A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection

Abstract

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

Keywords: TMED10; genome-wide screen; heparan sulfate; macropinocytosis; phosphatidylserine; poxvirus; vaccinia virus.

FIG 1

Genome-wide haploid genetic screen identifies host factors required for MVA infection. Genes enriched for retroviral insertion sites in MVA-exposed cells (left) compared to an unexposed control population (right). The percentage of sense orientation gene-trap insertions is plotted on the y axis. The total number of insertions in a particular gene is plotted on the x axis. Genes indicated by larger red dots were further characterized in this study.

FIG 2

EXT1, TM9SF2, TMED10, and SACM1L are essential genes for MVA infection. Validation of hits in MJS cells. Wild-type MJS cells (wt) and MJS cells transfected with Cas9 and indicated gRNAs (see Table 1) were infected with MVA-eGFP (MOI, 50). After 7 days of infection, cells were harvested and quantified by flow cytometry. Data are represented with standard errors of the means (SEMs) from three independent infection experiments.

FIG 3

Efficiency of MVA infection depends on HepS surface levels. (A) Wild-type MJS cells (wt) and MJS cells transfected with Cas9 and gRNA TAP1, EXT1#2, EXT1#4, TM9SF2#1, or TM9SF2#2 were cloned and infected with MVA-eGFP (MOI, 10). After 5 h of infection, cells were harvested and the amount of infected cells (GFP positive) was quantified by flow cytometry. SEMs from three independent infections are indicated. Three representative clones of five are shown for EXT1, four of these clones were protected from MVA infection. Three representative clones of eleven are shown for TM9SF2. (B) Clonal lines indicated in panel A were analyzed for HepS expression levels by flow cytometry. (C) Eleven clonal lines obtained from MJS cells transfected with gRNA TM9SF2#1 or TM9SF2#2 were infected with MVA-eGFP (MOI, 10). In addition, the cells were stained for HepS surface expression, and the mean fluorescence intensity (MFI) was quantified by flow cytometry.

FIG 4

TMED10 restricts infection of MVA- and VACV-eGFP. (A) HeLa cells expressing Cas9 only or coexpressing TMED10#1 and TMED10#5, and MJS cells expressing Cas9 only or coexpressing TMED10#1 or TMED10#5 were cloned, and transferrin receptor and TMED10 levels were determined by immunoblotting. The immunoblot is representative of three independent experiments. (B) MJS cell clones indicated in panel A or EXT1 clone 1 (see Fig. 3A) were infected with MVA-eGFP, MVA-eGFP^late, or VACV-eGFP. After 5 h, cells infected with MVA-eGFP and VACV-eGFP were harvested to determine early promoter-driven eGFP expression of MVA-eGFP (MVA-early) or VACV-eGFP (VACV-early). After 20 h of infection with MVA-eGFP^late, cells were harvested to determine eGFP expression driven from a late promoter (MVA-late). SEMs from four independent experiments are shown. ns, not significant; *, P < 0.05; **, P < 0.0005. Significance was calculated using a two-tailed unpaired t test. (C) MJS cells and HeLa cells indicated in panel A were infected with VACV-eGFP at an MOI of either 10 or 20, and the percentage of infected (eGFP positive) cells was quantified by flow cytometry. SEMs from three independent experiments are shown. (D) Wild-type MJS cells or MJS clones indicated in panel A were transduced with an empty vector (+EV) lentivirus or a lentivirus encoding TMED10 (+TMED10). Transferrin receptor and TMED10 expression levels were assessed by immunoblotting. The immunoblot shown is representative of two independent experiments. (E) Wild-type MJS cells or clones indicated in panel D were infected with VACV-eGFP. The percentage of infected (eGFP positive) cells was quantified by flow cytometry 5 h after infection. SEMs from four independent experiments are shown. (F) Flow cytometric analysis of wild-type MJS and MJS TMED10 clones 1 and 2 stained for HepS surface expression or with the secondary antibody only. (G) VACV-WR was allowed to bind control cells or indicated clones on ice. After 1 h, cells were washed, fixed, and stained with an antibody specific for the viral H3 protein. Bound antibody was quantified by flow cytometry. The mean fluorescence intensity of the control cells was set at 100%. SEMs from three experiments are shown.

FIG 5

Virus-induced formation of blebs is dependent on TMED10 expression. Control MJS cells expressing an empty vector (EV) or TMED10 clone 2 expressing an EV or TMED10 (see Fig. 4D) were incubated with VACV-WR (MOI, 100) (+VACV) or left untreated (−VACV) for 1 h on ice. Subsequently, the cells were incubated at 37°C for 45 min to allow bleb formation. Blebs were visualized by staining the cells for actin using phalloidin-iFluor 488; nuclei were counterstained using TO-PRO-3. A representative image field of 20 different image fields is presented. Bar, 7.5 μm.

FIG 6

Uptake of LUVs is mediated by macropinocytosis and depends on PS and serum components. (A) Microscopic tracing of cells exposed to 3 μM fluorescent LUVs composed of PC and PS. (B) Flow cytometric analysis of MJS cells incubated with fluorescent LUVs for 45 min. 1, uptake using different concentrations of LUVs (6 to 0.2 μM); 2, LUV uptake (3 μM) in the absence or presence of the specific macropinocytosis inhibitor EIPA; 3, LUV uptake (3 μM) in the presence or absence of serum; 4, uptake of LUVs (3 μM) composed of PC only or PC and PS.

FIG 7

Phosphatidylserine-mediated macropinocytotic uptake is dependent on TMED10. (A) Flow cytometric analysis of control HeLa and MJS cells (WT+LUV) and HeLa TMED10 clone 1 and MJS TMED10 clone 1 incubated for 40 min at 37°C degrees with 3 μM fluorescent LUVs composed of PC and PS. Cells were harvested and fluorescence was determined by flow cytometry. (B) Mean fluorescence intensity (MFI) of LUV uptake was measured as in panel A in the indicated HeLa and MJS clones transduced with an empty control (EV) or TMED10-expressing lentiviral vector. LUV uptake was normalized to wt cells incubated with LUV. SEMs from four independent experiments are shown. (C) Uptake of 70-kDa dextran Texas Red beads in parental MJS cells and indicated clonal cells. SEMs from three independent experiments are shown (D). Cell surface expression of the TAM receptor Axl on control MJS cells and TMED10 KO cells transduced with an empty control (EV) or TMED10-expressing lentiviral vector. SEMs from three independent experiments are shown.