Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Several studies have indicated that a higher display of appropriate receptors leads to 75 increased infectivity [15,21,22,27], while others indicated that only low amounts of 76 receptors are required for infection [28][29][30]. Based on our studies, we concluded that 77 above a required threshold, a greater number of suitable glycan receptors for H3N2 78 IAVs does not result in increased infection efficiency. 79

Generation of stable B3GNT2 and B4GALT1 knock-in MDCK and hCK cell lines 81
Rearrangement of the sialyltransferase expression in hCK cells supported increased 82 replication of many human H3N2 viruses [15]. However, only small quantities of 83 glycans with multiple LacNAc repeating units appeared to be present on both MDCK 84 and hCK cells [24]. Recently, we and others have shown that poly-LacNAc containing 85 N-glycans are critical for the binding of contemporary H3N2 viruses [8,12]. Therefore, 86 we used the glycosyltransferases B3GNT2 and B4GALT1 to increase the biosynthesis 87 of LacNAc repeating units to produce extended N-glycans [12,25,26,31]. We 88 hypothesized that the overexpression of B3GNT2 and/or B4GALT1 in MDCK and hCK 89 cells would produce appropriate glycan receptors for recent H3N2 (subclade 3C.2a) 90

IAVs. 91
To accomplish the overexpression of these genes in MDCK and hCK cells, lentiviral 92 transfer plasmids encoding the human B3GNT2 and/or B4GALT1 genes, together with 93 the Hygromycin B resistance gene, were constructed. The genes were expressed from 94 one human EF-1α promoter [32] and separated by P2A (and for double 95 glycosyltransferase knock-ins also T2A) self-cleaving peptides. Lentiviruses were 96 produced with a transfer plasmid and packaging plasmids, after which the viruses were 97 used to transduce MDCK and hCK cells (Fig. 1A). Cells in which the genes were 98 inserted in the genome were selected with Hygromycin B. 99 Stable overexpression of B3GNT2 and B4GALT1 was confirmed by RT-qPCR 100 analysis on isolated cellular RNA. Primers for the B3GNT2, B4GALT1, and ST6GAL1 101 genes were used, and the obtained values were normalized to the reference gene 102 GAPDH (Fig. 1B) 0019/2016 were used. The titration indicated that there is indeed binding to MDCK WT 220 cells but to a much lesser extent than to hCK WT (or hCK-B3GNT2 cells) (Fig. S3). 221 showed a substantial increase in the relative abundance of glycans with a higher 251 number of LacNAc repeating units, up to even nine LacNAcs (Table S7) (Fig. 2). Surprisingly, no substantial difference between the titers in hCK 294 WT and hCK-B3GNT2 cells was observed, while the glycans on the latter cell line were 295 extended as observed in the flow cytometry (Fig. 2) and glycan mass spectrometry 296 experiments (Fig. 3). Furthermore, no difference was observed in the titers for the 297 3C.3a viruses (Fig. 5C), not even between MDCK and hCK cells. Therefore, we 298 concluded that additional binding does not necessarily lead to a higher infection 299 efficiency. 300  Three transfer plasmids were constructed (pCF-B3GNT2, pCF-B4GALT1, and pCF-416 B3GNT2-B4GALT1). The region between the P2A and WPRE was removed and 417 replaced by either the B3GNT2 or B4GALT1. When the genes of both 418 glycosyltransferases were cloned into the plasmid they were connected with a T2A 419 self-cleaving peptide. The B3GNT2 and B4GALT1 genes were always proceeded by 420 the signal sequence of the human GalT, which we copied from the EGFP-GalT 421 plasmid (gift from Jennifer Lippincott-Schwartz, Addgene plasmid # 11929) [

