Glucosylceramide in bunyavirus particles is essential for virus binding to host cells

Abstract

Hexosylceramides (HexCer) are implicated in the infection process of various pathogens. However, the molecular and cellular functions of HexCer in infectious cycles are poorly understood. Investigating the enveloped virus Uukuniemi (UUKV), a bunyavirus of the Phenuiviridae family, we performed a lipidomic analysis with mass spectrometry and determined the lipidome of both infected cells and derived virions. We found that UUKV alters the processing of HexCer to glycosphingolipids (GSL) in infected cells. The infection resulted in the overexpression of glucosylceramide (GlcCer) synthase (UGCG) and the specific accumulation of GlcCer and its subsequent incorporation into viral progeny. UUKV and several pathogenic bunyaviruses relied on GlcCer in the viral envelope for binding to various host cell types. Overall, our results indicate that GlcCer is a structural determinant of virions crucial for bunyavirus infectivity. This study also highlights the importance of glycolipids on virions in facilitating interactions with host cell receptors and infectious entry of enveloped viruses.

Keywords: Alphavirus; Glucosylceramide synthase; Lipidomics; UGCG; Virus–receptor interactions.

Fig. 1

A label-free quantitative lipidomic analysis led to the identification of hexosylceramide (HexCer) as a major component of Uukuniemi virus (UUKV) particles. A BHK-21 cells were infected with UUKV at a multiplicity of infection of 0.1 for 48 h and then subjected to a lipidomic analysis. Lipid classes and subclasses are presented as mol % fractions. Unpaired t test with Welch correction was applied (n = 3). **, p < 0.01. B Schematic overview showing HexCer metabolism. The dashed colored lines indicate intracellular trafficking, while the dark lines show enzymatic reactions. β4GALT5/6, LacCer synthases β4GALT5 and β4GALT6; ER, endoplasmic reticulum; GalCer, galactosylceramide; GlcCer, glucosylceramide; GSL, glycosphingolipid; UGCG, UDP − GlcCer glucosyltransferase; UGT8, UDP − GalCer galactosyltransferase. Created with BioRender.com. C Supernatant from infected BHK-21 cells was harvested 48 h post-infection, and UUKV particles were purified before lipid MS analysis (n = 3). D Lipidome of UUKV-infected BHK-21 cells (n = 3). E Lipidome of uninfected BHK-21 cells (n = 3). C–E The phospholipids included all glycerophospholipids such as phosphatidylethanolamine plasmalogen (PE P), SM, and lysophosphatidylcholine (LPC). The neutral lipids were cholesteryl ester (CE), diacylglycerol (DAG), and triacylglycerol (TAG). F Chemical formulas of GlcCer and GalCer. Created with BioRender.com

