Abstract
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Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in 21 a variety of chemically modified forms, including those with acetyl modifications at the 22 C7, C8, and C9 positions. Here, we analyzed the distribution and amounts of these 23 acetyl modifications in different human and canine cells. As Sia or their variant forms 24 are receptors for influenza A and influenza C viruses, we examined the effects of these 25 modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl 26 modified Sia are widely but variably expressed across cell lines from both humans and 27 canines. While they were expressed on the cell surface of canine MDCK cell lines, they 28 were located primarily within the Golgi compartment of human HEK-293 and A549 cells. 29
The O-acetyl modified Sia were expressed at low levels of 1-2% of total Sia in these cell 30 lines. We knocked out and over-expressed the sialate O-acetyltransferase gene 31 (CasD1), and knocked out the sialate O-acetylesterase gene (SIAE) using 32 CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O-and 9-O-acetyl Sia 33 expression, confirming previous reports. However, over-expression of CasD1 and 34 knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no 35 change in 7,9-O-acetyl levels, indicating that there are complex regulations of these 36 modifications. These modifications were essential for influenza C infection, but had no 37 obvious effect on influenza A infection. 38

4 from which it has been observed to migrate to the C8 and C9 position under 66 physiological conditions, allowing the possible addition of another acetyl group to C7 (6, 67 7). A migrase enzyme has been proposed to aid in the transfer of the acetyl group from 68 C7 to C9, however a specific enzyme has yet to be identified (5, 8, 9) . CasD1 is 69 localized in the late Golgi, so acetyl modifications are added during the later stages of 70 protein glycosylation. The regulatory processes that control the number of acetyl groups 71 added or their positions have not been well defined, although clear differences in 72 expression of 7,9-O-Ac and 9-O-Ac have been reported in mouse and human tissues, 73 chicken embryos, and some other animals (10, 11). CasD1 uses acetyl-CoA and likely 74 has a preference for CMP-Neu5Ac as a substrate, so that it is less active on CMP-75 Neu5Gc (7). The existence of an enzyme to facilitate the migration of acetyl groups from 76
C7 has been proposed, but not identified (5). 77
At least one sialate O-acetylesterase (SIAE) enzyme regulates the display of 7,9-78 O-and 9-O-Ac in many cells and tissues (Fig. 1B) . The SIAE gene encodes two 79 isoforms that vary in the presence of a proposed C-terminal localization tag in the 80 lysosomal (Lse) form that is absent from the cytosolic form (Cse) (12). However, the 81 processes that regulate the expression and activity of these two isoforms remain poorly 82 defined. Lse has been found to localize to the Golgi and/or ER and on the surface of 83 cells when over-expressed, with the majority being secreted into the supernatant (12). 84
Antibody staining shows that Cse is found diffusely throughout the cytosol, where it is 85 thought to remove 9-O-and 7-O-acetyl groups to recycle the Sia for reuse in 86 glycosylation (12). Despite the reports of distinct protein expression in mouse tissues, 87 bioinformatic analysis of RNA expression in human cells and tissues show mRNA 88
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint corresponding to the Lse form of SIAE and none responding to the Cse form (13). It is 89 therefore still unclear how these two isoforms are regulated in humans or other animals, presence of 9-O-Ac can also reduce the activity of sialidases, including human 102 neuraminidases (18). Incorrect regulation of 9-O-Ac, 7,9-O-Ac, and SIAE activity has 103 been linked to autoimmune disorders through the development of auto-antibodies (17, 104 19). 9-O-Ac and 7,9-O-Ac and their regulation by SIAE also appear to play important 105 roles during early stages of embryonic development, spermatogenesis, and in different 106 forms of cancer including acute lymphoblastic leukemia, colon cancer, and breast 107 cancer (20) (21) (22) (23) . 108
