Mechanisms of late restriction induced by an endogenous retrovirus

Abstract

The host has developed during evolution a variety of "restriction factors" to fight retroviral infections. We investigated the mechanisms of a unique viral block acting at late stages of the retrovirus replication cycle. The sheep genome is colonized by several copies of endogenous retroviruses, known as enJSRVs, which are highly related to the oncogenic jaagsiekte sheep retrovirus (JSRV). enJS56A1, one of the enJSRV proviruses, can act as a restriction factor by blocking viral particles release of the exogenous JSRV. We show that in the absence of enJS56A1 expression, the JSRV Gag (the retroviral internal structural polyprotein) targets initially the pericentriolar region, in a dynein and microtubule-dependent fashion, and then colocalizes with the recycling endosomes. Indeed, by inhibiting the endocytosis and trafficking of recycling endosomes we hampered JSRV exit from the cell. Using a variety of approaches, we show that enJS56A1 and JSRV Gag interact soon after synthesis and before pericentriolar/recycling endosome targeting of the latter. The transdominant enJS56A1 induces intracellular Gag accumulation in microaggregates that colocalize with the aggresome marker GFP-250 but develop into bona fide aggresomes only when the proteasomal machinery is inhibited. The data argue that dominant-negative proteins can modify the overall structure of Gag multimers/viral particles hampering the interaction of the latter with the cellular trafficking machinery.

FIG. 1.

Kinetics of JSRV/enJS56A1 Gag staining pattern. (A and B) Quantification of JSRV and enJS56A1 Gag staining pattern in confocal microscopy of HeLa cells at various time points posttransfection. The number of cells in which Gag accumulation was observed diffused, dispersed, and concentrated was counted. Approximately 100 cells were evaluated at each time point. Graphs represent the average of two independent experiments. (C) HeLa cells expressing JSRVHA-MA and enJS56A1Flag-MA were fixed at the 4-h time point and analyzed by confocal microscopy using antibodies to the Flag (green) and Ha (red) epitopes and appropriate secondary conjugated antibodies. Scale bars, 10 μm.

FIG. 2.

Effects of myristoylation in Gag intracellular distribution and enJS56A1-induced restriction. (A) HeLa cells expressing JSRV-G2A were fixed at 24 h posttransfection and stained with anti-JSRV MA (green) and anti-γ-tubulin (red) antibodies. Scale bars, 10 μm. (B) Quantification of Gag staining patterns in confocal microscopy of HeLa cells expressing JSRV-G2A at various times posttransfection. Approximately 100 cells were evaluated at each time point. Graphs represent the average of two independent experiments. (C) Quantification of Gag staining pattern in JSRV and enJS56A1 myristoylation-defective mutants by confocal microscopy in HeLa cells at 24 h posttransfection. The graph represents the number of cells in which Gag staining was observed concentrated as opposed to diffused and dispersed. The number of cells counted in each experiment is represented on top of the bars. (D) SDS-PAGE and Western blotting of viral pellets (upper panel) and cell lysates (lower panel) of cells transfected with the indicated plasmids. Filters were incubated with antibodies to the JSRV CA.

FIG. 3.

The targeting of the JSRV Gag to the centrosomal region is dependent on dynein and intact microtubule network. (A) Quantification of JSRV, JSRV-G2A, enJS56A1, and JSRVHA-MA Gag staining patterns by confocal microscopy in HeLa cells after treatment with nocodazole or with the solvent dimethyl sulfoxide used as a negative control. Gag proteins were detected by using antibodies to JSRV MA or HA. The graph represents the number of cells in which Gag staining was observed concentrated as opposed to diffused or dispersed. The number of Gag-positive cells counted in each experiment is indicated above each bar. Note that the use of anti-HA antibodies, compared to polyclonal anti-JSRV-MA serum, results normally in a relatively higher percentage of cells with concentrated Gag staining and a lower percentage of cells displaying diffuse Gag. These differences are probably due to the different sensitivity of the two antisera. (B) The effect of p50-GFP on Gag staining pattern was evaluated as in panel A. The number of Gag-positive cells counted in each experiment is indicated above each bar. Note that only cells double positive for Gag and p50-GFP were counted. (C) Graph representing the quantification of three independent SDS-PAGE and Western blotting experiments of viral particles released in the supernatant of 293T cells transfected with a JSRV expression plasmid in the presence or absence of p50-GFP. Filters were incubated with an antiserum to the JSRV CA. Signals were quantified by chemifluorescence as described in Materials and Methods. Bars indicate the standard errors.

FIG. 4.

