Cellular SLC35B4 promotes internalization during influenza A virus entry

Abstract

SLC35B4, a nucleotide sugar transporter that mediates the transport of UDP-GlcNAc and UDP-xylose, was found to be required for the replication of influenza A virus (IAV) of the H5N1 subtype in our genome-wide siRNA library screen. We found that defective IAV replication in SLC35B4-deficient A549 cells was independent of virus strain specificity, and the virulence of IAV in Slc35b4 knockdown mice was also decreased. By examining the individual stages of the IAV replication cycle, we discovered that the amount of internalized IAV was significantly reduced in SLC35B4-knockout A549 cells. Mechanistically, SLC35B4 facilitated IAV replication by transporting UDP-xylose, which attaches to the serine residue of heparan sulfate proteoglycans (HSPGs) in the heparan sulfate (HS) biosynthesis pathway. Knockdown of associated host factors (i.e., XYLT2, B4GALT7, EXT1, and EXT2) in the HS biosynthesis pathway also impaired IAV replication. Furthermore, we revealed that AGRN, a unique HSPG family member, was important for the endocytosis of IAV in A549 cells. Moreover, we found that the homeostasis of the AGRN protein was regulated by HS modification mediated by the initial UDP-xylose transporter SLC35B4, thereby affecting the expression level of endocytic adapter AP2B1 to influence IAV internalization. Collectively, these findings establish that SLC35B4 is an important regulator of IAV replication and uncover the underlying mechanisms by which SLC35B4 employs UDP-xylose transport activity to promote IAV internalization.IMPORTANCEThe entry process of IAV represents a favorable target for drug development. In this study, we identified SLC35B4 as an important host factor for the efficient replication of different subtypes of IAV in vitro and for the virulence of IAV in mice. We revealed that SLC35B4 employed its UDP-xylose transport activity to promote the HS biosynthesis pathway, thereby assisting IAV internalization into target cells in the early stage of viral infection. Consistently, several downstream factors in the HS biosynthesis pathway, i.e., XYLT2, B4GALT7, EXT1, and EXT2, as well as a specific HSPG member AGRN were also important for the replication of IAV. Furthermore, the UDP-xylose-transporting activity of SLC35B4 was involved in the regulation of the homeostasis of the AGRN protein by HS modification, which influenced virus internalization by affecting the expression levels of AP2B1. Together, the identification of the SLC35B4-XYLT2-B4GALT7-EXT1-EXT2-AGRN-AP2B1 axis may shed light on the development of potential anti-IAV therapeutics.

Keywords: AGRN; AP2B1; SLC35B4; heparan sulfate modification; influenza A virus; internalization.

Fig 1

SLC35B4 positively regulates IAV and VSV replication in A549 cells. (A) Venus expression in SLC35B4-knockdown A549 cells infected with H5N1 NA-Venus reporter viruses (MOI = 0.1) was visualized by using the Operetta high-content imaging system at 24 h p.i. The inhibitory effect of SLC35B4 knockdown on reporter virus replication was calculated by normalizing the average fluorescence intensity of the SLC35B4 siRNA-treated cells with that of the scrambled siRNA-treated cells (n = 3 biologically independent samples). ***, P < 0.001. (B) Viability of A549 cells transfected with SLC35B4 siRNA or scrambled siRNA for 48 hours (n = 3 biologically independent samples). (C) SLC35B4 mRNA level in SLC35B4 siRNA-treated A549 cells determined by using RT-qPCR (n = 3 biologically independent samples). ***, P < 0.001. (D) SLC35B4 protein level in SLC35B4 siRNA-treated A549 cells detected by Western blotting with a rabbit anti-SLC35B4 pAb. (E to I) Virus titers in SLC35B4 siRNA- or scrambled siRNA-transfected A549 cells infected with WSN (H1N1) (MOI = 0.01) (E), SH13 (H9N2) (MOI = 0.1) (F), AH05 (H5N1) (MOI = 0.1) (G), FZ09 (H1N1) (MOI = 0.1) (H), or VSV-EGFP (100 TCID₅₀) (I) virus were determined by use of plaque assays (E to H) or TCID₅₀ (I) on MDCK cells at the indicated time points (n = 3 biologically independent samples). **, P < 0.01; ***, P < 0.001. (J to N) Virus titers in SLC35B4_KO or control A549 cells infected with WSN (H1N1) (MOI = 0.01) (J), SH13 (H9N2) (MOI = 0.1) (K), AH05 (H5N1) (MOI = 0.1) (L), FZ09 (H1N1) (MOI = 0.1) (M), or VSV-EGFP (100 TCID₅₀) (N) virus were determined by plaque assays (J to M) or TCID₅₀ (N) at the indicated time points (n = 3 biologically independent samples). **, P < 0.01; ***, P < 0.001. (O and P) SLC35B4 siRNA- or scrambled siRNA-transfected A549 cells were left untreated or were treated with IFN-α (O) or IFN-β (P) for 24 hours. The cell lysates were then subjected to Western blotting with a rabbit anti-MX1 pAb for the detection of the MX1 protein.