Effects of different Sia modifications have also been suggested for the binding of 109 pathogens or on the activities of their sialidases (neuraminidases). However, in general 110 these are still not well documented, with the exception of those that use the modified 111
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint 6 forms as receptors. Both influenza A (IAV) and influenza C (ICV) viruses use Sia as 112 their primary receptors for host recognition and cell entry, but with different effects of Sia 113 modification. IAV interacts with Sia through two surface glycoproteins, hemagglutinin 114 (HA) and neuraminidase (NA). HA binds Sia to initiate the endocytic uptake of the virus 115 by the cell, leading to fusion between the viral envelope and the endosomal membrane 116 at low pH (24). NA is a sialidase which cleaves Sia off glycan chains when it is present 117 in mucus or on the surface of cells, allowing the virus to penetrate to the epithelial cells, 118
and also preventing aggregation of newly produced virus after budding (25, 26). In 119 contrast to IAV, ICV has one surface glycoprotein, the hemagglutinin-esterase fusion 120 protein (HEF), which serves similar purposes to both HA and NA. HEF binds specifically 121 to 9-O-acetyl Sia to initiate uptake of the virus into cells, while the esterase domain 122 removes 9-O-acetyl modifications, releasing the virus from mucus, and disassembling 123 virus aggregates after budding (27). While the role of O-acetyl modified Sia for ICV 124 infection is well documented, how these modifications affect IAV is not well 125 characterized. Previous limited studies have suggested that the presence of 9-O-Ac on 126 cells may be inhibitory for NA activity and HA binding of IAV (28, 29) . 127
Here we examine and more closely define the expression and distribution of 9-O-128 acetyl and 7,9-O-acetyl Sia on cells in culture, define the effects of SIAE and CasD1 on 129 display of these Sia modifications, and perform an initial examination of the effects of 130 these modified Sia on infection by IAV and ICV. We used CRISPR-Cas9 for gene 131 7 quantifying the different Sia forms, we provide a more detailed understanding of the 135 expression and localization of these modifications in cells, and examine their affects on 136 two host-virus interactions as examples. 137
RESULTS. 138
Expression of 7,9-O-and 9-O-acetyl Sia in cells. There is currently only 139 sporadic information about the expression of modified Sia on commonly used cell lines, 140
or an understanding of how that compares to the expression in animal tissues. We 141 examined cell lines that are widely used in many experimental systems: A549 human 142 type II alveolar epithelial cells, HEK-293 human kidney derived cells (possibly 143 embryonic adrenal precursor cells (30)), and MDCK canine kidney epithelial cells. 144
Additionally, we tested MDCK-type I and type II cells that were previously sub-cloned 145 from the ATCC MDCK line (MDCK-NBL2) and have been extensively characterized 146 (31-33). We used probes derived from porcine torovirus (PToV) and bovine coronavirus 147 (BCoV) hemagglutinin-esterase proteins (HE) that were fused to human IgG1 Fc and 148 had the esterase active site inactivated (HE-Fc). The PToV HE-Fc probe recognizes 9-149 O-Ac, while BCoV HE-Fc recognizes primarily 7,9-O-Ac but also has a low affinity for 9-150 O-Ac (10, 11). By immunofluorescence microscopy, the different forms were present at 151 variable levels, with 10 to 70% of the cells showing staining under standard culture 152 conditions ( Fig. 2A,B) . MDCK-NBL2 and MDCK-type I cells showed both strong surface 153 and internal staining for 7,9-O-and 9-O-Ac forms, while both were mostly found in 154 intracellular locations in A549 and HEK-293 cells, with an occasional cell showing bright 155 surface staining. MDCK-type II cells showed staining only for 9-O-Ac and none for 7,9-156 O-Ac, indicating that these modifications are regulated independently. In HEK-293 and 157
