Foamy virus envelope glycoprotein is sufficient for particle budding and release

Abstract

Foamy viruses (FVs) are classified in the family Retroviridae, but recent data have shown that they are not conventional retroviruses. Notably, several characteristics of their particle replication strategies are more similar to those of hepatitis B virus (HBV) than those of typical retroviruses. Compared to conventional retroviruses, which require only Gag proteins for budding and release of virus-like particles (VLPs), both FV and HBV require Env proteins. In the case of HBV, Env (S protein) alone is sufficient to form subviral particles (SVPs). Because FVs also depend on Env for budding, we tested whether FV Env alone could produce SVPs. The Env proteins of FV and murine leukemia virus (MuLV) were both released into cell culture supernatants and migrated into isopycnic gradients; however, unlike MuLV Env, FV Env displayed characteristics of SVPs. FV Env particles were of greater density than those of MuLV (1.11 versus 1.07 g/ml, respectively), which strongly suggested that the released proteins of FV Env were particulate. When we examined FV SVPs by immunoelectron microscopy, we found particles that were consistent in morphology, size, and staining with gold beads, similar to FV VLPs and unlike the particle-like structures of MuLV Env, which were more consistent with vesicles produced from nonspecific membrane "blebbing." Taken together, our results demonstrated that FV Env alone is sufficient for particle budding. This finding is unique among retroviruses and further demonstrated the similarities between FV and HBV.

FIG. 1.

Autoradiography of SFVcpz(hu) and MuLV proteins from transiently transfected 293T cells. (A) SFVcpz(hu) proteins. Results for cell lysates, supernatants, and viral pellets from cells transfected with no DNA (mock) or vectors expressing SFVcpz(hu) Gag and Pol (pCgp1) alone, Gag and Pol with WT Env (pCE-K), Gag and Pol with an ERRS mutant Env (pCE-S), or ERRS mutant Env alone and immunoprecipitated with chimp serum are shown. Viral pellets were centrifuged through 20% sucrose. (B) MuLV proteins. Results for cell lysates and supernatants from cells transfected with no DNA (mock) or a vector expressing MuLV Env (pCME-wt) and immunoprecipitated with goat anti-MuLV antisera are shown. (C) NP-40 sensitivity of MuLV and SFVcpz(hu) Env complexes collected from the supernatant of cells transfected with pCME-wt or pCE-S, spun through 20% sucrose, and immunoprecipitated with goat anti-MuLV antiserum or chimp serum, respectively. (D) Viral pellets from COS-1 cells transfected with pCE-K or pCE-S. (E) Cell supernatants from 293T cells transfected with pCE-K or pCE-K/Clv (pCE-K containing the cleavage mutant R572T). PRE, precursor envelope glycoprotein (gp).

FIG. 2.

Comparison of densities of MuLV Env complexes and SFVcpz(hu) Env complexes from Optiprep gradient. The Env complexes from cell culture supernatants were collected from 293T cells transiently transfected with pCME-wt or pCE-S, concentrated through 20% sucrose, layered over the same Optiprep gradient, and spun to equilibrium. Sequential aliquots were removed from the top and immunoprecipitated, first with goat anti-MuLV antiserum and secondwith chimp serum. (A) Autoradiographies of gradient fractions (numbered sequentially above each lane) revealing banding pattern of MuLV Env proteins (top panel) and SFVcpz(hu) Env proteins (bottom panel). Densities (grams per milliliter) were determined for the indicated fractions and appear over the corresponding fraction numbers. (B) Quantitative protein measurements of SU and TM proteins from each gradient fraction made by phosphorimaging analysis and plotted as digital light units (DLU).

FIG. 3.

Electron micrographs of immunogold-labeled 293T cells (A to H) and cell-free, Optiprep-purified Env particles (I to O). 293T cells were transiently transfected with no DNA (A), pCE-S (B to E), pCE-S and pCgp1 (F), or pCME-wt (G and H), incubated with rabbit anti-HFV antiserum for FV proteins or goat anti-MuLV antiserum for MuLV proteins, incubated with appropriate immunogold (10-nm particle size)-labeled secondary antibody, fixed, and thin sectioned. Cell-free Env particles were collected from 293T cells transiently transfected with empty plasmid (I), pCE-S (J to M), pCE-S and pCgp1 (N), or pCME-wt (O), concentrated on a 50% Optiprep cushion, and purified through an Optiprep gradient. The particles were collected from the appropriate fractions, concentrated, and resuspended in 100 μl of PBS. Twenty microliters of sample was applied to a nickel grid, incubated with primary antiserum, incubated with appropriate immunogold-labeled secondary antibody as described above, and negatively stained with 1% uranyl acetate. Magnification: panels A to D and F to H, 30,000; panels E and I to O, 70,000. Bars, 100 nm.

FIG. 4.

Expression and release of WT SFVcpz(hu) Env in the absence or presence of FV Gag and Pol. Autoradiographies demonstrating cellular expression levels (A) and 20% sucrose pellets of cell culture supernatants (B) collected from 293T cells transiently transfected with pCE-K alone or with pCgp1 are shown. (C) Released Env complexes in 20% sucrose pellets, as shown in panel B, quantified by phosphorimaging analysis and displayed as digital light units (DLUs).

FIG. 5.

Detection of SVPs in SFVcpz(hu)-infected cells by sucrose density gradient analysis of particles collected from infected BHK-21 cells. (A) Autoradiography of sucrose gradient. BHK-21 cells were infected with SFVcpz(hu) at a multiplicity of infection of 1.5, and particles were collected from the culture supernatant at 5 days postinfection. The SFVcpz(hu) particles were concentrated through a 20% sucrose cushion, layered over a 20 to 50% continuous sucrose gradient, and spun to equilibrium. Sequential aliquots were removed from the top and immunoprecipitated with chimp serum. Densities (grams per milliliter) of the indicated fractions appear over the corresponding fraction numbers. (B) Quantitation by phosphorimager of Env (SU and TM) and Gag proteins from each fraction displayed as digital light units (DLUs).