Fig 2

Slc35b4 is important for IAV pathogenicity in mice. (A) Schematic illustration of two 2′-O-methoxylated (2′-Ome) and 3’cholesterol (3’chol)-conjugated Slc35b4 siRNAs used to generate Slc35b4_KD mice. (B) Decreased Slc35b4 expression in the lungs of Slc35b4_KD mice verified by Western blotting with a rabbit anti-SLC35B4 pAb. (C, D) Survival (C) and body weight changes (D) of mice (n = 5/group) treated with the modified scrambled siRNA and Slc35b4 siRNA after intranasal infection with WSN (H1N1) virus (5 MLD₅₀/mouse).

Fig 3

SLC35B4 affects the early stage of the IAV replication cycle. (A) SLC35B4_KO or control A549 cells were infected with WSN (H1N1) (MOI = 5). At 2, 3, 4, and 5 h p.i., the infected cells were fixed and stained with a mouse anti-NP mAb, followed by incubation with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). (B) SLC35B4_KO or control A549 cells were infected with WSN (H1N1) (MOI = 5) in the presence of cycloheximide (CHX). The levels of NP-specific vRNA in the infected cells were analyzed by RT-qPCR at 2 h p.i. and 3 h p.i. (n = 3 biologically independent samples). ***, P < 0.001. (C) Schematic diagram of two sgRNAs targeting sites at the SLC35B4 gene loci and the corresponding truncated mutant validated by sequencing. (D) Viability of SLC35B4_KO HEK293T cells (n = 3 biologically independent samples). (E) SLC35B4_KO or control HEK293T cells were transfected with the four vRNP protein expression plasmids (PB2, PB1, PA, and NP) of the WSN (H1N1) virus, together with pHH21-SC09NS F-Luc and pRL-TK. At 36 hours post-transfection, a dual-luciferase reporter assay was performed in which the firefly luciferase activity was normalized to the activity of the internal Renilla luciferase control (n = 3 biologically independent samples).

Fig 4

SLC35B4 is important for IAV internalization. (A to C) SLC35B4_KO, SLC35A1_KO, or control A549 cells were stained with WGA (A), MAL lectins (for α-2,3-SA) (B), or SNA lectins (for α-2,6-SA) (C) and then analyzed by flow cytometry. (D) SLC35B4_KO, SLC35A1_KO, or control A549 cells were infected with WSN (H1N1) virus (MOI = 5) at 4℃ for 1 hour. The cells were then fixed, stained with a rabbit anti-HA pAb and Alexa Fluor 488 goat anti-rabbit IgG (H+L), and analyzed by flow cytometry. (E) SLC35B4_KO or control A549 cells were infected with WSN (H1N1) virus (MOI = 5) at 4℃ for 1 hour, and then the temperature was shifted to 37°C. At 10 minutes or 20 minutes post temperature shift (p.t.s.), the cells were lysed after being washed with ice-cold PBS-HCl (pH 1.3), and the level of NP-specific vRNA was analyzed by RT-qPCR (n = 3 biologically independent samples). **, P < 0.01; ***, P < 0.001. (F) SLC35B4_KO, SLC35A1_KO, or control A549 cells were infected with WSN (H1N1) virus (MOI = 5) on ice at 4°C for 1 hour, and then the temperature was shifted to 37°C for 30 minutes. After being washed with ice-cold PBS-HCl (pH 1.3), the cells were lysed, and the amount of internalized virus particles was detected by Western blotting with a rabbit anti-NP pAb. (G) SLC35B4_KO or control A549 cells were infected with WSN (H1N1) virus (MOI = 10) on ice at 4°C for 1 hour and cultured at 37°C for 0 minutes, 10 minutes, or 20 minutes. The cells were fixed and stained with a rabbit anti-HA pAb (orange) and a HRP-labeled goat anti-rabbit IgG antibody according to the tyramine signal amplification protocol. The membrane was stained with CellBrite cytoplasmic membrane dyes (green). The ratio of internalized virus particles showing cytoplasmic localization was calculated from total viral particles (> 600) and is indicated at the bottom of each panel of images. (H, I) SLC35B4_KO or control A549 cells were infected with WSN (H1N1) virus (MOI = 1) on ice at 4°C for 1 hour and then cultured in F-12K medium (H) or acidic F-12K medium (pH 5.0) (I). After three washes with ice-cold PBS, the cells were incubated with the F-12K medium for 12 hours at 37°C. Virus titers in the supernatant were determined by use of plaque assays. ***, P < 0.001; ns, not significant.

