The universal suppressor mutation restores membrane budding defects in the HSV-1 nuclear egress complex by stabilizing the oligomeric lattice

Figures

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  Fig 1. The SUPUL31 mutation restores budding activity to budding-deficient oligomeric interface NEC mutants. a-c) HSV-1 NEC hexameric and interhexameric interfaces highlighting the locations of residues mutated for this study. d) A cartoon representation of the GUV budding assay showing the NEC (purple circles and pink rectangles) binding to red fluorescent GUVs and undergoing negative curvature to form an NEC-coated intraluminal vesicle (ILV). Free NEC continues to bud the GUVs until only fully budded vesicles containing NEC on the interior remain. Cascade blue, a membrane impermeant dye, is used to monitor budding. e) SUP-R229LUL31 rescues budding in both hexameric and interhexameric budding-deficient NEC mutants in vitro. The percentage of in vitro budding was determined by counting the number of ILVs within the GUVs after the addition of NEC220 or the corresponding NEC mutant. A background count, the number of ILVs in the absence of NEC, was subtracted from each condition. Each construct was tested in three biological replicates, each consisting of three technical replicates. Symbols show the average budding efficiency of three technical replicates compared to NEC220 (100%; grey).…

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  Fig 2. The SUPUL31 mutation complements the growth defects of HSV-1 containing oligomeric interface mutations. (a-b) WTUL31 can only complement the growth of the D35AUL34 and F252YUL31 oligomeric interface mutants whereas SUPUL31 can complement more (c-d). For all experiments, Hep-2 cells were transfected with the corresponding UL34 or UL31 mutant plasmid (x-axis labels) and infected with either a UL34-null (a), a UL31-null virus (b), a UL34-null/UL31R229L virus (c), or a UL31-null virus with the SUPUL31 mutation added in trans (d). Each bar represents the mean of three independent experiments. Statistical significance was determined by performing a one-way ANOVA on log-converted values using the Method of Tukey for multiple comparisons of each mutant against the WT in GraphPad Prism. P < 0.01 = **; P < 0.0001 = ****; ns, not significant.

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  Fig 3. The SUPUL31 mutation restores budding to heterodimeric interface mutants and complements viral growth defects. a) Locations of intramolecular NEC residues mutated for this study. Inset shows interactions between various residues at the heterodimeric interface thought to be important for NEC heterodimer stabilization. b) NEC-SUPUL31 rescues budding of NEC heterodimeric interface mutants in vitro. The percentage of in-vitro budding was determined by counting the number of ILVs within the GUVs after the addition of NEC220 or the corresponding NEC mutant. A background count, the number of ILVs in the absence of NEC, was subtracted from each condition. Each construct was tested in three biological replicates, each consisting of three technical replicates. Symbols show the average budding efficiency of three technical replicates compared to NEC220 (100%; grey). Significance was calculated using an unpaired Student’s t-test with Welch’s correction (P < 0.05 = *; P < 0.01 = **; ns = not significant) in GraphPad Prism 9.0. c) WTUL34 can only complement the growth of the R139AUL34 heterodimeric interface mutant whereas SUPUL31 (d) can partially complement more. For both experiments, Hep-2…

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  Fig 4. The SUPUL31 mutation partially restores budding to a membrane interface mutant. a-b) Location of membrane interface residues mutated for this study. c) NEC-SUPUL31 partially rescues budding in the membrane interface mutant in vitro. The percentage of in-vitro budding was determined by counting the number of ILVs within the GUVs after the addition of NEC220-His or the corresponding NEC mutant. A background count, the number of ILVs in the absence of NEC, was subtracted from each condition. Each construct was tested in three biological replicates, each consisting of three technical replicates. Symbols show the average budding efficiency of three technical replicates compared to NEC220-His8 (100%; grey). The NEC-SE6UL31-His8 data were previously reported in [31]. Significance was calculated using an unpaired Student’s t-test with Welch’s correction (P < 0.01 = **; ns = not significant) in GraphPad Prism 9.0. d) CryoEM of NEC-SE6UL31-His8 and large unilamellar vesicles (LUVs) shows that the SE6UL31 mutations perturb NEC oligomerization when bound to membranes.

