Abstract
Arenaviruses cause acute hemorrhagic fevers with high mortality. Entry of the virus into the host cell is mediated by the viral envelope glycoprotein, GPC. In contrast to other class I viral envelope glycoproteins, the mature GPC complex contains a cleaved stable signal peptide (SSP) in addition to the canonical receptor-binding (G1) and transmembrane fusion (G2) subunits. SSP is critical for intracellular transport of the GPC complex to the cell surface and for its membrane-fusion activity. Previous studies have suggested that SSP is retained in GPC through interaction with a zinc-binding domain (ZBD) in the cytoplasmic tail of G2. Here we used NMR spectroscopy to determine the structure of Junín virus (JUNV) ZBD (G2 residues 445-485) and investigate its interaction with a conserved Cys residue (Cys-57) in SSP. We show that JUNV ZBD displays a novel fold containing two zinc ions. One zinc ion is coordinated by His-447, His-449, Cys-455, and His-485. The second zinc ion is coordinated by His-459, Cys-467, and Cys-469 and readily accepts Cys-57 from SSP as the fourth ligand. Our studies describe the structural basis for retention of the unique SSP subunit and suggest a mechanism whereby SSP is positioned in the GPC complex to modulate pH-dependent membrane fusion.
Highlights
Arenavirus species are endemic in rodent populations worldwide [1], and some viruses can be transmitted to humans to cause hemorrhagic fevers with up to 30% mortality [2, 3]
Using a recombinant fusion protein containing the cytoplasmic domain of G2, we showed that three of the G2 residues are involved in high-affinity zinc binding
Isothermal titration calorimetry (ITC) experiments demonstrated that Junín virus (JUNV) zinc-binding domain (ZBD) binds 1.8 mol equivalents of Zn2ϩ with Kd in the nM range (Fig. 2A and Table 2)
Summary
Synthetic Peptides—N-Acetylated synthetic peptides corresponding to the JUNV GPC residues 56 –58 (acetyl-SCT) and 445– 485 (JUNV zinc-binding domain, JUNV ZBD) were obtained from GenScript Corp. at Ͼ98% and Ͼ95% purity, respectively. 10-l aliquots of 20 M ZnCl2 in ITC buffer were injected into the calorimeter cell containing 1.43 ml of 0.85 M JUNV ZBD in ITC buffer, and the heats of binding were recorded. 10-l aliquots of 20 M ZnCl2 in ITC buffer or 450 M acetyl-SCT in Zn-ITC buffer were injected into the calorimeter cell containing 1.43 ml of ITC or Zn-ITC buffer respectively to measure the heats of dilution. Calculations were performed with Aria1.2 [38] in conjunction with CNS 1.0 [39], using crosspeak volumes from the two-dimensional NOESY spectrum with manual assignments either retained, or discarded. All calculation protocols yielded similar structures that contained two distinct clusters of residues posed to coordinate zinc. Geometrical constraints were included to coordinate zinc ions by these two clusters. Inclusion of the hydrogen bond and zinc coordination restraints had negligible effect on the calculated structures, as judged by comparison of the final
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