R-Peptide Cleavage Potentiates Fusion-Controlling Isomerization of the Intersubunit Disulfide in Moloney Murine Leukemia Virus Env

Abstract

Fusion of the membrane of the Moloney murine leukemia virus (Mo-MLV) Env protein is facilitated by cleavage of the R peptide from the cytoplasmic tail of its TM subunit, but the mechanism for this effect has remained obscure. The fusion is also controlled by the isomerization of the intersubunit disulfide of the Env SU-TM complex. In the present study, we used several R-peptide-cleavage-inhibited virus mutants to show that the R peptide suppresses the isomerization reaction in both in vitro and in vivo assays. Thus, the R peptide affects early steps in the activation pathway of murine leukemia virus Env.During maturation of the Moloney murine leukemia virus (Mo-MLV), the viral protease cleaves a 16-residue-long peptide, the R peptide, from the cytoplasmic tail of the TM subunit of the membrane fusion protein Env (5, 6, 17). The cleavage potentiates the receptor-induced fusion activation in Env, but the mechanism for this effect has remained obscure (7,8,15,16,18). Similar regulation has been observed in other gammaretroviruses and in the Mason-Pfizer monkey betaretrovirus (2, 3). Recently, it was shown that the activation of MLV fusion is also controlled by receptor-induced isomerization of the intersubunit disulfide of the Env SU-TM subunit complex (14,19). Here, we studied whether R-peptide cleavage facilitates the isomerization.R-peptide cleavage site mutants, shown to inhibit cleavage in earlier studies, were created at the P1 (L649V, L649R, and L649I) and P1Ј (V650I) positions by PCR mutagenesis, using Mo-MLV proviral DNA (4,8,16,18). Corresponding particles were produced and labeled with 50 Ci/ml [ 35 S]Cys in calcium phosphate precipitate-transfected HEK 293T cells. Analysis by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of sedimentation-purified viruses that had been lysed in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 2 mM EDTA and immunoprecipitated with anti-MLV polyclonal antibody (pAb) (HE863; Viromed Biosafety Laboratories) showed that the mutant Env's, with the exception of V650I, incorporated with wild-type (wt) efficiency and were significantly inhibited in R-peptide cleavage, i.e., in cleavage of the Pr15E form of TM into the p15E form (Table 1). Incorporation of Env-V650I was reduced to 63%, and the R peptide was cleaved as in the wt. The infectivities of the mutants were analyzed in XC cells by using MLV p12 monoclonal antibody (Ab) 548 (B. W. Chesebro, Rocky Mountain Laboratories). Cell binding was augmented by centrifugation of the virus-cell sample at 850 ϫ g for 1 h at 4°C in the presence of 8 g/ml polybrene. The infectivities of the mutants were found to correlate with the biochemical findings (Table 1). This was also the case with their fusion efficiencies, which were tested in an XC cell-to-cell fusion-from-without assay (19). Thus, all mutants but V650I were inhibited in R-peptide cleavage and membrane fusion. Notably, the cleavage inhibition was only partial, and the L649V mutant contained a significant fraction (about 30%) of R-pept...

Maturation cleavage of the murine leukemia virus Env precursor separates the transmembrane subunits to prime it for receptor triggering

“…Earlier, we showed that the R-peptide Env was able to undergo early activation steps, although at a reduced rate (17). In particular, it isomerized its intersubunit disulfide when triggered in vitro by Ca 2+ depletion.…”

“…Earlier, we showed that the R-peptide Env could be triggered in vitro by Ca 2+ depletion and in vivo by the receptor on the cell surface, but that the activation process was slower than that of the mature Env and did not proceed to completion (17). To find out how the R-peptide impedes Env activation, we studied the structure of the R-peptide Env in its isomerization-arrested state.…”

“…Fusion was possible only if the R-peptide was deleted. Similarly, virus, where the R-peptide cleavage has been inhibited, cannot fuse with cells (17). It was suggested that the R-peptide ties the TM subunits together in the endodomain and that this prevents conformational changes in the Env that are necessary for fusion.…”

Cooperative Cleavage of the R Peptide in the Env Trimer of Moloney Murine Leukemia Virus Facilitates Its Maturation for Fusion Competence

“…XC cells were maintained as 293T cells, but the medium included 1 g/liter glucose and 20 mM HEPES. Plasmids containing the provirus DNA for wild-type (wt) and mutant viruses have been described previously (9). The anti-R peptide antibody, ␣R, is an affinity-purified polyclonal antibody from rabbits immunized with a 17-amino-acid-long peptide corresponding to the MoMLV R peptide, NH 2 -CVLTQQYHQLKPIEYEP-CO NH 2 .…”

“…Consequently, we along with others have created L 649 R (LR), L 649 I (LI), and L 649 V (LV) Env mutants and shown that these were inhibited in R peptide cleavage when assembled into virus particles, which were also shown to be noninfectious (8,15,20). Earlier, we used such mutant virus to characterize the mechanism of fusion facilitation by the R peptide cleavage (9). In MoMLV Env the fusion activation process is controlled by the disulfide that links the SU subunit with the TM (21).…”