TMEM41B is a pan-flavivirus host factor

Abstract

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection. Based on our mechanistic studies we hypothesize that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.

Highlights: TMEM41B and VMP1 are required for both autophagy and flavivirus infection, however, autophagy is not required for flavivirus infection.TMEM41B associates with viral proteins and likely facilitates membrane remodeling to establish viral RNA replication complexes.TMEM41B single nucleotide polymorphisms (SNPs) present at nearly twenty percent in East Asian populations reduce flavivirus infection.TMEM41B-deficient cells display an exaggerated innate immune response upon high multiplicity flavivirus infection.

Figure 1.. Genome-wide CRISPR-Cas9 screens for Zika and yellow fever viruses identify TMEM41B and VMP1 as required host factors.

(A) Bubble plot of data from ZIKV screen (top) and YFV screen (bottom). Red lines denote z = ± 2. (B) Heatmap of z scores for genes in the autophagy pathway ordered sequentially by functional role: L, lipid mobilization; 1, initiation; 2, nucleation; 3, elongation; 4, sequestration; 5, tethering/fusion. (C) Scatter plot of gene-wise log₂ fold change (LFC) from this study (ZIKV) versus Moretti et al. autophagy screen. (D) HAP1 wildtype (WT) and (n=3) individual knockout (KO) clones for VTT domain-containing proteins infected with ZIKV (MOI = 0.5 PFU/cell) for 48 h. (E) WT and TMEM41B KO HAP1 cells overexpressing individual VTT domain proteins infected with ZIKV (MOI = 0.25 PFU/cell) for 48 h. (F) WT and VMP1 KO HAP1 cells overexpressing individual VTT domain proteins infected with ZIKV ((MOI = 0.25 PFU/cell) for 48 h. (G) HAP1 WT and (n=3–5) individual KO clones for autophagy genes infected with ZIKV (MOI = 0.5 PFU/cell) for 48 h. (H-K) Same as panels D-G but infected with YFV Asibi (MOI = 0.1 PFU/cell) for 72 h. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Dots in panels D, G, H, and K represent the average of n=3 replicates from individual single cell clones. Error bars in panels E, F, I, and J depict standard deviation (SD) of n = three replicates. See also Figure S1 B–I.

Figure 1.. Genome-wide CRISPR-Cas9 screens for Zika and yellow fever viruses identify TMEM41B and VMP1 as required host factors.

(A) Bubble plot of data from ZIKV screen (top) and YFV screen (bottom). Red lines denote z = ± 2. (B) Heatmap of z scores for genes in the autophagy pathway ordered sequentially by functional role: L, lipid mobilization; 1, initiation; 2, nucleation; 3, elongation; 4, sequestration; 5, tethering/fusion. (C) Scatter plot of gene-wise log₂ fold change (LFC) from this study (ZIKV) versus Moretti et al. autophagy screen. (D) HAP1 wildtype (WT) and (n=3) individual knockout (KO) clones for VTT domain-containing proteins infected with ZIKV (MOI = 0.5 PFU/cell) for 48 h. (E) WT and TMEM41B KO HAP1 cells overexpressing individual VTT domain proteins infected with ZIKV (MOI = 0.25 PFU/cell) for 48 h. (F) WT and VMP1 KO HAP1 cells overexpressing individual VTT domain proteins infected with ZIKV ((MOI = 0.25 PFU/cell) for 48 h. (G) HAP1 WT and (n=3–5) individual KO clones for autophagy genes infected with ZIKV (MOI = 0.5 PFU/cell) for 48 h. (H-K) Same as panels D-G but infected with YFV Asibi (MOI = 0.1 PFU/cell) for 72 h. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Dots in panels D, G, H, and K represent the average of n=3 replicates from individual single cell clones. Error bars in panels E, F, I, and J depict standard deviation (SD) of n = three replicates. See also Figure S1 B–I.

Figure 2.. TMEM41B is a pan-flavivirus host factor.

