Intersubunit interactions modulate pH-induced activation of membrane fusion by the Junin virus envelope glycoprotein GPC

Abstract

The mature arenavirus envelope glycoprotein GPC is a tripartite complex comprising a stable signal peptide (SSP) in addition to the receptor-binding (G1) and transmembrane fusion (G2) subunits. We have shown previously that SSP is a key element in GPC-mediated membrane fusion, and that GPC sensitivity to acidic pH is modulated in part through the lysine residue at position 33 in the ectodomain loop of SSP (J. York and J. H. Nunberg, J. Virol. 80:7775-7780, 2006). A glutamine substitution at this position stabilizes the native GPC complex and thereby prevents the induction of pH-dependent membrane fusion. In efforts to identify the intersubunit interactions of K33, we performed alanine-scanning mutagenesis at charged residues in the membrane-proximal ectodomain of G2 and determined the ability of these mutations to rescue the fusion deficiency in K33Q GPC. Four second-site mutations that specifically complement K33Q were identified (D400A, E410A, R414A, and K417A). Moreover, complementation was also observed at three hydrophobic positions in the membrane-spanning domain of G2 (F427, W428, and F438). Interestingly, all of the complementing mutations restored wild-type pH sensitivity to the K33Q mutant, while none themselves affected the pH of membrane fusion. Our studies demonstrate a specific interaction between SSP and G2 that is involved in priming the native GPC complex for pH-induced membrane fusion. Importantly, this pH-dependent interaction has been shown to be vulnerable to small-molecule compounds that stabilize the native complex and prevent the activation of membrane fusion. A detailed mechanistic understanding of the control of GPC-mediated membrane fusion will be important in guiding the development of effective therapeutics against arenaviral hemorrhagic fever.

FIG. 1.

Arenavirus GPC complex. The JUNV GPC open reading frame is illustrated at the top. Amino acids are numbered from the initiating methionine, and the SSP, G1, and G2 subunits are indicated. Membrane-spanning regions in SSP (hφ1 and hφ2; 1) and in G2 (TM) are shaded dark gray, and the N- and C-terminal heptad repeat regions in G2 (43) are in light gray. The amino acid sequence of the G2 membrane-proximal ectodomain and transmembrane domain (shaded) of JUNV is detailed below; positions studied in this report are indicated by dots. At the lower right is a diagram of the proposed subunit organization in the tripartite GPC complex. Thickened lines represent membrane-spanning domains in SSP and G2 and the heptad repeat regions in G2. The cytosolic N terminus of SSP is myristoylated (thin line) (48), and an intersubunit zinc finger (ball) is thought to link the C terminus of SSP with the cytoplasmic domain of G2 (46). Lysine 33 (K33) in SSP is marked. The drawing is representational and not to scale. In the lower left, we show the alignment of the arenavirus membrane-proximal G2 ectodomain regions. Accession numbers are the following: JUNV, D10072; LASV-Nigeria (LASV-N), P17332; Tacaribe virus (TCRV), NP_694849; Pichinde virus (PICV), AAB58484; Machupo virus (MACV), AAX99337; Sabiá virus (SABV), AAC55091; LASV-Josiah (LASV-J), AAG41802; Mopeia virus (MOPV), AAV54108; and lymphocytic choriomeningitis virus-Armstrong (LCMV-A), NP_694851. Charged residues are highlighted in blue (basic) or red (acidic).

FIG. 2.

Membrane fusion activity of G2 mutants. The wild-type (wt) and G2 mutants in CD4sp-GPC were expressed in trans with wild-type SSP (open bars) or K33Q SSP (filled bars). The ability of the single and double mutants to mediate cell-cell fusion at pH 5.0 was determined as detailed in Materials and Methods. Error bars representing ±1 standard deviation are calculated for all points (n = 6) and may not be visible on the scale of the graph. These data represent one complete study (of at least three to six complete and partial experiments). All comparisons are reliably reproducible between experiments, although absolute percentages may vary. The average for K33Q GPC (6.1% ± 2.5%) differs from that previously cited (47) due to technical changes in the assay. The D400A, E405A, E410A, K417A, and D424A mutants have been reported previously (44).

FIG. 3.

Membrane fusion activity of charge mutants at R414. These studies were performed in the same fashion as those described in the legend to Fig. 2. The amino acid changes at R414 are indicated by single-letter abbreviations. Note that the vertical axis is truncated. wt, wild type.

FIG. 4.

pH sensitivity of membrane fusion. Cells expressing either wild-type SSP (black circles) or K33Q SSP (black squares) with the G2 mutant were pulsed at the indicated pH. The lightly shaded curves, repeated in all panels, depict fusion of the wild-type CD4sp-GPC with wild-type (gray circles) and K33Q (gray squares) SSP. The results shown in this study are representative of at least three independent experiments. Fusion activity is normalized to the maximum of each mutant for this comparison. Error bars represent ±1 standard deviation.

FIG. 5.

Complementation by transmembrane domain G2 mutants. The G2 mutants were expressed in trans with wild-type SSP (open bars) or K33Q SSP (filled bars), and membrane fusion activity was determined. These data represent one complete study (of at least three complete and partial experiments).