CLCC1 promotes membrane fusion during herpesvirus nuclear egress

Abstract

Herpesvirales are an ancient viral order that infects species from mollusks to humans for life. During infection, these viruses translocate their large capsids from the nucleus to the cytoplasm independently from the canonical route through the nuclear pore. Instead, capsids dock at the inner nuclear membrane and bud into the perinuclear space. These perinuclear enveloped virions fuse with the outer nuclear membrane releasing the capsids into the cytoplasm for maturation into infectious virions. The budding stage is mediated by virally encoded proteins. But the mediator of the subsequent fusion stage is unknown. Here, using a whole-genome CRISPR screen with herpes simplex virus 1, we identified CLCC1 as an essential host factor for the fusion stage of nuclear egress. Loss of CLCC1 results in a defect in nuclear egress, accumulation of capsid-containing perinuclear vesicles, and a drop in viral titers. In uninfected cells, loss of CLCC1 causes a defect in nuclear pore complex insertion. Viral homologs of CLCC1 are present in herpesviruses that infect mollusks and fish. Our findings uncover an ancient cellular membrane fusion mechanism important for the fundamental cellular process of nuclear envelope morphogenesis that herpesviruses hijack for capsid transport.

Keywords: CLCC1; CRISPR screen; HSV-1; chloride channel; herpes simplex virus; herpesviruses; membrane fusion; nuclear budding; nuclear egress; nuclear pore insertion; nucleocytoplasmic transport.

Extended Data Figure 1.. Development of the flow-cytometry based assay to measure nuclear egress.

a, b) Confocal images of HeLa cells infected with either WT HSV-1 (a) or HSV-1 ΔUL34 mutant deficient in nuclear egress (b). Cells were either partially permeabilized with digitonin (top) or fully permeabilized with Triton X-100 (bottom) and then stained with a capsid-specific primary antibody and Alexa-488-conjugated secondary antibody (green). Nucleus was stained with DAPI (blue). tdTomato signal indicates infection (red). Scale bar = 10 mm. c) Flow cytometry data for uninfected HeLa cells (UIC), or HeLa cells infected with WT HSV-1 (top) or HSV-1 ΔUL34 mutant. Infected cells were either partially permeabilized with digitonin or fully permeabilized with Triton X-100 and the stained with capsid-specific primary antibody and Alexa-488-conjugated secondary antibody. “2^ry” samples were partially permeabilized with digitonin and incubated with secondary antibody only. Each image is a representative of three biological replicates.

Extended Data Figure 2.. CLCC1 depletion causes a defect in HSV-1 nuclear egress.

a) Bulk CLCC1-KO (cko3_bulk and cko6_bulk), single-clone CLCC1-KO (cko3_2, cko3_4 and cko6_1, and cko6_2), HeLa, bulk intergenic site targeting (Int_bulk), or single-cell intergenic site targeting (Int_3 and Int_4) cell lines were infected with WT HSV-1 at an MOI of 5. As a positive control, HeLa cells were infected with HSV-1 ΔUL34 mutant virus, defective in nuclear egress, at an MOI of 10. Nuclear egress was measured at 24 hpi by the flow cytometry nuclear egress assay and normalized to HSV-1-infected HeLa cells. Each experiment had three biological replicates, each containing two technical replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.0001 = ****. Significance was calculated using one-way ANOVA, with multiple comparisons. Data for HeLa, Int_4, cko3_4, cko6_1, and HSV-1 ΔUL34 are the same as in Fig 2a. b) Western Blot analysis of CLCC1 levels in cell lines used in (a). Each image is a representative of three biological replicates. The bands in the second blot from the top were cut from the same gel and rearranged for better visualization.

Extended Data Figure 3.. CLCC1 depletion causes a defect in HSV-1 nuclear egress, visualized by confocal microscopy.

Confocal images of Int_4 and bulk CLCC1-KO (cko3_bulk) cell lines infected with WT HSV-1. Cells were either partially permeabilized with digitonin or fully permeabilized with Triton X-100 and then stained with a capsid-specific primary antibody and Alexa488-conjugated secondary antibody (green). Nucleus was stained with DAPI (blue). tdTomato signal indicates infection (red). Scale bar = 10 mm. Each image is a representative of one biological replicate.