(A) WT and TMEM41B KO HAP1 cells infected for 48 h with encephalitic tick-borne flaviviruses POWV at MOI = 0.02 PFU/cell, and for 24h with TBEV (strain: Hypr) at MOI = 0.02 PFU/cell, TBEV (strain: Sofjin) at MOI = 0.02 PFU/cell. (B) WT and TMEM41B KO HAP1 cells infected for 24 h with hemorrhagic fever tick-borne flaviviruses OHFV at MOI = 0.02 PFU/cell, KFDV at MOI = 0.02 PFU/cell, AHFV at MOI = 0.02 PFU/cell. (C) WT and TMEM41B KO Huh-7.5 clones infected with HCV-RFP for 72 h at MOI = 0.05 PFU/cell. (D) TMEM41B KO MDBK clones infected with BVDV for 48 h at MOI = 0.01 PFU/cell. Dots represent the average of n=3 replicates from individual single cell clones. (A-D) Virus strain is indicated in parenthesis. Error bars in panels A and B show SD for n=6 replicates. Error bars in panels C and D show SD for n=3 replicates (WT) and individual KO clones. (E) Table summarizing results shown in Figure S2 (panels A, B, D, F and H-K) from infection experiments with multiple viruses in various mammalian and mosquito cell lines. TBFV = tick-born flaviviruses of panel A and B, +++ = infection comparable to WT cells; +/− = reduced but detectable infection; − = negligible infection. (F) WT, TMEM41B KO and reconstituted HAP1 cells infected with (−) sense RNA viruses. Influenza A virus (IAV), MOI = 0.1 PFU/cell for 24 h; bunyamwera virus (BUNV-GFP), MOI = 0.025 IU/cell for 48 h; human parainfluenza virus 3 (h-PIV3-GFP), MOI = 0.02 IU/cell for 48 h; lymphocytic choriomeningitis virus (LCMV-GFP), MOI = 0.01 IU/cell for 48 h; vesicular stomatitis virus (VSV-GFP), MOI = 0.01 PFU/cell for 24 h. (G) WT, TMEM41B KO and reconstituted HAP1 cells infected with VSV-G-pseudotyped HIV-1 lentivirus (LV-GFP), MOI = (1:25 dilution of virus stock) for 48 h. (H) WT, TMEM41B KO and reconstituted HAP1 cells infected with (+) sense RNA viruses. Cells infected with SARS-CoV-2 were prior reconstituted to stably express ACE2 and TMPRSS2, MOI = 1 PFU/cell for 24 h; Coxsackie virus B (CVB-3-GFP), MOI = (1:100 dilution of virus stock) for 8 h; chikungunya virus (CHIKV-GFP), MOI = 0.025 PFU/cell for 24 h. (I) WT, TMEM41B KO and reconstituted HAP1 cells infected with DNA viruses. Adenovirus 5 (AdV5-GFP), MOI = 200 p/cell for 24 h; herpes simplex virus 1 (HSV-1-GFP), MOI = 2.5 PFU/cell for 8 h; vaccinia virus (VacV-GFP), MOI = 0.005 PFU/cell for 48 h. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells or GFP/RFP positive cells for reporter viruses expressing a fluorescent protein. Error bars depict SD for three replicates or the indicated number of clones.

Figure 2.. TMEM41B is a pan-flavivirus host factor.