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint 8 A549 cells, both 7,9-O-and 9-O-Ac appeared to be localized within the Golgi 158 compartment as confirmed by co-staining with the Golgi marker GM130 (Fig. 3) . Similar 159 localization differences between cell lines have been seen previously using the ICV 160 HEF as a probe (34). In many of the cell lines, there was inherent variability within 161 populations in terms of level of staining and localization. For example, in MDCK-NBL2 162 not all cells were positive for 9-O-Ac, while in MDCK-type I some cells retained more 9-163 O-Ac and 7,9-O-Ac internally and others displayed more of these modified Sia on their 164 surface ( Fig. 2A) . This heterogeneity was consistent between different passages of 165 each cell line. 166
The expression levels of those modified Sia variants were also quantified by 167 HPLC analysis using DMB labeling and fluorescence detection (35). The cells were 168 treated at 80˚C for 3h in the presence of 2M acetic acid, and likely represent the total 169
Sia present in the cells. Even though some cell lines showed strong staining by the HE-170
Fc probes, levels of 9-O-Ac were 1-2% of the total collected Sia from all cells while 7,9-171 O-Ac was not detected in any cell lines ( Fig. 2; Table 1; Supplemental Fig. 3 ). This 172 includes cells from other species including cat, mouse, horse, and swine. Unique 173 among the cells tested, A549 cells are secretory and express significant levels of mucin 174 (including MUC1 and MUC5B) (36). We found that A549 cells were able to secrete 175 MUC5B into conditioned media (Supplemental Fig. 1A ), although we found that the 176 amount secreted was inconsistent between collections. Analysis of conditioned media 177 from A549 cells showed approximately 2% of Sia was 9-O-Ac on secreted proteins with 178 no 7,9-O-Ac detected, indicating that at least in these cells, secreted proteins were not 179 enriched for O-acetylated Sia (Supplemental Fig. 1B) . However, it would be worth 180
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint
4B, C, D). This agrees with previous findings that loss of CasD1 leads to loss of both 204

7,9-O-and 9-O-Ac modifications in haploid HAP1 cells (7). HPLC analysis of total Sia 205
showed a significant decrease in 9-O-Ac expression compared to wild-type (WT) cells, 206 however very low levels (<1%) were still detectable, despite a lack of staining with the 207 HE-Fc probes (Fig. 4E) . This could be due to exogenous Sia from the fetal bovine 208 serum in the growth media, as is the case for Neu5Gc which is also detectable at 209 similarly low levels on cells even though the gene for Neu5Gc synthesis, CMAH, is not 210 functional in humans or canines (Supplemental showed significantly higher levels of CasD1 mRNA compared to WT cells, indicating 218 that the CasD1 expression plasmid was being transcribed at high levels (Fig. 4E) . 219
However, only a modest increase in average 9-O-and 7,9-O-Ac expression across the 220 population in HEK-293 cells was seen by flow cytometry, while fluorescent microscopy 221 showed heterogeneity of expression in the population (Fig. 4B, C, D) . The transfected 222 MDCK and A549 CasD1-OX cells showed recovery of 9-O-and 7,9-O-Ac synthesis 223 when analyzed by flow cytometry and immunofluorescence microscopy, but levels were 224 not as high as seen in WT cells (Fig. 4B, C, D) . HPLC analysis confirmed the 225 expression levels for HEK-293, A549, and MDCK cells (Fig. 4F) . Additionally, 226
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint heterogeneity was still seen in these populations for 9-O-Ac. This suggests that 7,9-O-227 and 9-O-Ac expression is not directly regulated by the levels of CasD1 gene expression, 228 but may be affected by post-translational regulation of CasD1 activity or removal of the 229 modifications by SIAE or other enzymes. In addition, these modifications are not 230 regulated the same across individual cells as evidenced by the population 231 heterogeneity. SIAE transcripts were seen to be consistently expressed in HEK-293 232 WT, ΔCasD1, and CasD1-OX cell populations by qPCR analysis, although SIAE mRNA 233 levels in CasD1-OX cells were lower than those seen in WT (Fig. 4E) . 