Extended Data Figure 4.. Expression of CLCC1 in trans can rescue the HSV-1 nuclear egress defect due to CLCC1 depletion.

a) Single-clone CLCC1-KO (cko3_4 and cko6_1), CLCC1 overexpressing (Int_4_IEF1a and Int_4_R_bulk, with CLCC1 under the control of a strong and weak promoter, respectively), and bulk CLCC1-R (cko3_4_IEF1a, cko6_1_IEF1a, cko3_4_R_bulk, and cko6_1_R_bulk, with CLCC1 under the control of a strong weak promoter, respectively), HeLa, and single-cell intergenic site targeting (Int_4) cell lines were infected with WT HSV-1 at an MOI of 5. Nuclear egress was measured at 24 hpi by the flow cytometry nuclear egress assay normalized to HSV-1-infected Int_4 cells. Each experiment had three biological replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.0001 = ****, P < 0.01 = **, P < 0.05 = *. Significance was calculated using one-way ANOVA, with multiple comparisons. Data for HeLa, Int_4, cko3_4, and cko6_1 are the same as in Fig 2a. b) Western Blot analysis of CLCC1 levels in single-clone CLCC1-KO (cko3_4 and cko6_1), control Int_4, CLCC1 overexpressing (Int_4_IEF1a and Int_4_R_bulk), and bulk CLCC1-R (cko3_4_IEF1a, cko6_1_IEF1a, cko3_4_R_bulk, and cko6_1_R_bulk) cell lines. Each image is a representative of at least two biological replicates. The bands in the top blot were cut from the same gel and rearranged for better visualization. c) Single-clone CLCC1-KO (cko3_4 and cko6_1), bulk CLCC1-R (cko3_4_R_bulk and cko6_1_R_bulk), Intergenic-site targeting (Int_4), single-clone CLCC1-R (cko3_4_R_1, cko3_4_R_6, cko6_1_R_1, and cko6_1_R_6), and control Int_4 cell lines were infected with WT HSV-1 at an MOI of 5. Nuclear egress was measured at 24 hpi by the flow cytometry nuclear egress assay normalized to HSV-1-infected Int_4 cells. Each experiment had three biological replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.0001 = ****, P < 0.01 = **, P < 0.05 = *. Significance was calculated using one-way ANOVA, with multiple comparisons. Data for HeLa, Int_4, cko3_4, and cko6_1 are the same as in Fig 2a. Data for Int_4, cko3_4, and cko6_1 are the same as in Fig 2a. Data for cko3_4_R_bulk and cko6_1_R_bulk are the same as in (a). d) Western Blot analysis of CLCC1 levels in single-clone CLCC1-KO (cko3_4 and cko6_1), single-clone CLCC1-R (cko3_4_R_1 and cko6_1_R_1), and control Int_4 cell lines. Each image is a representative of three biological replicates.

Extended Data Figure 5.. CLCC1 role in nuclear egress is independent of its role in ER stress response.

a) Western Blot analysis of BiP (ER stress marker). (Left) HeLa or cko6_1 cells were either pre-treated with 1.5 mM DTT for 4 h or left untreated, and then either infected with WT HSV-1 at an MOI of 5 or uninfected. Following infection, cells pre-treated with DTT were incubated in the presence of 0.38 mM DTT for an additional 24 h. Each image is a representative of three biological replicates. (Right) quantifications of three replicate Western Blots, normalized to the HeLa or cko6_1 untreated and uninfected group. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. b) HeLa or cko6_1 cells, treated as in (a). Nuclear egress was measured at 24 hpi by the flow cytometry nuclear egress assay. Each experiment had three biological replicates, each with three technical replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. c) Cell viability of HeLa or cko6_1 cells, following treatment with different DTT for 24 hours. Viability was measured and normalized to HeLa or cko6_1 mock treatment group. Each symbol is the mean of three biological replicates, and bars show SEM.

Extended Data Figure 6.. CLCC1 depletion causes MLF2 accumulation in the nuclear envelope, visualized by confocal microscopy.

Confocal images of single-cell CLCC1-KO (cko3_4 and cko6_1), single-cell CLCC1-R (cko3_4_R_1 and cko6_1_R_1), and control HeLa and Int_4 cell lines overexpressing MLF2-GFP (green). Nucleus was stained with DAPI (blue). Scale bar = 10 mm. Each image is a representative of one biological replicate.

Extended Data Figure 7.. Residues of potential functional importance in human CLCC1.