(A) WT and TMEM41B KO HAP1 cells infected for 48 h with encephalitic tick-borne flaviviruses POWV at MOI = 0.02 PFU/cell, and for 24h with TBEV (strain: Hypr) at MOI = 0.02 PFU/cell, TBEV (strain: Sofjin) at MOI = 0.02 PFU/cell. (B) WT and TMEM41B KO HAP1 cells infected for 24 h with hemorrhagic fever tick-borne flaviviruses OHFV at MOI = 0.02 PFU/cell, KFDV at MOI = 0.02 PFU/cell, AHFV at MOI = 0.02 PFU/cell. (C) WT and TMEM41B KO Huh-7.5 clones infected with HCV-RFP for 72 h at MOI = 0.05 PFU/cell. (D) TMEM41B KO MDBK clones infected with BVDV for 48 h at MOI = 0.01 PFU/cell. Dots represent the average of n=3 replicates from individual single cell clones. (A-D) Virus strain is indicated in parenthesis. Error bars in panels A and B show SD for n=6 replicates. Error bars in panels C and D show SD for n=3 replicates (WT) and individual KO clones. (E) Table summarizing results shown in Figure S2 (panels A, B, D, F and H-K) from infection experiments with multiple viruses in various mammalian and mosquito cell lines. TBFV = tick-born flaviviruses of panel A and B, +++ = infection comparable to WT cells; +/− = reduced but detectable infection; − = negligible infection. (F) WT, TMEM41B KO and reconstituted HAP1 cells infected with (−) sense RNA viruses. Influenza A virus (IAV), MOI = 0.1 PFU/cell for 24 h; bunyamwera virus (BUNV-GFP), MOI = 0.025 IU/cell for 48 h; human parainfluenza virus 3 (h-PIV3-GFP), MOI = 0.02 IU/cell for 48 h; lymphocytic choriomeningitis virus (LCMV-GFP), MOI = 0.01 IU/cell for 48 h; vesicular stomatitis virus (VSV-GFP), MOI = 0.01 PFU/cell for 24 h. (G) WT, TMEM41B KO and reconstituted HAP1 cells infected with VSV-G-pseudotyped HIV-1 lentivirus (LV-GFP), MOI = (1:25 dilution of virus stock) for 48 h. (H) WT, TMEM41B KO and reconstituted HAP1 cells infected with (+) sense RNA viruses. Cells infected with SARS-CoV-2 were prior reconstituted to stably express ACE2 and TMPRSS2, MOI = 1 PFU/cell for 24 h; Coxsackie virus B (CVB-3-GFP), MOI = (1:100 dilution of virus stock) for 8 h; chikungunya virus (CHIKV-GFP), MOI = 0.025 PFU/cell for 24 h. (I) WT, TMEM41B KO and reconstituted HAP1 cells infected with DNA viruses. Adenovirus 5 (AdV5-GFP), MOI = 200 p/cell for 24 h; herpes simplex virus 1 (HSV-1-GFP), MOI = 2.5 PFU/cell for 8 h; vaccinia virus (VacV-GFP), MOI = 0.005 PFU/cell for 48 h. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells or GFP/RFP positive cells for reporter viruses expressing a fluorescent protein. Error bars depict SD for three replicates or the indicated number of clones.

Figure 3.. Functional TMEM41B is conserved across mammalian and vector species.

(A) Graphical representation of human TMEM41B isoforms and deletion construct. VTT domain is shaded pink. Amino acid length of each isoform is indicated. Isoform 1, UniProt ID: Q5BJD5–1; isoform 2, UniProt ID: Q5BJD5–2; isoform 3, UniProt ID: Q5BJD5–3; isoform 4, UniProt ID: E9PJ42. (B) WT, TMEM41B KO, and TMEM41B KO cells expressing human TMEM41B isoforms or (C) mosquito and tick TMEM41B isoforms infected with YFV Asibi, MOI = 0.1 PFU/cell for 72 h, or ZIKV, MOI = 0.25 PFU/cell for 48 h. (D) Table comparing homology between human TMEM41B (isoform 1) and mosquito and tick TMEM41B homologues. Numbers indicate percent amino acids identity for the full-length protein (first number) vs VTT domain only (second number). Protein sequences were aligned using Geneious Software (Geneious 8.1.9. https://www.geneious.com). Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Error bars depict SD for three replicates.

Figure 4.. Naturally occurring TMEM41B SNPs negatively impact flavivirus infection but are able to maintain normal lipid distribution in cells.

(A) Table shows the frequency of a SNP (rs78813996) in TMEM41B in several human populations. (B) WT, TMEM41B KO, and TMEM41B KO HAP1 cells expressing WT or TMEM41B SNP variants infected with YFV 17D, MOI = 0.005 PFU/cell for 48 h. Left, cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Right, supernatants were collected and titrated by tissue culture infectious dose (TCID₅₀/ml) assay on Huh-7.5 cells. (C) Same as panel (B, left) with ZIKV at MOI = 0.25 PFU/cell for 48 h; DENV-GFP at MOI = 0.1 PFU/cell for 96 h; WNV-GFP at MOI = 1 PFU/cell for 72 h; and hPIV-3-GFP at MOI = 0.02 IU/cell for 48 h. (D) WT and TMEM41B KO Huh-7.5 cell clones stained with Nile red to visualize lipid droplets. (E) Cumulative frequency of droplets plotted vs. droplet area (μm²) for six independent single cell clones generated with two independent sgRNAs. Inset shows the mean lipid droplet area (μm²) for the six TMEM41B KO clones compared to WT Huh-7.5 cells. (F) Mean lipid droplet area (μm²) for WT, TMEM41B KO, and TMEM41B KO Huh-7.5 cells (clone 1–1) expressing the indicated TMEM41B variants. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Error bars depict SD for three replicates.

Figure 5.. TMEM41B colocalizes with flavivirus NS4A and NS4B proteins.