Isolation of influenza viruses in hCK-B3GNT2 cells is not improved as compared
434 Lentiviral integration of the B3GNT2 and B4GALT1 genes 435 Lentiviral particles were produced using HEK293T cells [58]. One of the transfer 436 plasmids as described above, together with the packaging plasmids pMDLg/pRRE, 437 pRSV-Rev, and pMD2.G were used, which were kind gifts from Didier Trono   were washed once using PBS supplemented with 1% FCS and 2 mM EDTA, after 541 which they were resuspended in 100 µl of the same buffer. Flow cytometry was 542 performed using the BD FACSCanto II (BD Biosciences) using appropriate laser 543 voltages. Data were analyzed using FlowLogic (Inivai Technologies) and gated as 544 described in Fig. 2A to consecutively select cells, single cells, and cells that are not 545 dead. Mean fluorescence values of triplicates were averaged and standard deviations 546 were calculated. Curves for titration experiments were smoothed using the standard 547 settings. 548

Identification of N-glycans on cells by mass spectrometry 549
Cell lysates of WT and B3GNT2/B4GALT1 knock-in MDCK and hCK cells were 550 obtained as described above. The total protein concentration in the cell lysates was 551 determined using a BCA assay. The glycans in 400 µg of total protein were released 552 by PNGaseF treatment. Proteins were first denatured in DTT/SDS (40 mM DTT, 0.5% 553 v/v SDS) for 8 minutes at 95°C, after which they were cooled on ice. Subsequently, 554 NP-40 (1% v/v) and glycobuffer G7 (50 mM sodium phosphate at pH 7.5) were added, 555 together with 30 µg of PNGaseF. The samples were incubated in a shaking incubator 556 overnight at 37°C. Samples were centrifuged (4700 rcf, 3 min) to remove potential 557 precipitate, after which they were loaded on separate C18 SPE cartridges (Avantor™ 558 7020-02 BAKERBOND™ SPE Octadecyl), which were beforehand conditioned with 1 559 ml acetonitrile (MeCN) and 1 ml MQ water. The flow-through was collected and the 560 remaining glycans were eluted from the C18 cartridges with 1 ml of 5% MeCN and Full glycan composition feature lists for the different cell lines are presented in Table  624 S1-8. 625 Analysis of the number of LacNAc repeating units was performed on the complex and 626 hybrid N-glycans with at least one LacNAc repeating unit. A glycan with one LacNAc 627 repeating unit was defined as a glycan with 4 hexoses and a minimum of 3 HexNAcs 628 or 3 HexNAcs and at least 4 hexoses. A glycan with two LacNAc repeating units was 629 defined as a glycan with 5 hexoses and a minimum of 4 HexNAcs or 4 HexNAcs and 630 at least 5 hexoses. This pattern was continued for the higher numbers of LacNAc 631 repeating units. The total absolute abundance of all selected glycans was added up, 632 after which the relative abundance of a given number of LacNAc repeating units was 633 calculated from this total. Additionally, the percentage of these glycans with at least 634 one SIA was calculated. 635 Chromatograms of the N-glycans with two to seven LacNAc repeating units, calculated 636 as described above, from hCK WT and hCK-B3GNT2 cells were constructed using 637 Agilent's Masshunter Qualtitative Analysis 10.0 software (Fig. 3A). The shown 638 chromatograms are the summed extracted-ion-count (EIC) for the ten most abundant 639 glycan features per LacNAc repeating unit group. The EIC for a glycan was set as the 640 observed m/z value with a symmetrical 10 ppm expansion. Different ionization states 641 of the same glycan that were found as a separate feature by the feature-finding 642 software were also included in the summed EIC chromatogram. 643

Sugar nucleotide analysis 644
Cells were grown to 60-70% confluency in a 6-wells plate, after which the medium was 645 removed and the cells were washed twice with wash buffer (75 mM ammonium 646 carbonate in MQ water, pH 7.4 (corrected with glacial acetic acid), at 4°C). The cells 647 were then treated with 700 µl of extraction buffer (40% acetonitrile, 40% methanol, 648 20% MQ water, at 4°C) per well for 2 minutes, after which the supernatant was 649 transferred to a vial. This extraction step is repeated for 3 minutes, after which the two 650 extracts were pooled and centrifuged at 18000 rcf for 3 min. The supernatant was 651 taken and dried in the vacuum concentrator. Samples were frozen at -80°C until 652 analysis using an ion-pair UHPLC-QqQ 1290-6490 Agilent mass spectrometer by 653 Glycomscan BV (Oss, the Netherlands) [41]. 654