Fig 5

SLC35B4 mediates IAV endocytosis without using its UDP-GlcNAc transporter function. (A and B) A549 cells transfected with plasmids expressing SLC35B4-Myc, EOGT-Myc, or OGT-Myc were fixed at 24 hours post-transfection; incubated with a mouse anti-Myc mAb and a rabbit anti-CANX pAb or a rabbit anti-GM130 pAb; and stained with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (green). The nuclei were stained with DAPI (blue). (C) A549 cells co-transfected with plasmids expressing EOGT-HA and SLC35B4-Myc were fixed at 24 hours post-transfection, incubated with a mouse anti-HA mAb and a rabbit anti-Myc pAb, and stained with Alexa Fluor 488 goat anti-mouse IgG (H+L) (green) and Alexa Fluor 633 goat anti-rabbit IgG (H+L) (red). The nuclei were stained with DAPI (blue). (D to G) Plasmids expressing EOGT-Myc (D and E) or SLC35B4-Myc (F and G) were transfected individually or co-transfected with plasmids expressing Notch1(1-715)-V5 (D and F) or Notch2(1-719)-V5 (E and G) into HEK293T cells. At 36 hours post-transfection, the cell lysates were immunoprecipitated with a rabbit anti-V5 pAb, followed by Western blotting with a mouse anti-V5 mAb and a mouse anti-O-GlcNAc mAb for the detection of Notch1, Notch2, and their O-GlcNAc-modified forms, respectively. (H to K) Plasmids expressing SLC35B4-Myc (H and J) or EOGT-Myc (I and K) were transfected individually or co-transfected with plasmids expressing IGDCC4-V5 (H and I) or FFAR2-V5 (J and K) into HEK293T cells. At 36 hours post-transfection, the cell lysates were immunoprecipitated with a rabbit anti-V5 pAb, followed by Western blotting with a mouse anti-V5 mAb and a mouse anti-O-GlcNAc mAb for the detection of IGDCC4, FFAR2, and their O-GlcNAc-modified forms, respectively. (L and M) Virus titers in EOGT-knockdown or EOGT_KO A549 cells infected with the WSN (H1N1) virus (MOI = 0.01) at the indicated time points, as determined by using plaque assays on MDCK cells (n = 3 biologically independent samples).

Fig 6

O-xylose-associated factors promote IAV replication in A549 cells. (A) Schematic diagram of HS chain elongation of heparan sulfate proteoglycans (HSPGs). Xyl, xylose; Gal, galactose; GlcA, glucuronic acid; GlcNAc, N-acetylglucosamine. (B and C) A549 cells transfected with plasmids expressing XYLT2-Myc or B4GALT7-Myc (B) or co-transfected with plasmids expressing EXT1-Myc and EXT2-HA (C) were fixed at 24 hours post-transfection, incubated with a mouse anti-Myc mAb and a rabbit anti-CANX pAb or a rabbit anti-GM130 pAb (B) or with a mouse anti-HA mAb and a rabbit anti-Myc pAb (C), and stained with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (green). (D) The XYLT2 protein level was detected by Western blotting in A549 cells transfected with XYLT2 siRNA or scrambled siRNA for 48 hours. (E) Virus titers in XYLT2-knockdown A549 cells infected with the WSN (H1N1) virus (MOI = 0.01) at the indicated time points (n = 3 biologically independent samples). ***, P < 0.001. (F) A549 cells were transfected with B4GALT7 siRNA or scrambled siRNA for 12 hours and further transfected with plasmids expressing B4GALT7-HA for 24 hours. The B4GALT7 protein level was evaluated by Western blotting. (G) Virus titers in B4GALT7-knockdown A549 cells infected with the WSN (H1N1) virus (MOI = 0.01) at the indicated time points (n = 3 biologically independent samples). ***, P < 0.001. (H, I) EXT1 (H) or EXT2 (I) protein levels detected by Western blotting in A549 cells transfected with EXT1 siRNA, EXT2 siRNA, or scrambled siRNA for 48 hours. (J) Virus titers in EXT1- or EXT2-knockdown A549 cells infected with WSN (H1N1) virus (MOI = 0.01) at the indicated time points (n = 3 biologically independent samples). *, P < 0.05; **, P < 0.01. (K) A549 cells co-transfected with plasmids expressing SLC35B4-Myc and XYLT2-HA, B4GALT7-HA, or EXT1/EXT2-HA were fixed at 24 hours post-transfection, incubated with a mouse anti-HA mAb and a rabbit anti-Myc pAb, and stained with Alexa Fluor 633 goat anti-mouse IgG (H+L) (red) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (green).