(A) TMEM41B KO HAP1 cells expressing RFP-TMEM41B visualized in uninfected cells (mock) and YFV- and ZIKV-infected cells for 24 h at MOI = 1 and 2.5 PFU/cell, respectively). Anti-NS4A (ZIKV) and anti-NS4B antibodies detect viral antigens. Yellow-orange color in the merged column shows the colocalization of TMEM41B with viral antigens. DAPI stains cell nuclei. (B) Western blot shows that an anti-RFP antibody which recognizes RFP-TMEM41B (but not an IgG antibody control) co-immunoprecipitates ZIKV NS4A in HAP1 cells (MOI = 2.5 PFU/cell for 24 h). (C) Same as (B) but with YFV infection (MOI = 1 PFU/cell for 24 h) and visualized with an antibody that detects YFV NS4B. WCL = whole cell lysate. Of note, the heavy chain of the capture antibodies is detected by the secondary antibody protein A-HRP as indicated ~ 52 kDa. See also Figure S5.

Figure 6.. NS4A and NS4B mutations bypass TMEM41B deficiency.

(A) Graphical schematic of virus adaptation experiment. Top, WT Aag2 and two TMEM41B KO clones were infected with ZIKV (MOI = 0.5 PFU/cell) in triplicate. Cells were passaged every 2–3 days for 15 passages after which supernatants were collected and viral RNA was reverse transcribed and sequenced with next generation sequencing (NGS). Bottom, A549 WT cells together with two TMEM41B KO clones, and HAP1 WT cells together with one TMEM41B KO clone, were infected with ZIKV (MOI = 5 PFU/cell) in triplicate. Supernatants were diluted 1:2 and used to inoculate naïve cells every 3–4 days for 20 passages after which supernatants were collected and viral RNA was reverse transcribed and sequenced with next generation sequencing (NGS). (B) Summary of ZIKV adaptive mutations. Top, graphical representation of the ZIKV genome with mature (proteolytically processed) viral proteins shown as arrows and nucleotide positions listed below. NS4A and NS4B, are labeled and colored in light blue. Bottom, table of the amino acids where missense mutations were identified after passaging. Only mutations that were present in at least three independent passages (irrespective of cell type) and that did not appear in virus populations passaged in WT cells are shown. Boxes show the number of replicates a given mutation was identified over the total number of replicates analyzed for that cell type. Of note, out of six initial replicates for the A549 TMEM41B KO clones, only five were able to be carried over the course of 20 passages. (C) Missense mutations I42T and F91Y were independently engineered in a ZIKV infectious clone and used to generate virus stocks to infect WT (left) or TMEM41B KO (right) Aag2 cells (MOI = 0.001 PFU/cell for 72 h). The virus stocks used for infection are listed below the bars. (D) Same experiment as in (C) in WT and TMEM41B KO A549 cells (MOI = 0.05 PFU/cell for 48 h). (E-F) YFV 17D and Asibi chimeric viruses were generated and used to infect WT and TMEM41B KO A549 cells. (E) 17D backbone with Asibi NS4A and NS4B proteins (MOI = 0.1 PFU/cell for 48 h). (F) Asibi backbone with 17D NS4A and NS4B proteins (MOI = 0.1 PFU/cell for 48 h). Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Error bars depict SD for three replicates.

Figure 7.. TMEM41B KO cells mount an exaggerated innate immune response to flavivirus inoculum.

(A) Viral RNA quantified by qRT-PCR from WT and TMEM41B KO HAP1 cells infected with YFV 17D MOI = 0.4 PFU/cell for 24 h. (B) OAS1 mRNA quantified by q-RT-PCR from WT and TMEM41B KO HAP1 cells infected with YFV 17D (with and without UV-inactivation) MOI = 0.4 PFU/cell for 24 h or with IAV (ΔNS1) MOI = 0.1 PFU/cell for 24 h. (C) WT and TMEM41B KO HAP1 cells untreated (mock), infected with YFV 17D MOI = 4 PFU/cell for 24 h, or treated with 250 nM staurosporine (STS) and assayed to detect apoptotic cells via Annexin-V staining. Cells were analyzed by flow cytometry and plotted as a percentage of viral antigen positive cells. Error bars depict standard deviation (SD) of n = three replicates. (D) Model for the role of TMEM41B in the flavivirus life cycle. The model is described in the main text.