Fig. 2

Glucosylceramide (GlcCer) synthase (UGCG) depletion inhibits Uukuniemi virus (UUKV) infection. A BHK-21 cells and hamster brain tissues were lyzed and total RNA was extracted before cDNA synthesis. The levels of mRNA encoding UGT8, UGCG, β4Galt5, and β4Galt6 were quantified by RT-qPCR (n = 2). B UGCG synthase was silenced in BHK-21 cells with two nonoverlapping short interfering RNAs (si1_UGCG and si2_UGCG; 20 nM) and assayed by western blotting 72 h later using the pAb against UGCG from LSBio. SiCtrl (Scrambled_1), negative-control siRNAs. C GlcCer levels in BHK-21 cells silenced for UGCG were examined by dot blotting using the same samples as in B. D The efficiency of UGCG knockdown was semi-quantified under the conditions described in B. UGCG protein levels are reported as the percentage of UGCG levels in cells treated with siRNAs against UGCG and normalized to levels of actin and UGCG in BHK-21 cells treated with negative-control siRNAs (si_Ctrl) (n = 2). E GlcCer levels were semi-quantified based on the dot blots shown in C and expressed as a percentage of GlcCer levels in cells treated with siRNAs against GlcCer and normalized to GlcCer levels in BHK-21 cells treated with negative control siRNAs (si_Ctrl, Scrambled_1) (n = 6). F BHK-21 cells were treated with siRNAs against UGCG (20 nM) for 72 h and then exposed to UUKV (multiplicity of infection (MOI) ~ 0.1). Infection was determined by flow cytometry after immunostaining for the viral nucleoprotein N 24 h post-infection. G The values obtained in F were normalized to the infection level in samples treated with siRNA controls (si_Ctrl) (n = 3). H BHK-21 cells were transfected with siRNAs (20 nM) to silence β4Galt5 (si_β4Galt5) or β4Galt6 (si_β4Galt6). Cells were lyzed 72 h post-transfection and total RNA was extracted and purified. The levels of mRNA encoding β4Galt5 or β4Galt6 were quantified by RT-qPCR and expressed as fold change over the values obtained for BHK-21 cells treated with negative control siRNAs (si_Ctrl, Scrambled_2) and set to 1 (n = 2). I BHK-21 cells silenced for β4Galt5, β4Galt6, or β4Galt5 and β4Galt6 (si_β4Galt5 + 6, 20 nM each) were infected with UUKV at an MOI of 0.1 for 24 h. Infection was then analyzed by flow cytometry as shown in F, and the values were normalized to the infection level in samples treated with scrambled siRNAs (n = 2)

Fig. 3

Uukuniemi virus (UUKV) relies on glucosylceramide (GlcCer) in viral particles for infection. A BHK-21 cells were pretreated with 2.5 µM of DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and then infected with UUKV (multiplicity of infection (MOI) ~ 0.1) in the presence of PPMP, which inhibits UGCG activity. Infected cells were subjected to lipidomics analyses 24 h post-infection. Unpaired t test with Welch correction was applied (n = 4). *, p < 0.05. B Specific analysis of HexCer and Hex2Cer levels in the samples processed as described in A. Unpaired t test with Welch correction was applied (n = 4). *, p < 0.05; ns not significant. C GlcCer levels were analyzed in BHK-21 cells after PPMP treatment by dot blotting using an identical number of cells for each sample. D GlcCer levels in C were semi-quantified and expressed as a percentage of the GlcCer level measured in the absence of PPMP (n = 3). E 5 × 10⁸ BHK-21 cells in a total of 20 T175 flasks were treated with 2.5 µM PPMP for 16 h before the infection with UUKV. Supernatant from UUKV-infected BHK-21 cells was harvested 24 h post-infection, and UUKV particles were purified before quantitative MS-based lipid analysis. F BHK-21 cells were first treated with PPMP for 16 h and then infected with UUKV (MOI ~ 0.5) in the continuous presence of the drug. Infection was detected by flow cytometry up to 24 h later. Note that the standard error of mean (SEMs) of some data series are not visible on the graph. An one-way ANOVA with Dunnett’s multiple comparison test was applied for the time point at 24 hpi (n = 3). **, p < 0.01; ****p < 0.0001. G and H BHK-21 cells were pretreated with three other UGCG inhibitors, namely, N-(2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl)-decanamide (PDMP), N-Butyldeoxynojirimycin (NB-DNJ), or N-Butyldeoxygalactonojirimycin (NB-DGJ) at the indicated concentrations for 16 h (PDMP) or 24 h (NB-DNJ and NB-DGJ) and were then infected with UUKV (MOI ~ 0.1) in the continuous presence of the drugs. Infection was quantified by flow cytometry after immunostaining for the viral nucleoprotein, and the data were normalized to those in control samples without inhibitor treatment (n ≥ 2)