Fig 7

AGRN is important for IAV endocytosis. (A and B) Detection of the AGRN mRNA level or protein expression in AGRN-knockdown A549 cells by using RT-qPCR (A) (n = 3 biologically independent samples) or Western blotting (B). ***, P < 0.001. (C) Viability of A549 cells transfected with AGRN siRNA or scrambled siRNA for 48 hours (n = 3 biologically independent samples). (D) Virus titers in AGRN-knockdown A549 cells infected with the WSN (H1N1) virus (MOI = 0.01) at the indicated time points (n = 3 biologically independent samples). **, P < 0.01. (E) AGRN protein level detected by Western blotting in AGRN_KO A549 cells. (F to I) Virus titers in AGRN_KO or control A549 cells infected with WSN (H1N1) (MOI = 0.01) (F), SH13 (H9N2) (MOI = 0.1) (G), AH05 (H5N1) (MOI = 0.1) (H), or FZ09 (H1N1) (MOI = 0.1) (I) virus at the indicated time points (n = 3 biologically independent samples). **, P < 0.01; ***, P < 0.001. (J) Expression of viral proteins in AGRN_KO A549 cells infected with WSN (H1N1) virus (MOI = 5) was detected by Western blotting at 3 h p.i., 6 h p.i., and 9 h p.i. (K) AGRN_KO and control A549 cells were infected with the WSN (H1N1) virus (MOI = 5). At 2 h p.i. and 3 h p.i., the cells were fixed and stained with a mouse anti-NP mAb and Alexa Fluor 633 goat anti-mouse IgG (H+L) (red). (L) AGRN_KO or control A549 cells were stained with WGA and analyzed by flow cytometry. (M) AGRN_KO or control A549 cells were infected with WSN (H1N1) virus (MOI = 5) at 4°C for 1 hour. The cells were fixed and stained with a rabbit anti-HA pAb and Alexa Fluor 488 goat anti-rabbit IgG (H+L) and analyzed by flow cytometry. (N) AGRN_KO or control A549 cells were infected with the WSN (H1N1) virus (MOI = 5) on ice at 4°C for 1 hour, and then the temperature was shifted to 37°C for 30 minutes. After being washed with ice-cold PBS-HCl (pH 1.3), the cells were lysed, and the amount of internalized virus particles was evaluated by detecting the viral NP protein by Western blotting. (O) AGRN_KO1 or control A549 cells were infected with the WSN (H1N1) virus (MOI = 10) on ice at 4°C for 1 hour and cultured at 37°C for 0 or 20 minutes. The cells were fixed and stained with a rabbit anti-HA pAb and HRP-labeled goat anti-rabbit IgG antibody according to the tyramine signal amplification protocol.

