AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity

Abstract

Flaviviruses strategically utilize the endoplasmic reticulum (ER) in their replication cycles. However, the role of ER autophagy (ER-phagy) in viral replication process remains poorly understood. Here, we reveal that prolonged Zika virus (ZIKV) infection results from the degradation of ER-phagy receptor FAM134B, facilitated by viral NS2A protein. Mechanistically, ER-localized NS2A undergoes K48-linked polyubiquitination at lysine (K) 56 by E3 ligase AMFR. Ubiquitinated NS2A binds to FAM134B and AMFR orchestrates the degradation of NS2A-FAM134B complexes. AMFR-catalyzed NS2A ubiquitination not only targets FAM134B degradation but also hinders the FAM134B-AMFR axis. Notably, a recombinant ZIKV mutant (ZIKV-NS2A_K56R), lacking ubiquitination and ER-phagy inhibition, exhibits attenuation in ZIKV-induced microcephalic phenotypes in human brain organoids and replicates less efficiently, resulting in weakened pathogenesis in mouse models. In this work, our mechanistic insights propose that flaviviruses manipulate ER-phagy to modulate ER turnover, driving viral infection. Furthermore, AMFR-mediated flavivirus NS2A ubiquitination emerges as a potential determinant of viral pathogenecity.

Fig. 1. FAM134B undergoes degradation during several flavivirus infections.

a Workflow for TMT-based quantitative proteomic analysis. b Median abundance of individual ZIKV proteins. c Cellular component analysis of significantly up-changing proteins (−log₁₀ (P value)). d Western blotting analysis of A549 cells either mock-infected or infected with ZIKV at a MOI of 0.1. e Quantification of relative protein level of ER-phagy receptors. f Cells (A549, Huh7.0, and HeLa) stably expressing GFP-FAM134B were either mock-infected or infected with ZIKV at different MOIs (A549[0.1]; Huh7.0[1]; HeLa[1]) for 72 h, determined by flow cytometry. g–j Western blotting analysis of Huh7.0 (g) or A549 (i) were either mock-infected or infected with ZIKV at multiple time points with a MOI of 0.1 (g) or at multiple MOIs for 24 h (i) and the corresponding quantification of relative FAM134B level (h, j). k Cells (A549 and Huh7.0) stably expressing GFP-FAM134B_R142A were either mock-infected or infected with ZIKV at different MOIs (A549[0.1]; Huh7.0[1]) for 72 h, determined by flow cytometry. l A549 stably expressing GFP-FAM134B_R142A were either mock-infected or infected with ZIKV at multiple time points with an MOI of 0.1, determined by flow cytometry. m Viral titers of supernatants from Huh7.0 FAM134B WT cells, Huh7.0 FAM134B KO cells, Huh7.0 FAM134B KO cells stably expressing FAM134B_WT or Huh7.0 FAM134B KO cells stably expressing FAM134B_R142A infected with ZIKV at a MOI of 1 for 72 h, quantified by plaque assay. n Huh7.0 cells stably expressing GFP-FAM134B_R142A were either mock-infected or infected with ZIKV, DENV, or JEV at a MOI of 1 for 72 h, determined by flow cytometry. o, p Viral titers of supernatants from Huh7.0 FAM134B WT, Huh7.0 FAM134B KO cells, or Huh7.0 FAM134B KO cells stably expressing FAM134B_WT infected with DENV or JEV at a MOI of 1 for 72 h, quantified by plaque assay. Error bars of (e, f, h, j–p) present as the mean ± SEM (n = 3 independent experiments); Statistical analysis of (e, f, k, n) was performed using two-way ANOVA with Sidak’s multiple comparisons test and (h, j, l, m, o, p) was performed using one-way ANOVA with Tukey’s multiple comparisons test; Source data are provided as a Source Data file.

Fig. 2. Flavivirus-NS2A targets FAM134B for degradation.

