PTAP Gag alone (lanes 1, 3, and 5) or were cotransfected with Gag vectors and the TSG-5′ expression vector at a 1:1 DNA ratio (lanes 2, 4, and 6). (A) Cell and viral lysates were radioimmunoprecipitated with horse anti-EIAV antiserum. (B) The cell lysates were immunoblotted with anti-HA antiserum to detect TSG-5′ expression. For panels A and B, the positions of the EIAV Gag precursor (Pr55^Gag) and TSG-5′ are indicated on the left, and the positions of molecular mass markers are shown on the right. (C) Virus release efficiency, calculated as described in the legend to Fig. 1. The data are averages from at least three independent experiments plus or minus the standard error.

FIG. 3.

EIAV/PTAP, but not EIAV/WT, VLPs incorporate TSG-5′. 293T cells were transfected with vectors expressing EIAV/WT or EIAV/PTAP Gag alone (lanes 1 and 3) or were cotransfected with the Gag plasmids and the TSG-5′ expression vector at a 1:1 DNA ratio (lanes 2 and 4). Cell and viral lysates were immunoblotted with horse anti-EIAV antiserum (top panel) or anti-HA antiserum (lower panel). The positions of Pr55^Gag and TSG-5′ are indicated at the left; the positions of molecular mass markers are shown on the right.

FIG. 4.

EIAV/PTAP and EIAV/PPPY, but not EIAV/WT, VLP release is inhibited by TSG-F and TSG-3′. 293T cells were transfected with vectors expressing EIAV/WT, EIAV/PPPY, or EIAV/PTAP Gag alone (lanes 1) or were cotransfected with Gag plasmids and vectors encoding TSG-3′ (A) or TSG-F (B) at 1:1 DNA ratios (lanes 2). Cell and viral lysates were radioimmunoprecipitated with horse anti-EIAV antiserum, and the cell lysates were subjected to immunoblotting with anti-HA antiserum for the detection of TSG-3′ and TSG-F. Virus release efficiency was calculated as described in the legend to Fig. 1. The data are averages from at least three independent experiments plus or minus the standard error. Molecular mass markers are indicated on the right.

FIG. 5.

EIAV/WT, EIAV/PPPY, and EIAV/PTAP VLP release is suppressed by Vps4EQ. 293T cells were transfected with vectors expressing EIAV/WT, EIAV/PPPY, EIAV/PTAP Gag, or pNL4-3/PR⁻ alone (lanes 1, 3, 5, and 7) or were cotransfected with these DNAs and a vector encoding an EGFP-Vps4EQ fusion protein (lanes 2, 4, 6, and 8). Cell and viral lysates were radioimmunoprecipitated with horse anti-EIAV antiserum (lanes 1 through 6) or anti-HIV Ig (lanes 7 and 8) (A) or were immunoblotted with an anti-eGFP antibody to detect eGFP-Vps4EQ (B). Molecular mass markers are indicated on the right. Virus release efficiency was calculated as described in the legend to Fig. 1 C. The data are averages from at least three independent experiments plus or minus the standard error.

FIG. 6.

The resistance of EIAV Gag to proteasome inhibitors is L domain dependent. (A) 293T cells were transfected in duplicate with EIAV/WT, EIAV/PTAP, or EIAV/PPPY. Twelve hours posttransfection, the cells were treated for 90 min with DMSO (lanes 1, 3, and 5) or with 10 μM MG132 (lanes 2, 4, and 6). The treated cells were metabolically labeled for 2.5 h in the presence or absence of MG132. Cell and viral lysates were radioimmunoprecipitated with horse anti-EIAV antiserum. Molecular mass markers are indicated on the right. (B) Virus release efficiency was calculated as described in the legend to Fig. 1. The data are averages from at least three independent experiments plus or minus the standard error.