234 SIAE knock out cells and display of modified Sia. To determine the role of 235 SIAE activity in regulating 7,9-O-and 9-O-Ac display, SIAE knockout (ΔSIAE) cells were 236 generated from WT HEK-293 and A549 cells by targeting early exons in the SIAE gene 237 (Fig. 5A) . Complete knockout of SIAE was confirmed both by genotyping to show 238 deletions in both alleles and by loss of mRNA by qPCR (Fig. 5A, D) . ΔSIAE cells 239 showed a small but significant increase in surface display and a large increase in 240 internal display of 9-O-Ac based on flow cytometry, but did not appear to show any 241 changes in surface or internal display of 7,9-O-Ac (Fig. 5B, C) . HPLC analysis also 242 showed a small increase of 9-O-Ac in HEK-293 cells, but no 7,9-O-Ac was detected in 243 either cell line (Fig. 5E) . When the overexpression CasD1 plasmid was transfected into 244 ΔSIAE cells (ΔSIAE+CasD1), cells showed an increase in surface and internal 9-O-Ac, 245 but no increase in surface or internal display of 7,9-O-Ac by either flow cytometry or 246 immunofluorescence microscopy (Fig. 5B,C) . HPLC confirmed the flow cytometry 247 results by showing a small increase of 9-O-Ac levels in HEK-293 cells that were similar 248 to those seen in CasD1-OX (Figs. 5E) . A549 cells showed a small but non-significant 249 All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint increase in 9-O-Ac levels compared to WT by HPLC analysis. Interestingly, HEK-293 250
ΔSIAE+CasD1 cells grew more slowly than either ΔSIAE or WT cells (Fig. 5F) . 251
However, A549 ΔSIAE+CasD1 showed increased growth rates compared to ΔSIAE or 252 WT cells, while ΔSIAE cells had slightly lower growth rates compared to WT. This 253 suggests that 7,9-O-and 9-O-Ac may affect cell metabolism and growth rates, and that 254 these effects may be cell type specific. Overall, these results show that SIAE regulates 255 levels of 9-O-Ac and 7,9-O-Ac, but that knocking out SIAE only leads to small increases 256 in these modifications. However, there appear to be mechanisms regulating the surface 257 display of 9-O-Ac and 7,9-O-Ac, as most of the modified Sia were specifically retained 258 in infection efficiency in any of these cells (Fig. 6A) . IAV strains also showed equal 265 infection efficiency on MDCK WT, ΔCasD1, and CasD1-OX (Fig. 6C) . However, ICV 266 strains C/Ann Arbor/1/50 and C/Taylor/1233/1947 showed no infectivity in MDCK 267 ΔCasD1 (Fig. 6B) . Only the Ann Arbor strain recovered infectivity in MDCK CasD1-OX 268 at much lower levels than in MDCK WT, likely due to the lower surface expression of 9-269 O-and 7,9-O-Ac in these cells. It therefore appears that 7,9-O-Ac and 9-O-Ac do not 270 strongly influence IAV infection, likely due to their low levels of expression. However, 271 We confirmed that 9-O-and 7,9-O-Ac are expressed on cells in culture and that 287 expression varies between cell lines, as has been previously reported (34). However, 288 when present these modified Sia made up only 1 to 2% of the total Sia when analyzed 289 by HPLC. Cells showed distinct population heterogeneity in 7,9-O-and 9-O-Ac display 290 and localization that was observed over many passages examined. Previous studies 291 using ICV HEF probes found similar staining patterns on some cell lines and also 292 showed that after sorting into high and low staining populations, both populations 293 returned to previous levels of heterogeneity within a few passages (11, 34). Here we 294
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint 14 saw that the modified Sia are retained in the Golgi of HEK-293 and A549 cells, although 295 an occasional cell displayed these modified Sia on the surface. It is not clear why these 296 modified Sia are localized in the Golgi, although perhaps the modifications could block 297 the onward trafficking of glycoproteins to the cell surface. In contrast to the human cells 298 lines, MDCK cells showed many cells expressing 7,9-O-Ac and 9-O-Ac on the cell 299 surface, raising the possibility that trafficking could be cell-type or species specific. A 300 summary of the synthesis and localization of 7,9-O-and 9-O-Ac is shown in Figure 7 . 301
