Distribution of O-Acetylated Sialic Acids among Target Host Tissues for Influenza Virus

Abstract

Sialic acids (Sias) are important glycans displayed on the cells and tissues of many different animals and are frequent targets for binding and modification by pathogens, including influenza viruses. Influenza virus hemagglutinins bind Sias during the infection of their normal hosts, while the encoded neuraminidases and/or esterases remove or modify the Sia to allow virion release or to prevent rebinding. Sias naturally occur in a variety of modified forms, and modified Sias can alter influenza virus host tropisms through their altered interactions with the viral glycoproteins. However, the distribution of modified Sia forms and their effects on pathogen-host interactions are still poorly understood. Here we used probes developed from viral Sia-binding proteins to detect O-acetylated (4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl) Sias displayed on the tissues of some natural or experimental hosts for influenza viruses. These modified Sias showed highly variable displays between the hosts and tissues examined. The 9-O-acetyl (and 7,9-) modified Sia forms were found on cells and tissues of many hosts, including mice, humans, ferrets, guinea pigs, pigs, horses, dogs, as well as in those of ducks and embryonated chicken egg tissues and membranes, although in variable amounts. The 4-O-acetyl Sias were found in the respiratory tissues of fewer animals, being primarily displayed in the horse and guinea pig, but were not detected in humans or pigs. The results suggest that these Sia variants may influence virus tropisms by altering and selecting their cell interactions. IMPORTANCE Sialic acids (Sias) are key glycans that control or modulate many normal cell and tissue functions while also interacting with a variety of pathogens, including many different viruses. Sias are naturally displayed in a variety of different forms, with modifications at several positions that can alter their functional interactions with pathogens. In addition, Sias are often modified or removed by enzymes such as host or pathogen esterases or sialidases (neuraminidases), and Sia modifications can alter those enzymatic activities to impact pathogen infections. Sia chemical diversity in different hosts and tissues likely alters the pathogen-host interactions and influences the outcome of infection. Here we explored the display of 4-O-acetyl, 9-O-acetyl, and 7,9-O-acetyl modified Sia forms in some target tissues for influenza virus infection in mice, humans, birds, guinea pigs, ferrets, swine, horses, and dogs, which encompass many natural and laboratory hosts of those viruses.

Keywords: host range; influenza; receptor-ligand interaction; respiratory viruses; sialic acid; virus-host interactions.

FIG 1

Expression and characterization of SGRPs. (A) Cartoon schematic of SGRPs. Nidovirus HE ectodomains were expressed with a C-terminal tag of the human IgG Fc domain and a 6-His sequence. The esterase active site Ser (denoted by red squares) was mutated to Ala to generate binding SGRPs. (B) Expressed and purified SGRPs resolved on SDS-PAGE. (C) MHV-S HE-Fc treated with various glycosidases and resolved on SDS-PAGE shows presence of N-linked (but not O-linked) glycosylation. (D) Solid-phase lectin binding assays of SGRPs. Plates were coated with horse serum (MHV-S) or bovine submaxillary mucin (PToV-P4, BCoV-Mebus) and probed with 2-fold dilutions of SGRPs (starting at 50 μg/ml). Parallel rows were treated during blocking with active-esterase forms of the proteins (Est+).

FIG 2

Glycan microarray screening of MHV-S HE-Fc, PToV-P4 HE-Fc, and BCoV-Mebus HE-Fc. The binding is presented in heat map form. The heat map was generated according to a method previously described (87). Binding was ranked and calculated as follows, where RFU represents relative fluorescence units: (glycan RFU/maximum glycan RFU) * 100. Binding data represent results determined at 40 µg/ml (n = 4, standard deviations [SD]).