A ribbon diagram of an AlphaFold3 model of human CLCC1. Structural elements and domains are colored as in Fig 5 and labelled. Mutated residues are shown in sphere representation and colored as follows: 10 residues that are invariant across 8 representative animal and 4 herpesviral homologs (red, except cysteines shown in purple), aromatic residues in the “knuckle” FD_h2 helix of FD (yellow orange), polar spine residues in TM2 (cyan). Residues 365–539 were removed, for clarity.

Extended Data Figure 8.. AlphaFold3 models of human and herpesviral CLCC1 multimers.

a) AlphaFold3 models of the core region of human CLCC1 (residues 161–360) as a hexamer (left), decamer (middle), or hexadecamer (right). b) AlphaFold3 models of the core region of OsHV-1 ORF57 (residues 66–268) as a hexamer (left), decamer (middle), or hexadecamer (right). Structural elements and domains are colored as in Fig 4: TM1 (blue), TM2 (deep teal), FD (magenta), AH (orange), and TM3 (teal).

Figure 1.. CLCC1 emerged as a top positive regulator of HSV-1 nuclear egress in a genome-wide CRISPR-Cas9 screen.

a) The flow-cytometry-based nuclear egress assay separates HSV-1 infected HeLa cells with vs. without nuclear egress based on two fluorescent signals: tdTomato (red, HSV-1 infection) and Alexa-488 (green, presence of cytoplasmic capsids). HeLa cells infected with HSV-1 encoding tdTomato, were partially permeabilized 24 hpi and stained with an anti-capsid mAb and an Alexa488-conjugated secondary mAb. Cells in the tdTomato+/Alexa488+ quadrant (Q2) are infected and have cytoplasmic capsids, indicating nuclear egress. Cells in the tdTomato+/Alexa488− quadrant (Q1) are infected but do not have cytoplasmic capsids, indicating no nuclear egress. Left: ~90% cells infected with WT HSV-1 are tdTomato+/Alexa488+. Right: only ~6% cells infected with HSV-1 ΔUL34 virus, which has a defect in nuclear egress, are tdTomato+/Alexa488+. b) Schematic of the genome-wide CRISPR screen. HeLa-Cas9 cells were transduced with Gattinara library lentivirus, containing ~40,000 sgRNAs, with 2 sgRNAs/gene, and after selection with puromycin, were infected with HSV-1 encoding tdTomato. 24 hpi, partially permeabilized cells were stained with a capsid-specific antibody and sorted by flow cytometry. c) Volcano plot of the screen results. Each dot represents a specific gene. The x-axis shows the fold change (FC) of sgRNAs, plotted as log(FC). Genes with log(FC) values >0 or <0 are candidate positive or negative regulators, respectively. The y-axis shows the significance score plotted as −log(p-value). The dotted line at y = 3 is the threshold for p-value < 0.001, indicating high confidence candidates. The dashed line at y = 1.3 is the threshold for p-value < 0.05. Red: top hit, CLCC1. Green: genes known to contribute to HSV-1 nuclear egress. High-confidence hit, EMD, is labelled. Gray: control sgRNAs (targeted, non-site, or intergenic sites).

Figure 2.. CLCC1 is essential for HSV-1 nuclear egress and HSV-1, HSV-2, and PrV replication.

a) Depletion of CLCC1 causes a defect in nuclear egress, measured by the flow cytometry nuclear egress assay. Single-clone CLCC1-KO (cko3_4 and cko6_1) or two control, HeLa and intergenic site targeting (Int_4) cell lines were infected with WT HSV-1 at an MOI of 5. As a positive control, HeLa cells were infected with HSV-1 ΔUL34 mutant virus, defective in nuclear egress, at an MOI of 10. Nuclear egress was measured at 24 hpi and normalized to HSV-1-infected HeLa cells. Each experiment had three biological replicates, each containing two technical replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.0001 = ****. Significance was calculated using one-way ANOVA, with multiple comparisons. b) Multiple-step growth curves for HSV-1 on single-clone CLCC1-KO (cko3_4 and cko6_1), single-clone CLCC1-R (cko3_4_R_1 and cko6_1_R_1), or two control, HeLa and Int_4, cell lines. Cells were infected with HSV-1 at MOI of 0.1, and supernatants were titrated in Vero cells using plaque assay. The y-intercept is set to 10° at time 0 for visual clarity. Each symbol shows the mean of three biological replicates, each containing two technical replicates, and bars show SEM. c) Expression of CLCC1 in trans rescues the defect in nuclear egress, measured by the flow cytometry nuclear egress assay as in a). Single-cell CLCC1-KO (cko3_4 and cko6_1), single-cell CLCC1-R (cko3_4_R_1 and cko6_1_R_1), or control Int_4 cell lines were infected with HSV-1 at an MOI of 5. Nuclear egress was measured at 24 hpi and normalized to Int_4 signal. Each data point represents a biological replicate. Each experiment had three biological replicates, each containing two technical replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.0001 = ****. Significance was calculated using one-way ANOVA, with multiple comparisons.

