Cytoplasmic tail of Moloney murine leukemia virus envelope protein influences the conformation of the extracellular domain: implications for mechanism of action of the R Peptide

Abstract

The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptide-truncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.

FIG. 1.

Schematic of Env proteins. (A) The 632-amino-acid MoMuLV Env protein comprises SU and TM polypeptides. The membrane-spanning region of the TM protein is shown (M), while the cytoplasmic tail consists of the R peptide (R) and the mature tail (T) remaining after R peptide removal. Construct GLA15E retains eight amino acids of the membrane-spanning region linked to a GPI anchor. (B) Amino acid sequence of the cytoplasmic tail of MoMuLV Env, showing the substitutions made in the indicated mutants. A putative endocytosis motif in the R peptide is boxed.

FIG. 2.

Relationship between cell surface levels of Env and cell-cell fusion activities for different Env constructs. (A) Effect on CSE of increasing the amount of plasmid DNA transfected into 10-cm plates of 293T cells. CSE was measured by FACS analysis by using antibody 273. (B) Relationship between CSE levels in 293T cells and cell-cell fusion activity in the 293T-XC cocultivation system. Fusion activity was expressed as the number of nuclei recruited into syncytia in a 20-mm² area. All data points are the averages of at least three independent experiments.

FIG. 3.

Biotinylation of SU and TM from truncated Env mutants. Env proteins were expressed in 293T cells by transient transfection. Total cellular proteins were radiolabeled with ³⁵S, or surface-expressed proteins were biotinylated with a membrane-impermeable reagent. Both SU and TM subunits were immunoprecipitated by using a polyclonal anti-SU antiserum and subjected to SDS-PAGE. Biotinylated proteins were detected by using ¹²⁵I-streptavidin.

FIG. 4.

Deglycosylated cell lysates and virions. 293T cells were transfected with MuLV Gag-Pol plasmids and the indicated Env constructs. Both cell lysates and pelleted viral supernatants were deglycosylated and subjected to SDS-PAGE. Specific proteins were detected by using appropriate antibodies. The various TM proteins run at different sizes due to the truncations they contain. In addition, the wild-type protein, CEE, is present in virions in both full-length and R- peptide-truncated forms. PrEnv is the uncleaved precursor Env protein, Pr85.

FIG. 5.

Truncation of TM enhances shedding of SU from virions. ³⁵S-labeled viral supernatants were harvested from transiently transfected 293T cells. Aliquots were retained (total SU) or centrifuged through 20% sucrose to give pelleted and supernatant fractions. SU was immunoprecipitated from each fraction by using an anti-SU antibody, and the extent of radioactivity in each fraction was determined by SDS-PAGE and PhosphorImager analysis. Shedding indexes were calculated as the ratio of the amount of SU in the cleared supernatant to that in the total SU fraction; the data are shown as the averages plus or minus standard deviations of results of three independent experiments, with P values calculated for each mutant relative to CEE by using a t test. Mutant L493V contains a substitution in the heptad repeat region of TM and has previously been characterized as a shedding mutant (79).