Fig. 4

Uukuniemi virus (UUKV) infection induces upregulation of glucosylceramide synthase (UGCG) expression. A and B BHK-21 cells were transfected with a simian virus 40 (SV40) promoter-driven expression vector encoding Gaussia luciferase (Gluc), which is naturally secreted. One day later, transfected cells were exposed to either UUKV (MOI ~ 1), brefeldin A (1 µM), or PPMP (2.5 µM). Supernatants were collected 24 h post-infection B, and cells were lyzed A. Luciferase activity was then measured with a Varioskan Lux multimode reader. Two-way ANOVA with Dunnett’s multiple comparison test was applied (n = 2). **, p < 0.01; ****, p < 0.0001. C and D BHK-21 cells were infected with UUKV (MOI ~ 1) for up to 24 h. Infected cells were collected at different time points and lyzed. Total RNA was then extracted and purified before quantification of mRNAs for UGCG, β4Galt5, β4Galt6, and UGT8 C or the S segment of UUKV D by RT-qPCR (n = 2). Note that the standard error of mean (SEMs) of some data series are not visible on the graph. E BHK-21 cells were exposed to UUKV at MOI ~ 0.1 for 24 h and assayed for UGCG and the viral N protein by western blotting using the antibodies 1E5 and 8B11A3, respectively. F Shows the semi-quantification of UGCG and N proteins from western blotting analyses as shown in (E) (n = 2)

Fig. 5

Uukuniemi viruses (UUKVs) with and without glucosylceramide (GlcCer) show no morphological differences. A Viral particles produced from BHK-21 cells treated with 2.5 µM of DL-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) were analyzed by SDS-PAGE and western blotting under nonreducing conditions with a polyclonal antibody that recognized UUKV N, Gn, and Gc. B UUKV structural proteins in Fig. 5A were semi-quantified. Values are shown as the ratio of the UUKV glycoproteins Gn and Gc level to nucleoprotein N level. Unpaired t test with Welch correction was applied (n = 3). ns, not significant. C UUKV derived from BHK-21 cells exposed to PPMP was digested with PNGase F (1000 units) or Endo H (2000 units) under reducing conditions for 4 h at 37 ℃. Proteins were separated by SDS-PAGE and analyzed by western blotting with a mAb against either UUKV glycoproteins Gn or Gc. ns, not significant. D Cryo-electron tomography of gradient-purified UUKV in the presence or absence of PPMP. (a, d) Slices of tomogram capturing UUKV virions. Scale bars correspond to 100 nm (a, d) and 50 nm (b, e). (c, f) Tomogram slices showing Gn and Gc virion surface arrangement (scale bar: 20 nm). E The diameter of UUKV particles produced in the presence or absence of PPMP was measured. Unpaired t test with Welch correction was applied (n = 30). ns not significant

