Fv-4: identification of the defect in Env and the mechanism of resistance to ecotropic murine leukemia virus

Abstract

Mice expressing the Fv-4 gene are resistant to infection by ecotropic murine leukemia viruses (MuLVs). The Fv-4 gene encodes an envelope (Env) protein whose putative receptor-binding domain resembles that of ecotropic MuLV Env protein. Resistance to ecotropic MuLVs appears to result from viral interference involving binding of the endogenously expressed Fv-4 env-encoded protein to the ecotropic receptor, although the immune system also plays a role in resistance. The Fv-4 env-encoded protein is processed normally and can be incorporated into virus particles but is unable to promote viral entry. Among the many sequence variations between the transmembrane (TM) subunit of the Fv-4 env-encoded protein and the TM subunits of other MuLV Env proteins, there is a substitution of an arginine residue in the Fv-4 env-encoded protein for a glycine residue (gly-491 in Moloney MuLV Env) that is otherwise conserved in all of the other MuLVs. This residue is present in the MuLV TM fusion peptide sequence. In this study, gly-491 of Moloney MuLV Env has been replaced with other residues and a mutant Env bearing a substitution for gly-487 was also created. G491R recapitulates the Fv-4 Env phenotype in cell culture, indicating that this substitution is sufficient for creation of an Env protein that can establish the interference-mediated resistance to ecotropic viruses produced by the Fv-4 gene. Analysis of the mutant MuLV Env proteins also has implications for an understanding of the role of conserved glycine residues in fusion peptides and for the engineering of organismal resistance to retroviruses.

FIG. 1

Amino acid sequence of the Fv-4 fusion peptide at the amino terminus of TM compared to those of other MuLVs (GenBank accession numbers 387149, 58830, 119478, 332083, and 332027, respectively). The residues that were mutated are in light gray.

FIG. 2

Analysis of processing (A) and incorporation into virus particles (B) of the various mutant Mo-MuLV Env proteins in gpGFP cells. gpGFP cells expressing the mutant Env proteins were labeled with [³⁵S]cysteine-[³⁵S]methionine, and the cell lysate (A) and viral supernatant (B) were immunoprecipitated with antibody against SU. An analysis of cell lysate and virus from gpGFP cells transfected with the wild-type (w.t.) Env plasmid or mock transfected (−) is also presented. At the left is indicated the position of SU (70 kDa).

FIG. 3

Normal processing of mutant Env proteins in NIH 3T3 cells. NIH 3T3 cells expressing mutant Env proteins labeled with [³⁵S]cysteine-[³⁵S]methionine and immunoprecipitated with antibody against SU. GP+E-86 cells were used as the positive control for wild-type (w.t.) Env protein expression, while NIH 3T3 cells were used as the negative control (−). At the left are indicated the positions of wild-type uncleaved SU plus TM (85 kDa) and SU (70 kDa). There was a cross-reactive protein in the cell lysate that migrated slightly slower than SU plus TM (85 kDa).

FIG. 4

Effect of G491R mutant Env on the ability of wild-type Env to promote transduction. A constant amount of plasmid penv1min (0.5 μg) and various amounts of penv1G491R were transfected into gpGFP cells, and the virus present in the supernatant medium was used to transduce NIH 3T3 cells. The level of transduction was determined by flow cytometry.