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  Fig 5. WT NEC, NEC-SUPUL31, and NEC-DNUL34/SUPUL31 form similar membrane-bound hexagonal coats. CryoET reconstruction of a) the WT NEC coat at 5.9 Å, b) the NEC-SUPUL31 coat at 13.1 Å, and c) the NEC-DNUL34/SUPUL31 coat at 5.4 Å. Only the three hexameric units marked with orange stars are shown in lower 90°-rotated panels, where the low-pass filtered transparent densities show the connection between NEC lattices and LUV membrane. The HSV-1 NEC crystal structure (PDB ID: 4ZXS) docks similarly into both the WT NEC (d) and NEC-DNUL34/SUPUL31 (f) cryoET densities. e, g) Docking of the ⍺4 helix from the PRV NEC crystal structure (PDB ID: 4Z3U) accounts for the additional density observed in both cryoET reconstructions which was originally unresolved in the HSV-1 NEC crystal structure [24]. All coats have the hexCD arrangement.

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  Fig 6. NEC-SUPUL31 and NEC-DNUL34/SUPUL31 form hexAB lattices in the crystals. a) The HSV-1 WT NEC hexAB (PDB ID: 4ZXS), b) the WT NEC hexCD (PDB ID: 4ZXS), c) the NEC-SUPUL31 and d) the NEC-DNUL34/SUPUL31 crystal lattices [24]. Hexameric (teal) and interhexameric (dimer1: coral, dimer2: gold, trimer1: red, and trimer2: light pink) interfaces are colored accordingly. Two distinct trimers formed in the NEC-SUPUL31 lattice (red and light pink). Due to resolution, an interface analysis was not performed on the NEC-DNUL34/SUPUL31 crystal lattice. The corresponding heterodimers within the lattice are labeled.

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  Fig 7. The SUPUL31 mutation eliminates a salt bridge near the heterodimeric interface and changes loop conformations. The crystal structures of (a) WT NECAB and (b) NEC-SUPAB heterodimers. The heterodimeric interface residues are colored blue (globular interface) or green (hook interface). Residue 229UL31 is colored magenta. Insets show close-up views of the globular heterodimeric interface. Residues forming salt bridges at the interface are shown as sticks and colored in blue (Rs) or red (Es and Ds). Residue 229UL31 is shown as sticks and colored in magenta. Salt bridges are shown as dashed green lines, with distances in Angstrom. Distances are also listed in the corresponding tables. The resolved portions of the dynamic loops 129UL31-134UL31 and 261UL31-268UL31 are shown in red and purple, respectively. The HSV-1 NEC crystal structure (PDB: 4ZXS) was used to generate the figure in (a).

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  Fig 8. The interhexameric interfaces in the NEC-SUPUL31 mutant have new salt bridges. Close-up views of the interhexameric interfaces in the (a) WT NEC crystal lattice (UL31 trimer; crimson) and (b) NEC-SUPUL31 crystal lattice [UL31 trimers (crimson and pink) and dimers (orange and yellow)]. The HSV-1 NEC crystal structure (PDB: 4ZXS) was used to generate the figure in panel a. Salt bridges are shown as green dashed lines, with distances in Angstrom. Residues forming salt bridges are shown as sticks and colored in blue (Rs) or red (Es and Ds). c) Distances of contacts at the highlighted interfaces. Contacts in bold are salt bridges.

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  Fig 9. A model of SUPUL31 budding restoration in the context of a lattice destabilizing mutation. a) WT NEC heterodimers are arranged into hexamers that assemble into a stable hexagonal lattice by forming contacts at the hexameric and interhexameric interfaces. The salt bridges located at the trimeric interhexameric interfaces (crimson) favor lattice association, rather than disassociation. (b) In the presence of a lattice destabilizing mutation such as NEC-DNUL34, hexamer formation and lattice assembly are perturbed. (c) The SUPUL31 mutation restores hexagonal lattice formation by promoting the formation of new contacts at both the dimeric and trimeric interhexameric interfaces (pink and salmon), resulting in a stable and functional NEC lattice despite the presence of a destabilizing mutation.