Fig. 6

Glucosylceramide (GlcCer) is critical for Uukuniemi virus (UUKV) infectivity. A BHK-21 cells were pretreated with DL-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) at the indicated concentrations for 16 h and exposed to UUKV (multiplicity of infection ~ 0.1). The same amount of supernatant of infected cultured cells were harvested 24 h post-infection and then allowed to infect freshly seeded naïve BHK-21 cells for 8 h. The infected cells were immunostained for the detection of UUKV N and analyzed by flow cytometry. The values were normalized to those in control samples where viruses were produced in the absence of the inhibitor (n = 3). B Infectious UUKV progeny in the supernatant collected from cells cultured in the presence of PPMP was evaluated by focus-forming assay. The data are presented as the percentage of the control samples where viruses were produced in the absence of the inhibitor (n = 9). C UUKV structural proteins were analyzed by western blotting as described in Fig. 5A and semi-quantified. The data were expressed as a percentage of the level of UUKV N, Gn, and Gc in the supernatant of PPMP-treated and infected BHK-21 cells to the level of N, Gn, and Gc in the supernatant of infected cells that had not been exposed to PPMP (n = 6). D Viral particles produced from BHK-21 cells in the presence of 2.5 µM of PPMP were analyzed by RT-qPCR using primers that selectively target the RNA segments S, M, and L. The values obtained for each segment were summed, and the data were expressed as a percentage of the RNA content in the supernatant of PPMP-treated and infected BHK-21 cells to the RNA content in the supernatant of infected cells that had not been exposed to PPMP (n = 5). E UUKV stocks, whether produced in the presence or absence of PPMP, were evaluated for their viral RNA content using RT-qPCR, as detailed in D. Their viral structural protein content was also evaluated using western blotting as described in C. The ratio of the relative unit of UUKV RNA segments to the relative unit of UUKV structural proteins is presented as the percentage of the value obtained for the control sample without any drug treatment. Ratio paired t test was applied (n = 3). ns not significant. F Same volumes of virus stocks produced in the presence or absence of PPMP were evaluated by both focus-forming assay and western blotting as detailed in B and C. The ratio of the number of focus-forming units (ffu) to the relative unit of UUKV structural proteins is presented as the percentage of the value obtained for the control sample without any drug treatment (n = 6). G Identical volumes of UUKV stocks produced in the presence or absence of PPMP were evaluated for their viral RNA content both by foci-forming assay and RT-qPCR as described in B and D. The values of the three segments were summed, and the relative unit of UUKV RNA segments (2^−∆Ct) was plotted against the number of ffu (n = 2). H The ratio of the number of ffu to the relative unit of UUKV RNA segments is presented as the percentage of the value obtained for the control sample without any drug treatment (n = 5)

Fig. 7

Glucosylceramide (GlcCer) in viral particles promotes Uukuniemi virus (UUKV) binding. A UUKV particles derived from BHK-21 cells in the presence of DL-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, 2.5 µM) were bound to freshly seeded naïve BHK-21 cells for 2 h on ice before fixation and western blot analysis with an antibody recognizing the UUKV N protein. B N was semi-quantified from the cells described in A, and the value is presented as a percentage of the N level measured in the sample corresponding to virus binding in the absence of PPMP (n = 8). C Alternatively, UUKV particles produced in the presence or the absence of PPMP were allowed to bind to BHK-21 cells for 2 h on ice, and binding was assessed by measuring the BHK-21 cell-associated S viral segment by RT-qPCR (n = 6). D Fluorescently labeled UUKV particles (UUKV-ATTO488) were bound to BHK-21 cells [multiplicity of infection (MOI) ~ 4] on ice for 1 h, and viral binding was evaluated by flow cytometry analysis. E and F BHK-21 cells, A549 human lung epithelial cells, and BHK-21 cells expressing the UUKV receptor DC-SIGN (BHK-21 DC-SIGN +) were preincubated with varying amounts of soluble C6-GlcCer E or C6-Cer F for 2 h and then exposed to UUKV-ATTO488 (MOI ~ 4) on ice for 1 h. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer or C6-Cer. An one-way ANOVA with Dunnett’s multiple comparison test was applied (n ≥ 3). *, p < 0.05; **, p < 0.01; ns, not significant; RFI, relative fluorescence intensity. G BHK-21 cells were transduced with a retroviral vector system to express DC-SIGN (BHK-21 DC-SIGN +). DC-SIGN expression was measured by flow cytometry analysis using phycoerythrin-conjugated anti-DC-SIGN mAb. H Soluble C6-GlcCer was allowed to bind BHK-21 cells on ice for 2 h before exposure to UUKV for 1 h (MOI ~ 0.5). After virus binding on ice, unbound UUKV particles were washed away, and the cells were incubated at 37 ℃ for 8 h. Infection was quantified by flow cytometry after immunostaining for UUKV N protein. Values are presented as the percentage of the control sample without prebinding of soluble C6-GlcCer (n = 4)