Virus titration on B3GNT2/B4GALT1 knock-in MDCK and hCK cells 655
Virus titers in the virus stocks in Table 3 were determined using end-point titration in 656 MDCK cells and inoculated cell cultures were tested for agglutination activity using 657 turkey red blood cells as an indicator of virus replication in the cells. For recent (2017-658 2019) H3N2 viruses, no binding to erythrocytes was observed and therefore virus titers 659 were determined using a nucleoprotein (NP) staining. The NP staining was performed 660 on the inoculated cells that were fixed with acetone for at least 20 minutes at -20°C. 661 Primary mouse anti-NP antibody (HB65, 2 mg/ml) was diluted 1:3000 and the 662 secondary goat anti-mouse IgG HRP antibody (A16702, 1 mg/ml, Thermo Fisher 663 Scientific) was used at a dilution of 1:30000, after which 50 µl per well was used for 664 both solutions. True Blue substrate (KPL) was then added to visualize positive wells 665 using an ImmunoSpot Analyzer (CTL Europe, Bonn, Germany). Based on the negative 666 control values and the highest positive values per plate, the cut-off for positivity was 667 determined. Infectious titers were calculated from five replicates using the Spearman-668 Kärber method [65]. 669 Table 3. Details of IAVs used in the experiment shown in Fig. 5A

Inoculation of hCK and hCK-B3GNT2 cells with influenza viruses 675
To evaluate whether IAVs that could not be isolated previously in hCK cells would 676 replicate in hCK-B3GNT2 cells, hCK and hCK-B3GNT2 cells were seeded at a density 677 of 20.000 cells per well in 96 wells plates at 24 hours before inoculation. The original 678 patient material (100 µl) containing influenza virus (details of viruses in Table 4) was 679 diluted in 700 µl infection medium (EMEM (Cambrex, Heerhugowaard, The 680 Netherlands) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2mM 681 glutamine, 1.5mg/ml sodium bicarbonate (Cambrex), 10mM Hepes (Cambrex), 682 nonessential amino acids (MP Biomedicals) and 20 μg/ml trypsin (Cambrex)), after 683 which a two-fold dilution series was made. After three days, the cytopathic effects in 684 the cells were evaluated and the mean (n=3) of the number of infected wells was 685 calculated. 686 Table 4. Details of IAVs used in the experiment shown in Fig. 5D Acknowledgments 692 We thank professor Yoshihiro Kawaoka for providing the hCK cells. We would like to 693 thank Frederik Broszeit for producing the glycans that are used in the glycan 694 microarray in Fig. S1. Balthasar Heesters is thanked for his advice on the flow 695 cytometry experiments and analysis. Monique van Scherpenzeel is thanked for the 696 sugar nucleotide analysis. We also thank Roosmarijn van der Woude for her technical 697 assistance. 698  The N-glycans from WT and B3GNT2/B4GALT1 knock-in MDCK and hCK cells were 982 measured using mass spectrometry. (A) Chromatograms of hCK WT and hCK-983 B3GNT2 cells were constructed for the glycans with at least two and at most seven 984 LacNAc repeating units. The extracted-ion-counts for the ten most abundant glycan 985 features per LacNAc repeating unit group were summed to yield a chromatogram. (B) 986 The N-glycans with at least one LacNAc repeating unit were analyzed for the number 987 of LacNAc repeating units present and the relative abundance was calculated. Further 988 analysis is presented in Figure S4. Full glycan feature lists for each cell line are 989 presented in Table S1-8. 990 The sugar nucleotides in the lysate of MDCK, hCK, and hCK-B3GNT2 cells were 993 analyzed by mass spectrometry (n=2). The normalized abundance of CMP-Neu5Ac, 994 UDP-Gal, and UDP-HexNAc are shown. Normalization was performed on the cell line 995 with the highest amount of each sugar nucleotide. Detailed information about all 996 measured sugar nucleotides is presented in Fig. S4. 997