An evolutionarily conserved splice generates a secreted env-Bet fusion protein during human foamy virus infection

Abstract

Foamy viruses (spumaretroviruses) represent a retroviral genus which exhibits unusual features relating it to pararetroviruses. Previously, we reported the existence of a protein species harboring Env, Bel, and Bet epitopes in human foamy virus (HFV)-infected cells (M. L. Giron, F. Rozain, M. C. Debons-Guillemin, M. Canivet, J. Périès, and R. Emanoil-Ravier, J. Virol. 67:3596-3600, 1993). Here, we identify this protein as a 160-kDa Env-Bet fusion glycoprotein (gp160) translated from an mRNA species harboring a highly conserved splice site which deletes the membrane anchor domain of Env and fuses the env open reading frame with that of bel1/bet. While gp160 and Bet proteins were both secreted into the supernatant, only Bet was taken up by recipient cells. Since Bet plays a key role in the switch from lytic to chronic infection, secretion of Bet and gp160, together with cellular uptake of Bet, could be highly relevant for both immune response and development of HFV infection in vivo.

FIG. 1

Identification of Env and Bel gene products by immunoprecipitation of protein extracts from HFV-infected cells. M, molecular mass markers. Rabbit anti-HFV polyclonal antiserum on protein extracts from noninfected cells (negative control) or from infected cells (positive control) were used as controls. Rabbit polyclonal anti-Bel1 and anti-Bel2 Abs and mouse monoclonal anti-Bet (D11) and anti-SU-Env (B4) Abs were used to detect the gp160 band.

FIG. 2

The gp160 band does not represent a protein complex. (A) Peptide mapping of gp130, gp160, and Bet. Several bands are shared by gp130 and gp160 and others are shared by gp160 and Bet (arrows). (B) Low-pH treatment of gp160 and gp130. M, molecular mass markers. The apparent molecular masses of the two proteins are not modified after 1 h of incubation at pH 4.

FIG. 3

(A) RT-PCR experiments on total RNA from HFV-infected cells. Four distinct classes of RNAs, which correspond to the four RNA species of 900, 780, 600, and 480 bp, are amplified. Plasmid pHSRV13 was used as a probe for PCR hybridization. (B) Schematic representation of the different mRNA species which can be detected between primer A and primer B. Note that three new amino acids are created by the splicing event (Asp-Cys-Ile in boldface type). The dilysine ER retention motif is also depicted in the env ORF. (C) Sequence comparisons of known and putative splice sites among different sequenced FV. The donor and acceptor splice sites flank the transmembrane anchor sequence of the Env TM protein and fuse the env ORF with the transactivator one. SFVcpz, simian FV from chimpanzee; FeFV, feline FV.

FIG. 4

Identification of the gp160 protein in pEB-transfected cells. Immunoprecipitation was performed with the rabbit anti-HFV antiserum. M, molecular mass markers. Cells were transfected with p2EB, p2EnvΔBel, p2EnvΔBel plus pSGBet, and p2EctB. Mutation of the splice acceptor site abolishes the formation of gp160. Note the absence of the gp160 species but the detection of Bet in p2EctB-transfected cells.

FIG. 5

Secretion and uptake of Env-Bet and Bet proteins. Cytoplasmic extracts from cells transfected with p2EB (lane 1), p2EBΔRGD (lane 2) and supernatants from p2EB (lane 3)- and p2EBΔRGD (lane 4)-transfected cells are shown. The upper (200-kDa) band probably represents a cellular protein. After 18 h of labeling, filtered culture medium from transfected cells was incubated with naive recipient COS-6 cells for 4 h and subsequent immunoprecipitations were performed with the rabbit anti-HFV antiserum on cytoplasmic extracts. Lane 5, extracts from p2EB-transfected cells; lane 6, extracts from p2EBΔRGD-transfected cells.