Fig. 8

GlcCer in target cells is not essential for Uukuniemi virus (UUKV) entry and fusion. A–D BHK-21 cells were treated with PPMP for 16 h at 37 ℃ before UUKV binding. A Cells were exposed to UUKV-ATTO488 (MOI ~ 4) in suspension at 4 ℃ and rapidly warmed to 37 ℃ to allow virus uptake for 30 min. Endocytic internalization of virions was determined by flow cytometry after trypan blue treatment. B UUKV-ATTO488 (MOI ~ 4) was allowed to bind to cells on ice before fixation and analysis by flow cytometry. Virus binding is expressed as the relative fluorescence intensity associated with the cells, as measured by flow cytometry. Unpaired t test with Welch correction was applied (n = 2). ns, not significant. C Internalization is given as the percentage of fluorescence quantified in samples treated with trypan blue as compared to that in untreated samples. The fluorescence signal measured in cells not exposed to UUKV-ATTO488 was considered the background signal and subtracted from the other values. Unpaired t test with Welch correction was applied (n = 2). ns, not significant. D UUKV was bound at MOI ~ 5 to cells on ice. Subsequently, cells were washed and subjected to pH ~ 5.0 at 37 ℃ for 90 s to trigger the virus fusion at the plasma membrane. Infected cells were then incubated for 7 h at 37 ℃ in the presence of 50 mM NH₄Cl to prevent viral penetration from endosomes, and thereby only monitor the release of viral genomes from the plasma membrane. Infection was quantified by flow cytometry, and the data were normalized to those from samples where cells were not treated with PPMP. Unpaired t test with Welch correction was applied (n = 2). ns not significant

Fig. 9

Glucosylceramide (GlcCer) is important for the attachment of viruses that bud from the Golgi. A BHK-21 cells were infected with Semliki forest virus (SFV) at a multiplicity of infection (MOI) of 0.01 and subjected to lipidomic analyses 14 h post-infection (n = 3). B SFV particles were harvested 24 h post-infection and purified from the supernatant of infected cells and included in the analysis (n = 3). C Ebola virus-like particles (EBOVLPs) were produced from HEK293T cells and purified before the lipidomic analysis with MS (n = 3). B, C Phospholipids include all glycerophospholipids, sphingomyelin (SM), and lysophosphatidylcholine (LPC). The neutral lipids were cholesteryl ester (CE), diacylglycerol (DAG), and triacylglycerol (TAG). D BHK-21 cells were first treated with DL-threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) at the indicated concentrations for 16 h and then infected with either TOSV (MOI ~ 0.1) or SFV (MOI ~ 0.01) in the continuous presence of PPMP. Infection was measured by flow cytometry 24 h later. An one-way ANOVA with Dunnett’s multiple comparison test was applied (n ≥ 2). *, p < 0.05; **, p < 0.01. E Infectious progeny of TOSV and SFV produced by BHK-21 cells in the presence of PPMP for 24 h was evaluated by focus-forming assay. The data are presented as the percentage of the control samples where viruses were produced in the absence of an inhibitor. Ratio paired t test was applied (n = 3). **, p < 0.01; ns, not significant. F BHK-21 cells were pretreated with PPMP at the indicated concentrations for 16 h and exposed to SFV (MOI ~ 1) for 5 h. Infected cells were immunostained for the SFV glycoprotein E2 and analyzed by flow cytometry. An one-way ANOVA with Dunnett’s multiple comparison test was applied (n = 3). ns not significant. G Cells were preincubated with the indicated amounts of soluble C6-GlcCer for 2 h on ice and then exposed to ATTO488-labeled TOSV (TOSV-ATTO488) and ATTO488-labeled Germiston virus (GERV-ATTO488), and EBOVLPs containing green fluorescent protein (EBOVLP-GFP) on ice for 1 h. TOSV-ATTO488 and GERV-ATTO488 were used to infect cells at MOIs of 4 and 15, respectively. Approximately 500 ng of EBOVLP-GFP total protein was used to bind to 1 × 10⁵ cells. Virus binding was measured by flow cytometry, and the data were normalized to those in control samples processed in the absence of soluble C6-GlcCer (n ≥ 3). RFI relative fluorescence intensity