Characterizing the Murine Leukemia Virus Envelope Glycoprotein Membrane-Spanning Domain for Its Roles in Interface Alignment and Fusogenicity

Abstract

The membrane-proximal region of murine leukemia virus envelope (Env) is a critical modulator of its functionality. We have previously shown that the insertion of one amino acid (+1 leucine) within the membrane-spanning domain (MSD) abolished protein functionality in infectivity assays. However, functionality could be restored to this +1 leucine mutant by either inserting two additional amino acids (+3 leucine) or by deleting the cytoplasmic tail domain (CTD) in the +1 leucine background. We inferred that the ectodomain and CTD have protein interfaces that have to be in alignment for Env to be functional. Here, we made single residue deletions to the Env mutant with the +1 leucine insertion to restore the interface alignment (gain of functionality) and therefore define the boundaries of the two interfaces. We identified the glycine-proline pairs near the N terminus (positions 147 and 148) and the C terminus (positions 159 and 160) of the MSD as being the boundaries of the two interfaces. Deletions between these pairs restored function, but deletions outside of them did not. In addition, the vast majority of the single residue deletions regained function if the CTD was deleted. The exceptions were four hydroxyl-containing amino acid residues (T139, T140, S143, and T144) that reside in the ectodomain interface and the proline at position 148, which were all indispensable for functionality. We hypothesize that the hydroxyl-containing residues at positions T139 and S143 could be a driving force for stabilizing the ectodomain interface through formation of a hydrogen-bonding network.

Importance: The membrane-proximal external region (MPER) and membrane-spanning domains (MSDs) of viral glycoproteins have been shown to be critical for regulating glycoprotein fusogenicity. However, the roles of these two domains are poorly understood. We report here that point deletions and insertions within the MPER or MSD result in functionally inactive proteins. However, when the C-terminal tail domain (CTD) is deleted, the majority of the proteins remain functional. The only residues that were found to be critical for function regardless of the CTD were four hydroxyl-containing amino acids located at the C terminus of the MPER (T139 and T140) and at the N terminus of the MSD (S143 and T144) and a proline near the beginning of the MSD (P148). We demonstrate that hydrogen-bonding at positions T139 and S143 is critical for protein function. Our findings provide novel insights into the role of the MPER in regulating fusogenic activity of viral glycoproteins.

FIG 1

Diagram of predicted trimer interfaces. (Left) Depiction of predicted ectodomain and CTD trimer interfaces highlighted as white segments within the trimer core of wild-type MLV Env. (Middle) Depiction of the +1L insertion within the MSD and the resulting disruption of the CTD interface by ∼103°. (Right) Depiction of the +3L insertions within the MSD and the subsequent restoration of orientation of the CTD interface. The introduction of three leucines nearly restores alignment of the CTD interface within the trimer core.

FIG 2

Infectivity of MLV Env deletion scan in the +1L background. (A) Diagram of the MPER, MSD, and CTD in MLV Env targeted for the −1 deletion scan. The arrow indicates the insertion of +1L within the MSD. The predicted α-helical structure of this region is depicted below (secondary structure prediction constructed using Phyre² protein fold recognition software). (B) The infectivity of the full-length Env with +1L insertion at position 154 is shown as a control, and its insertion position within the membrane-spanning domain is indicated with an arrow. All filled bars are full-length (HA-tagged) Env with the +1L insertion and the indicated residue deleted. The hashed open bar is full-length wild-type Env shown as a positive control (5.6 × 10⁵ IU/ml). All data are normalized relative to the wild-type HA-tagged Env control. The dashed line shown across the data indicates the relative level of infectivity of the +1L Env. The data shown here are the averages of three independent experiments.

FIG 3

Leucine insertions upstream and downstream of the respective glycine-proline pairs do not display a phasing effect. (Top) Diagram of the leucine insertions upstream of the glycine-proline pair positioned at the N terminus of the membrane-spanning domain and downstream of the glycine-proline pair positioned at the C terminus of the membrane-spanning domain. Leucine insertions in the middle of the MSD, between the glycine-proline pairs, are shown as a control. (Bottom) Infectivity of leucine insertion mutants shown relative to wild-type MLV Env (HA tag, 4.9 × 10⁵ IU/ml). The data shown in this figure are the average of three independent experiments.

FIG 4

Truncation of the CTD restores abrogated infectivity of certain deletion mutants. The CTD of the specified deletion mutants, in the context of the +1L glycoprotein background, was truncated (Δ25CTD), and the infectivity was assessed. Filled bars indicate full-length glycoprotein with +1L at position 154 along with indicated single amino acid deletions. Open bars indicate the same glycoproteins with the last 25 residues of the CTD deleted. The hashed open bar is wild-type Env Δ25CTD (HA tagged) shown as a positive control (3.0 × 10⁵ IU/ml). The dashed line between positions P148 and L157 indicates that these residues were not tested in this experiment. All data are normalized relative to the wild-type Env Δ25CTD control. The data shown here are the averages of three independent experiments.

FIG 5

The hydroxyl-containing amino acids in the MPER and MSD are crucial for promoting cell-to-cell fusion. (A) Stable cell lines were created in 293FT cells expressing constructs for the indicated Env mutants (GFP tagged). Cells were surface labeled with an anti-GFP Alexa Fluor 647 antibody (Sigma) and analyzed via flow cytometry. The mean fluorescence intensity was normalized to the WT Δ25CTD control. 293FT cells not expressing MLV Env were included as a negative control in the labeling process and are indicated as Env(−). (B) Stable cell lines were assayed for cell-to-cell fusogenicity. Stable cells lines were transfected with a tet-off expression plasmid and cocultured with a permissive TRE-Gluc cell line. The results are depicted as relative light units normalized to the WT Δ25CTD control. 293FT cells not expressing MLV Env were transfected with the tet-off expression plasmid and were included in the cell-to-cell fusion assay as a negative control [Env(−)]. (C) Western blot analysis of Env incorporation into viral particles. Stable cell lines were transfected with HIV GagPol, and supernatants and cell lysates were analyzed for Env incorporation and cellular expression, respectively.

FIG 6

Amino acid substitutions in the critical hydroxyl positions display defective functionality. (A) Infectivity of alanine substitutions in the context of full-length HA-tagged (filled bars) and truncated HA-tagged (open bars) Env relative to wild-type (2.1 × 10⁵ IU/ml). (B) Infectivity of amino acid substitutions in the context of full-length (HA-tagged) Env relative to wild-type Env (6.9 × 10⁵ IU/ml). The hydroxyl-containing positions were substituted to N (asparagine), Q (glutamine), Y (tyrosine), A (alanine), or F (phenylalanine), and the infectivity was assessed. The asterisks indicate a significant difference between the infectivity of tyrosine and phenylalanine substitutions at all four positions tested. (***, P < 0.001; **, P < 0.01).

FIG 7

Predicted hydrogen-bonding network in the core of the MLV Env trimer. MLV Env monomer structural predictions were generated using Phyre² protein fold recognition software. The monomers were assembled into trimers using the molecular modeling program Chimera based on our prediction. The trimer is depicted from both the side and the top. A space-filling model is shown to highlight the bulky side chains of the tyrosine substitutions. Black dotted lines in the first panel indicate theoretical hydrogen-bonding between polar residues T139 and S143 in the predicted trimer core.