To look more closely at the expression and roles of 7,9-O-and 9-O-Ac 302 modifications and their effects on viral infections, we prepared glyco-engineered cell 303 lines that lacked these modifications or that expressed higher levels of CasD1. A 304 deletion and frame shift in CasD1 completely removed both 9-O-and 7,9-O-Ac 305 expression, confirming this enzyme was responsible for creating both modifications, 306 likely through addition to the C-7 position, from which it migrates to the C-9 position (5, 307
8, 9). Adding CasD1 back into the cells by plasmid transfection restored modified Sia 308
expression, but none of the cell clones isolated showed universally higher levels of 309 modified Sia synthesis and population heterogeneity was still seen. The CasD1-310 transfected HEK-293 cells showed the greatest increase, while for A549 and MDCK 311 cells expression was similar to or lower than WT cells. However, even in the HEK-293 312
CasD1-OX cells, 9-O-Ac still only accounted for ~1.5% of total Sia and 7,9-O-Ac was 313 not detected. This indicates that the levels of these modifications are not only controlled 314 by the expression of CasD1, and could be regulated by other processes. Additionally, 315 there is clearly a differential regulation of 7,9-O-Ac compared to 9-O-Ac, as over-316
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint expressing CasD1 did not lead to an increase in 7,9-O-Ac, as it was not detectable by 317 HPLC and only very low fluorescence via staining with BCoV HE-Fc. 318
One candidate for control of these modifications is SIAE. When we knocked 319 SIAE out of HEK-293 and A549 cells, we saw an increase in 9-O-Ac that was still 320 retained in the Golgi but no increase in 7,9-O-Ac. Expressing CasD1 from a plasmid in 321 ΔSIAE HEK-293 and A549 cells resulted in an additional small increase in 9-O-Ac that 322 was still Golgi associated, but with little increase in expression on the cell surface. present at levels too low to affect IAV, they are expressed at much higher levels in 357 mucosal tissues and in secreted mucins of many animals and tissues, which may 358 provide a more effective barrier (10, 11, 43-45). In future work we are examining these 359 processes for mucus and other sources in different animals. 360 
MATERIALS AND METHODS 361
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4°C then diluted in Carbo-Free blocking solution to a final concentration of 5 µg/ml HE-385
Fc and 1:500 of secondary antibody. Cells were stained with HE-Fc/anti-IgG complex 386 for 1 hr at room temperature. Coverslips were mounted using Prolong Antifade-Gold 387 with DAPI (Invitrogen). Cell were imaged using a Nikon TE300 fluorescent microscope. 388
For flow cytometry, cells were seeded onto non-adherent cell culture dishes and left 389 overnight at 37°C and 5% CO 2 . Cells were collected using ice-cold PBS (HEK-293 and 390 conditioned media were compared to purified human Muc5B using a 8% SDS-PAGE gel 430
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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint and probed with an anti-human Muc5B antibody (both purified protein and antibody 431 were gifts from Dr. Stefan Ruhl, University of Buffalo). Conditioned media was analyzed 432 for total Sia using the HPLC methods listed above. 433 qPCR of SIAE and CasD1 expression. RNA from cells was extracted using 434 EZNA Total RNA Kit I (Omega Bio-Tek) and cDNA was synthesized using SuperScript II 435
Reverse Transcriptase (Invitrogen) standard protocol using oligo(dT) 12-18 primers 436 (Invitrogen). CasD1 and SIAE specific primers were designed using Geneious 437 (Biomatters, Ltd.). For CasD1, adequate primers could not be targeted around the 438 CRISPR-Cas9 edit site due to high G/C content. qPCR was performed on a Applied 439
Biosystems StepOnePlus Real-Time PCR System using Fast SYBR Green (Bio-Rad). The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint were permeabilized using 0.001% tween-20 to determine surface and internal 565 expression. 566

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