FIG 3

Presence of O-acetyl modified Sias on a selection of cell lines from different hosts commonly used for the growth and assay of influenza viruses. Cell lines of human (HEK-293, A549), canine (MDCK), and equine (NBL-6) origin were screened with the SGRPs for the display of O-acetylated Sias. The display of 4-O-Ac Sias was isolated to horse origin NBL-6 cells, while all screened cells displayed 7,9-O-Ac and 9-O-Ac Sias. Human cell lines appeared to predominantly display 7,9-O-Ac and 9-O-Ac Sias intracellularly, while MDCK cells displayed clearly on the membrane surface. Esterase treatment ablated probing for the modified Sia form (MDCK for 9-O-Ac inset box).

FIG 4

(A) Staining (red) of FFPE mouse respiratory tissues for the distribution of O-acetylated modified forms and Sia linkages. Controls with anti-human IgG Fc-specific antibody do not show background staining. Trachea and lung of mice were highly enriched in 9-O-Ac (and 7,9-O-Ac) Sias, including at the tracheal epithelium and lung pneumocytes. The α2-3-linked Sias are enriched in the mouse lung. (B) Staining (red) of frozen mouse respiratory tissues for O-acetylated modified Sias shows detection results similar to those seen with the FFPE tissue. Bars, 50 μm.

FIG 5

Staining (red) of mammalian respiratory tissues for the distribution of the 4-O-Ac Sia, detected with the MHV-S HE-Fc SGRP. Tracheal and lung sections were isolated from human, ferret, guinea pig, pig, horse, and dog. 4-O-Ac Sias were predominately found in the respiratory tissues of horses and guinea pigs, while trace signal was seen in ferret and dog tissues. Human and pig tissues appeared to contain no 4-O-Ac Sias. Bars, 50 μm.

FIG 6

(A) Staining (red) of mammalian respiratory tissues for the distribution of the 9-O-Ac Sia, detected with the PToV-P4 HE-Fc SGRP. (B) Staining of respiratory tissues for the distribution of 7,9-O-Ac Sia, detected with the BCoV-Mebus HE-Fc SGRP. Both Sia forms were identified in respiratory tissues of all species examined, with variations in distribution and display. The 9-O-Ac and 7,9-O-Ac Sias generally appeared to have similar patterns of distribution. Human tissues displayed 7,9- and 9-O-Ac Sias in the tracheal submucosal glands and throughout the lung. These Sia forms were displayed at the tracheal epithelia of pigs, horses, and dogs. Bars, 50 μm.

FIG 7

(A) Staining (red) of mammalian respiratory tissues by SNA for analysis of the distribution of α2-6-linked Sias. Human tissues show abundant staining at the tracheal epithelium, similarly to pig tissues. Ferret tissues display large amounts of α2-6-linked Sias in the submucosal glands and lung. (B) Staining (red) of mammalian respiratory tissues by MAH for analysis of the distribution of α2-3-linked Sias. These Sia linkages are displayed in the human lung alveolar tissue. They were found in all species examined but were particularly enriched in dog tissue such as the trachea. Bars, 50 μm.

FIG 8

The distribution of Sias in the tissues of a Pekin duck, a major waterfowl reservoir of avian influenza viruses. (A) Staining (red) of respiratory tissues with SGRPs for O-acetyl modified Sias. Both 4-O-Ac and 9-O-Ac Sias are displayed at tracheal epithelia and in lung alveolar tissue. Within the digestive tissues, trace 7,9- and 9-O-Ac Sias are displayed in enteric ganglia of the intestines (Sm intestine, small intestine), whereas epithelial cells of the cloaca display abundant mono-9-O-Ac Sias. (B) Staining (red) of respiratory tissues for Sia linkage chemistry. Respiratory tissue displayed both Sia linkages, whereas digestive tissues showed more predominant α2-3-linked Sias. Bars, 50 μm.

FIG 9

The distribution of Sias in tissues from 10-day postfertilization embryonated chicken eggs. Staining (red) of embryonic tissues and extraembryonic chorioallantoic membranes (CAM) is shown. Only mono-9-O-Ac Sias were detected in embryonic tissue and on CAM. There was significant staining for α2-3-linked Sias in the embryo and on membranes and limited presence of the α2-6-linked forms. Bars, 50 μm.