A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion

Abstract

The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation.

FIG. 1.

The H8R envelope protein mediates lipid mixing but not fusion pore formation. The cytoplasm of receptor-negative human 293 cells transiently transfected with Moloney Env R-less, histidine 8-changed-to-arginine (H8R Env) R-less, negative control 595* Env, influenza virus HA, glycine 1-changed-to-serine HA (G1S HA), or glycine 1-changed-to-valine HA (G1V HA) expression plasmids was labeled with CMAC CellTracker Blue, and the cytoplasm of target human 293 cells stably expressing ecotropic MLV receptor was labeled with calcein AM. In addition, the membrane lipids of target cells were labeled with DiI. Equal numbers of fluorescently labeled effector and target cells were mixed, applied to poly-l-lysine-coated plates to promote adherence, and incubated on ice for 30 min to allow cell attachment and binding, after which cells were shifted to 37°C for membrane fusion. Control wild-type and mutant HA-expressing effector cells were activated prior to mixing, and fusion was initiated by a brief exposure to low pH as described in Materials and Methods. Representative micrographs of live cells were captured after 30 min. Fluorescent images of Env-expressing effectors (blue) and receptor-positive target cells (red and green) are shown on the left side of each panel and are merged with a phase-contrast image on the right side. Below each set is a panel of the merged fluorescence images. Arrows point to example effector cells paired with target cells.

FIG. 2.

Lipid mixing is delayed with H8R virus. The membrane lipid of purified virions containing either wild-type Env or mutant Env were labeled to self-quenching levels with R-18, incubated at 4°C with receptor-positive cells (NIH 3T3 or XC) or cells lacking receptor (293) for 1 h, and then shifted to 37°C, and the RFI was monitored continuously. The data are representative of two independent experiments. Colors: blue, wild-type virus; magenta, H8R virus; purple, triple-mutant H8R QR DY virus; cyan, double-mutant QR DY virus.

FIG. 3.

Stability of the hemifusion intermediates caused by H8R Moloney Env and G1S HA. Cell-cell fusion assays were performed as described in the legend to Fig. 1 except that, after the initial set of micrographs were captured, effector-target cell mixtures were briefly exposed to CPZ and incubated an additional 30 min, and then a second set of micrographs were captured. The total number of cell pairs, the number of cell pairs showing lipid dye transfer alone, and the number of pairs showing lipid and content dye transfer were counted from the micrographs, with a total of at least 250 cell pairs scored for each sample. Values shown are the percent full fusion as defined in Qiao et al. (33) and calculated as follows: (the number of fully fused cell pairs/total number of cell pairs showing lipid transfer) × 100. The identity of the envelope protein expressed on the effector cells is indicated below the horizontal axis. Bars: ░⃞, no CPZ; ▪, 0.4 mM CPZ.

FIG. 4.

CPZ, DB, and TFP rescued infection by H8R virus. XC cells were exposed to 10-fold serial dilutions of H8R or wild-type virus for 1 h at 37°C and then washed and incubated briefly with the membrane-curving agents or with the solvent ethanol alone in BES-buffered medium at the indicated pH. Cells were scored 48 h later for virus infection, and the titers were calculated from the endpoint dilutions. The values shown are averages from two independent experiments of each type. No infection was detected in cells exposed to virions lacking Env (data not shown). (A) Dose response to CPZ and oleic acid. For comparison, wild-type virus was titrated for 0.2, 0.4, and 0.5 mM CPZ and for 10 and 50 μM oleic acid at pH 7.5. (B) DB and TFP also rescue H8R virus infection. (C) pH dependence of rescue by CPZ. For comparison, wild-type virus was titrated under all conditions. Bars (A and C): ▪, H8R virus; ░⃞, wild-type Moloney MLV. (D) Effect of CPZ on cell-cell fusion with XC target cells. Cell-cell fusion assays were performed as described in the legend to Fig. 3, except that XC cells were used as target cells. The values shown were calculated as follows: (number of cell pairs showing lipid and content dye transfer/the total number of cell pairs) × 100. Bars: , no CPZ; ▪, 0.4 mM CPZ.

FIG. 5.

CPZ rescued H8R virus infection of NIH 3T3 cells. Quadruplicate wells of NIH 3T3 cells were exposed to serial 10-fold dilutions of wild-type Moloney MLV or H8R Env psuedotype virus stock for 1 h at 37°C, after which fresh medium containing 0.3 mM CPZ was applied for 5 min at 37°C, followed by rapid washes with regular culture medium. Cells were scored 48 h later for virus transduction of β-galactosidase activity. Titers were calculated from the endpoint dilutions.

FIG. 6.

Schematic representation of a model for the block to membrane fusion in an H8R mutant. Receptor binding induces conformation changes that expose the fusion peptide on the N terminus of TM. Membrane fusion begins with local joining of the outer leaflets of juxtaposed membranes (22). As the outer leaflets merge, the inner leaflets remain separate, forming an intermediate called hemifusion that does not allow mixing of the contents of the fusing compartments. Additional conformation changes involve the conserved N-terminal histidine. These changes lead to dissociation of SU from TM, removing the physical constraint on six-helix bundle formation. Hairpin formation merges the inner membrane leaflets to complete fusion and open pores between the virion interior and the host cell cytoplasm. RBD, receptor-binding domain of SU; CTD, carboxy-terminal domain of SU.