Fig 8

The 30–350 and 1325–1548 regions of AGRN interact with IAV HA1. (A to C) Plasmids expressing AGRN-Flag were transfected individually or co-transfected with plasmids expressing HA (A) or Myc-tagged HA1 (B) or HA2 (C) of the WSN (H1N1) virus into HEK293T cells. At 36 hours post-transfection, cell lysates were immunoprecipitated with a mouse anti-Flag mAb and subjected to Western blotting with the indicated antibodies. (D) Schematic diagram of AGRN(1–2069) truncation mutants. (E to G) Plasmids expressing AGRN-Flag truncation mutants (E), AGRN(1–1102)-Flag truncation mutants (F), or AGRN(1103–2069)-Flag truncation mutants (G) were transfected individually or co-transfected with plasmids expressing HA1-Myc of the WSN (H1N1) virus into HEK293T cells. At 36 hours post-transfection, Co-IP and Western blotting were carried out as described in (A to C). (H to M) Plasmids expressing AGRN(30-350)-Flag (H to J) or AGRN(1325–1548)-Flag (K to M) were transfected individually or co-transfected with plasmids expressing Myc-tagged HA1 of AH05 (H5N1) (H and K), SH13 (H9N2) (I and L), or FZ09 (H1N1) (J and M) viruses into HEK293T cells. At 36 hours post-transfection, Co-IP and Western blotting were carried out as described in (A to C).

Fig 9

HS modification of AGRN promotes IAV internalization by regulating the stability of endocytic adapter AP2B1. (A) Virus titers in heparinase II (4U)-treated A549 cells infected with the WSN (H1N1) virus (MOI = 0.01) were determined by plaque assays at 24 h p.i. (n = 3 biologically independent samples). (B) Plasmids expressing AGRN-Flag or AGRN HS mutant-Flag were transfected into HEK293T cells. Cell lysates and supernatants were subjected to Western blotting with a mouse anti-Flag mAb at 36 hours post-transfection. (C) HEK293T cells were transfected with plasmids expressing AGRN-Flag or AGRN HS mutant-Flag. At 36 hours post-transfection, the cells were treated with CHX for 10 hours, and then cell lysates were subjected to Western blotting with a mouse anti-Flag mAb. (D) AGRN_KO1 or control A549 cells were infected with WSN (H1N1) virus (MOI = 0.01) that was pre-incubated at 4°C for 1 hour with the supernatant collected from HEK293T cells transfected with plasmids expressing AGRN-Flag, AGRN HS mutant-Flag or the pCAGGS vector. Virus titers were determined by plaque assays at 24 h p.i. (n = 3 biologically independent samples). ***, P < 0.001. (E) HEK293T cells were transfected with the indicated combinations of plasmids expressing AGRN-Flag and AP2B1-V5. At 36 hours post-transfection, cell lysates were immunoprecipitated with a mouse anti-Flag mAb and then subjected to Western blotting with the indicated antibodies. (F) A549 cells co-transfected with plasmids expressing AGRN-Flag and AP2B1-V5 were fixed at 24 hours post-transfection, incubated with a mouse anti-Flag mAb and a rabbit anti-V5 pAb, and stained with Alexa Fluor 488 goat anti-mouse IgG (H+L) (green) and Alexa Fluor 633 goat anti-rabbit IgG (H+L) (red). (G) Endogenous expressions of AGRN, AP2B1, clathrin subunits (CLTA, CLTB, and CLTC) in A549 cells and SLC35B4_KO A549 cells by Western blotting with the indicated antibodies. (H, I) HEK293T cells were transfected with plasmids expressing AP2B1-V5 individually or in combination with plasmids expressing SLC35B4-Myc (H), AGRN-Flag, or AGRN HS mutant-Flag (I). At 36 hours post-transfection, cell lysates were subjected to Western blotting. (J) HEK293T cells were transfected with plasmids expressing AP2B1-V5 individually or in combination with plasmids expressing AGRN HS mutant-Flag. At 36 hours post-transfection, the cells were treated with proteasome inhibitor (MG132) or autophagy inhibitors (wortmannin, rapamycin, bafilomycin A1 or 3-methyladenine) for 24 hours. Cell lysates were subjected to Western blotting.

Fig 10

SLC35B4-mediated HS modification of AGRN promotes IAV internalization into host cells. (A) SLC35B4 transports UDP-xylose from the cytoplasm to the endoplasmic reticulum (ER), XYLT2 catalyzes UDP-xylose coupling with the serine residue of HS-modified protein AGRN, and B4GALT7-EXT1/EXT2 catalyzes the HS chain elongation of AGRN. The HS-modified AGRN protein is transported to the plasma membrane, binds the viral HA protein, and further recruits the endocytic adapter AP2B1 to initiate virus endocytosis. (B) Excessive accumulation of the unmodified AGRN HS mutant due to the deficiency in SLC35B4 expression leads to AP2B1 degradation via the proteasome pathway, thereby inhibiting the endocytosis of IAV.