a, b Western blotting analysis of HEK293T cells co-transfected for 48 h with indicated Myc-FAM134B and Flag-tagged ZIKV viral protein-expressing plasmids or empty vector. The red font highlighted the ZIKV-NS2A protein. c Western blotting analysis of A549 cells transfected for 48 h with increasing amounts of Flag-NS2A. d Huh7.0-GFP-FAM134B transfected for 48 h with increasing amounts of Flag-NS2A, determined by flow cytometry. e Huh7.0-GFP-FAM134B_R142A cells were infected with recombinant virus ZIKV-HA-NS2A at a MOI of 1 for 72 h and assessed confocal microscopy analysis. The plus sign (+) represents virus-infected cells marked by HA-NS2A. Scale bars, 10 μm. f Quantification of mean intensity of GFP-FAM134B_R142A, related to (e). g HEK293T cells were co-transfected with Flag-NS2A and HA-tagged ER-phagic receptor-expressing plasmids or empty vector. Cells were harvested for coIP using anti-HA beads. The asterisk (*) represents the indicated ER-phagy receptors. h Huh7.0 cells were either mock-infected or infected with ZIKV-HA-NS2A at a MOI of 1 for 72 h, and then cells were harvested for coIP using anti-HA beads and analyzed by western blotting. The asterisk (*) represents the co-immunoprecipitated endogenous FAM134B, and the band of HA-NS2Aα detected by western blotting represented a truncated form of NS2A. i HEK293T cells were co-transfected with HA-FAM134B and Flag-tagged flavivirus-NS2A-expressing plasmids or empty vector. Cells were harvested for coIP using anti-flag beads. The asterisk (*) represents the indicated flavivirus-NS2A. j Schematic diagram of FAM134B mutants. k HEK293T cells were co-transfected with FAM134B_WT and mutants with Flag-NS2A or empty vector. Cells were harvested for coIP using anti-flag beads. The asterisk (*) represents the indicated FAM134B_WT and mutants. l, m Huh7.0-GFP-FAM134B_△NTD were transfected for 48 h with increasing amounts of Flag-NS2A (l) or infected with ZIKV at a MOI of 1 (m) for 72 h, determined by flow cytometry. Error bars of (d, f, l, m) present as the mean ± SEM (d, l, m: n = 3; f: n = 32); Statistical analysis of (d, l, m) was performed using one-way ANOVA with Tukey’s multiple comparisons test and (f) was performed using two-tailed unpaired t-test analysis; Source data are provided as a Source Data file.

Fig. 3. AMFR mediates flavivirus-NS2A ubiquitination which is required for NS2A-drived FAM134B degradation.

a, b, Western blotting analysis of HEK293T cells transfected for 30 h with the indicated plasmids and then treated with DMSO, MG132 (10 μM), and CQ (50 μM) for 8 h (a); Quantification of relative FAM134B level (b). c–f, h, j, HEK293T cells were transfected with the indicated plasmids. Cells were harvested for coIP using anti-Flag beads and analyzed by western blotting with a K48-linkage-specific polyubiquitin antibody to detect the ubiquitination of NS2A. g In vitro ubiquitination assays were measured by incubating purified Flag-NS2A with purified HA-AMFR_WT or HA-AMFR_C356G/H361A analyzed by western blotting with a K48-linkage specific polyubiquitin antibody to detect the ubiquitination of NS2A. i Huh7.0 cells were either mock-infected or infected with ZIKV-HA-NS2A at an MOI of 1 for 72 h and then cells were harvested for coIP using anti-HA beads and analyzed by western blotting. k A549 and Vero cells were mock-infected or infected with ZIKV-HA-NS2A_WT (MOI = 0.1) or ZIKV-HA-NS2A_K56R (MOI = 1) for 72 h and then harvested for coIP using anti-HA beads and analyzed by western blotting with a K48-linkage specific polyubiquitin antibody to detect the ubiquitination of NS2A and the input E was normalized for immunoprecipitation. l, m Western blotting analysis of HEK293T cells or HEK293T-AMFR knockdown cells transfected for 48 h with the indicated plasmids (l); Quantification of relative FAM134B level (m). n Huh7.0-GFP-FAM134B_R142A cells were infected with ZIKV-NS2A_WT (MOI = 1), ZIKV-NS2A_K56R (MOI = 10), or mock-infected for 72 h, determined by flow cytometry. Error bars of (b, m, n) present as the mean ± SEM (n = 3 independent experiments); Statistical analysis of (b) was performed using two-way ANOVA with Sidak’s multiple comparisons test and (m, n) was performed using one-way ANOVA with Tukey’s multiple comparisons test; Source data are provided as a Source Data file.

Fig. 4. ZIKV subverts FAM134B to restrict ER-phagy process.

a Diagram of mCherry-EGFP tandem tagging assays. b Diagram of GFP-cleavage assays. c Immunofluorescence analysis of U2OS cells expressing mCherry-eGFP- RAMP4 infected with ZIKV-HA-NS2A_WT (MOI = 1), ZIKV-HA-NS2A_K56R (MOI = 10), or mock-infected for 48 h. Scale bars, 10 μm. d ER-phagy flux was quantified by the number of mCherry+ eGFP– dots. Error bars present as the mean ± SEM (n = 21); Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; Source data are provided as a Source Data file. e, g Western blotting analysis of U2OS cells transfected for 12 h with GFP-SEC61B and Myc-FAM134B (e) or Myc-FAM134B (g) and then infected with ZIKV-NS2A_WT (MOI = 1), ZIKV-NS2A_K56R (MOI = 10), or mock-infected for 48 h. f, i Western blotting analysis of U2OS cells transfected for 48 h with GFP-SEC61B and Myc-FAM134B and Flag-NS2A_WT or Flag-NS2A_K56R or empty vector (f) or Myc-FAM134B and Flag-NS2A_WT or Flag-NS2A_K56R or empty vector (h). h, j Quantification of relative CLIMP63 and REEP5 protein level, h corresponds to (g, j) corresponds to (i); Error bars of (h, j) present as the mean ± SEM (n = 3 independent experiments); Statistical analysis of (h, j) was performed using two-way ANOVA with Sidak’s multiple comparisons test; Source data are provided as a Source Data file.