Figure 3.. Depletion of CLCC1 causes accumulation of PEVs in HSV-1-infected cells and formation of nuclear enveloped blebs (NEBs) in uninfected cells.

a, c) TEM images of Int_4, CLCC1-KO (cko3_4 and cko6_1), and CLCC1-R (cko6_1_R_1) cell-lines either infected with HSV-1 at an MOI of 5 (a) or uninfected (c). Scale bar = 800 nm. Zoomed-in views of features of interest are shown on the right. b, d) Quantification of PEVs in infected cells (b) and NEBs in uninfected cells (d). In (b), data were combined from two biological replicates. Each dot represents the number of events in a single cell. Bars represent mean values, and the error bars represent SEM. P < 0.001 = ***; P < 0.0001 = ****. Significance was calculated using one-way ANOVA, with multiple comparisons.

Figure 4.. Herpesviral and cellular CLCC1 homologs share sequence and structural similarity.

a) Domain architecture of human CLCC1 and two herpesviral CLCC1 homologs, IcHV1 ORF16a and OsHV-1 ORF57. Structural elements and domains were assigned based on structural predictions and secondary structure assignments and colored as follows: transmembrane helix 1 (TM1, blue), TM2 (deep teal), fist domain (FD, magenta), amphipathic helix (AH, orange), and TM3 (teal). S-S = predicted disulfide bond (purple). Transmembrane domains were predicted by TMHMM 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/). b) Ribbon diagrams of AlphaFold3 models of human CLCC1, IcHV1 ORF16a, and OsHV-1 ORF57. Structural models were generated using the AlphaFold 3.0 online server (https://golgi.sandbox.google.com/) and displayed using Pymol. Structural elements and domains are colored as in (a) and labelled. c) Multiple sequence alignment of human CLCC1, its herpesviral homologs IcHV1 ORF16a and OsHV-1 ORF57, and CLCC1 homologs from the respective hosts, Ictalurus punctatus (Channel catfish) and Crassostrea gigas (Pacific oyster). Similar residues are highlighted in yellow. Identical residues are highlighted in red. Structural elements and domains are colored and labelled as in (a) and (b). Sequence alignment was generated using Clustal Omega and rendered using ESPript 3.0 (https://espript.ibcp.fr).

Figure 5.. A highly conserved CLCC1 residue and residues involved in chloride channel activity are important for HSV-1 nuclear egress.

A) Schematic representation of the locations of CLCC1 mutations and their functions (if known). Structural elements and domains were assigned as in Fig 4 and colored as follows: TM1/TM2/TM3 (light blue), FD (light pink), and AH (light orange). S-S = predicted disulfide bond (purple). Approximate locations of mutated residues are shown as dots. b) A ribbon diagram of an AlphaFold3 model of human CLCC1. Structural elements and domains are colored as in (a) and labelled. Mutated residues are shown in sphere representation and colored as in a). Residues 365–539 were removed, for clarity. c, d) Several CLCC1 mutants rescue a defect in nuclear egress due to CLCC1 depletion whereas others do not, as measured by the flow cytometry nuclear egress assay. Single-clone CLCC1-KO (cko6_1) (c) or control Int_4 cell line (d) were either mock-transduced (Mock) or transduced with lentiviral constructs encoding WT CLCC1 or mutants D152R/D153R, E175R/D176R, D181R, D25E/D181R, S263R, W267R, D277R, K298E in the CLCC1-CR background. Bulk pools were infected with WT HSV-1 at an MOI of 5. Nuclear egress was measured at 24 hpi and normalized to the Int_4 mock. Each experiment had at least two biological replicates, each containing two technical replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.01 = **, P < 0.05 = *. Significance was calculated using one-way ANOVA, with multiple comparisons. The color scheme is as in (a) and (b).