JSRV Gag colocalizes with pericentriolar Rab11-positive endosomes. (A) HeLa cells were transfected with JSRV- or enJS56A1-expressing plasmids. At 24 h posttransfection, cells were transferred to serum-free medium for 1 h and incubated with fresh medium containing transferrin conjugated to Alexa Fluor 568 (red). After 30 min of incubation, the cells were fixed and analyzed by confocal microscopy using antibodies to the JSRV MA and a secondary antibody conjugated with Alexa Fluor 488. (B) HeLa cells transfected with JSRV or enJS56A1 expression plasmids were fixed and analyzed by confocal microscopy using antibodies to JSRV MA and Rab11a with the appropriate conjugates. Panels in black and white show colocalization using the plug-in of the Image J program. Scale bars, 10 μm.

FIG. 5.

JSRV exit from the cells is facilitated by functional recycling endosomes. (A) HeLa cells were transfected with plasmids expressing the GFP-tagged DIIIΔ2 or EΔ95/295. At 24 h posttransfection, cells were transferred to serum-free medium for 1 h. Cells were then washed and incubated in fresh medium containing transferrin conjugated to Alexa Fluor 568 (Red). After 30 min of incubation, cells were fixed and images acquired. Scale bars, 10 μm. (B) Western blot analysis of JSRV in presence of D3Δ2 or EΔ95/295. Viral pellets of cells transfected with the indicated plasmids were resolved by SDS-PAGE and immunoblotted with an antiserum to JSRV CA. The graph shows the quantification by chemifluorescence of three independent Western blotting experiments as described in Materials and Methods. (C) Western blot analysis of Rab11 expression after siRNA treatment. Lysates of cells transfected with a scrambled siRNA or Rab11a and Rab11b siRNAs were resolved by SDS-PAGE and immunoblotted with antibodies to Rab11a (upper panel) or γ-tubulin (lower panel). Three independent experiments were quantified as in panel B. (D) Western blot analysis of JSRV viral particles release in cells treated with the indicated siRNAs using antibodies to the JSRV CA. The graph shows the quantification of signal intensities given by the virus pellets in three independent experiments as described in Materials and Methods (bars indicate the standard errors).

FIG. 6.

enJS56A1 Gag colocalizes with GFP-250 and polyubiquitin-rich residues. (A) HeLa cells were cotransfected with plasmids expressing either enJS56A1 or JSRV with the aggresome marker GFP-250. At 24 h after transfection, cells were fixed and analyzed by confocal microscopy using an antiserum to the JSRV MA and the appropriate conjugate. Black and white pictures showing colocalization were obtained with the Image J program. (B) HeLa cells were transfected with JSRV- or enJS56A1-expressing plasmids. At 24 h after transfection, cells were fixed and analyzed by confocal microscopy using antibodies to the JSRV MA and polyubiquitin residues (Fk1) with the appropriate fluorescent conjugates. Black and white pictures showing colocalization were obtained with the Image J program. Scale bars, 10 μm.

FIG. 7.

enJS56A1 Gag forms an aggresome in the presence of lactacystin. (A) HeLa cells were cotransfected with GFP-250 and either JSRV or enJS56A1 expression plasmids. At 24 h posttransfection cells were treated with lactacystin for 6 h and then fixed and analyzed by confocal microscopy using an antiserum to JSRV MA with the appropriate labeled secondary antibody. Colocalization between enJS56A1 Gag and GFP-250 is shown also in black and white images obtained with the plug-in of the Image J program. (B) Quantification of JSRV and enJS56A1 Gag staining patterns by confocal microscopy in HeLa cells after treatment with lactacystin (6 h) (L), lactacystin plus nocodazole (L+N) for 4 h, or EtOH. EtOH was used as a solvent for lactacystin and was also used as a negative control. Gag was detected by using antibodies to JSRV MA and the appropriate conjugate. The graph represents the number of cells in which Gag staining was observed concentrated as opposed to diffused and dispersed. The number of Gag-positive cells counted in each experiment is indicated above each bar. (C) HeLa cells were transfected with plasmids expressing GFP-250 or enJS56A1. At 24 h after transfection, some of the cells (as indicated) were treated with lactacystin or ethanol as a negative control for further 12 h. Cells were then fixed and analyzed by confocal microscopy using antibodies to vimentin antibody labeled with Cy3 (Red), γ-tubulin, or JSRV Gag in the presence of appropriate secondary antibodies. Scale bars, 10 μm. (D) SDS-PAGE and Western blotting analysis of cells expressing JSRV and enJS56A1 in the presence or absence of lactacystin. Two different exposures of the same gel (indicated as “Long” and “Short”) are shown in the figure to better appreciate the different levels of Gag in the presence or absence of lactacystin. Filters were incubated with antibodies to the JSRV CA or γ-tubulin to control for loading uniformity.

FIG. 8.

Model of JSRV Gag trafficking and enJS56A1 restriction. See Discussion for details.