Fig. 5. ZIKV mutant (ZIKV-NS2A_K56R) with ER-phagy inhibition-deficiency exhibits impaired microcephaly in human brain organoids.

a Diagram of viral exposure of brain organoids. b qPCR analysis of ZIKV-E RNA in organoids exposed to ZIKV-NS2A_WT or ZIKV-NS2A_K56R at 4 and 8 dpi. c Immunofluorescence analysis of Vero cells incubated with supernatants of ZIKV-infected organoids at 4 and 8 dpi. Scale bars, 75 μm. ZIKV-E (green), DAPI (blue). d, e Images and area measurements of organoids exposed to ZIKV-NS2A_WT, ZIKV-NS2A_K56R, or mock-treated at 4 and 8 dpi. Scale bars, 500 μm. f, g qRT-PCR analysis of SOX1 (f) and SOX2 (g) RNA in organoids exposed to ZIKV-NS2A_WT, ZIKV-NS2A_K56R or mock-treated at 4 and 8 dpi. h Immunofluorescence analysis of organoids exposed to ZIKV-NS2A_WT, ZIKV-NS2A_K56R or mock-treated. Scale bars, 75 μm. The area of two dashed lines represents ventricular zone (VZ)-like structures. ZIKV-E (green), SOX2 (red). i The measurement of VZ-like structures of (h). j Western blotting analysis of organoids exposed to ZIKV-NS2A_WT (7 × 10⁴ PFU/organoid), ZIKV-NS2A_K56R (7 × 10⁵ PFU/organoid) or mock-treated at 8 dpi. Error bars of (b, e, f, g, i) present as the mean ± SEM (b, f, g: n = 3; e: n = 13; i: n = 6); Statistical analysis of (b, e, f, g) was performed using two-way ANOVA with Sidak’s multiple comparisons test and (i) was performed using one-way ANOVA with Tukey’s multiple comparisons test; Source data are provided as a Source Data file.

Fig. 6. ZIKV-NS2A_K56R infection causes weakened pathogenicity and lethality in mice.

a Daily weights of mice treated with PBS (n = 6), ZIKV-NS2A_WT (n = 7), or ZIKV-NS2A_K56R (n = 7) (i.p., 10⁴ PFU/ mouse); Error bars present as the mean ± SEM; Source data are provided as a Source Data file. b Survival of mice from (a); Log-rank (Mantel-Cox) test. c–g qRT-PCR analysis of ZIKV-E RNA in different tissues of mice infected with ZIKV-NS2A_WT or ZIKV-NS2A_K56R (n = 6); Error bars of (c–g) present as the mean ± SEM; Statistical analysis of (c–g) was performed using two-tailed unpaired t-test analysis; Source data are provided as a Source Data file. h Immunofluorescence analysis of brains of mice infected with ZIKV-NS2A_WT or ZIKV-NS2A_K56R. Scale bars, 1000 μm. ZIKV-E (green), DAPI (blue). i Hematoxylin and eosin staining analysis of brains of mice infected with PBS, ZIKV-NS2A_WT, or ZIKV-NS2A_K56R. The black arrows represent the inflammation damage in the brain tissues. Scale bars, 100 μm. j Western blotting analysis of mFAM134B expression in mouse tissues infected with ZIKV-NS2A_WT or ZIKV-NS2A_K56R.

Fig. 7. Model for AMFR-mediated Flavivirus NS2A ubiquitination subverts ER-phagy to augment viral pathogenicity.

Left: The ER-resident E3 ligase AMFR-mediated ubiquitination of ER-phagy receptor FAM134B promotes the dynamic flux of ER-phagy process to degrade ER sheets; Middle: During flavivirus infection, ZIKV hijacks AMFR to catalyze the polyubiquitination of ER-localized viral NS2A protein at K56 through K48-linked manner. Ubiquitinated NS2A binds to FAM134B, and AMFR orchestrates the degradation of NS2A-FAM134B complexes, ultimately impeding the flux of ER-phagy for ER accumulation and hindering the FAM134B-AMFR axis. Right: The ubiquitination and ER-phagy inhibition-deficient ZIKV-NS2A_K56R infection exhibits weakened microcephaly and reduced replicative potential and decreased pathgenicity in mice.