Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase

Abstract

BackgroundInfluenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.MethodsWe tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity.ResultsIAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM.ConclusionIAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.

Pandemic Swine H1N1 Influenza Viruses with Almost Undetectable Neuraminidase Activity Are Not Transmitted via Aerosols in Ferrets and Are Inhibited by Human Mucus but Not Swine Mucus

“…This is an important host defense mechanism, as pathogens trapped in mucus are expelled via mucociliary clearance before they are able to bind to cells and initiate an infection (27,29). The interaction between NA and mucins was hypothesized as early as 1943; more recently, by studying viral binding to MDCK cells, A549 cells, erythrocytes, and isolated respiratory mucus, NA activity has been shown to be important in initiating infection and viral penetration of mucus in vitro (18,20,(30)(31)(32). Mucus overlying frozen human trachea/bronchial tissue sections bound to viruses in a manner which appeared to be mediated by sialic acids (20).…”

“…The interaction between NA and mucins was hypothesized as early as 1943; more recently, by studying viral binding to MDCK cells, A549 cells, erythrocytes, and isolated respiratory mucus, NA activity has been shown to be important in initiating infection and viral penetration of mucus in vitro (18,20,(30)(31)(32). Mucus overlying frozen human trachea/bronchial tissue sections bound to viruses in a manner which appeared to be mediated by sialic acids (20). In in vivo experiments performed in mice, overexpression of MUC5AC, one of the major mucins in respiratory mucus, significantly impaired viral replication in the lung (18).…”

“…Further, mice that do not express MUC5B, the other major secreted respiratory mucin, showed severely impaired mucociliary clearance and perturbed immune responses and succumbed to bacterial lung infections (33). Interestingly, a previous study showed that an overlay of human mucus on MDCK cell monolayers seemed to be more inhibitory to viruses than an overlay of porcine mucus, despite similar sialic acid content, although this effect showed some strain specificity (20). The porcine and human respiratory glycomes show similarities, including the expression of ␣2,3 and ␣2,6 sialylated glycoproteins and large complex N-glycans with sialylated poly-LacNAc chains, but differences have been characterized (34,35).…”

“…We hypothesized that mucus may have played a role in the differences observed between these viruses in vitro and in vivo. NA is important in the release of viruses from cells, but there is also evidence that it plays a role early in infection, by freeing viruses bound to sialic acids present on mucus in the airway (18)(19)(20). To determine if pH1N1 low -1 was inhibited by mucus, we used washed and unwashed NHBE cells.…”

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection

“…Candidate vaccine viruses (CVVs) of A(H7N9) have been generated and tested for safety and immunogenicity (4)(5)(6) and could be authorized for use if needed. The most recent outbreak of A(H7N9) infection, i.e., the fifth epidemic wave, has increased the concern regarding the potential pandemic threat of this virus because the incidence rate has increased, A(H7N9) strains with hemagglutinins (HAs) that are antigenically distinct from the tested CVVs have emerged, and highly pathogenic strains have been isolated (7,8). There is therefore a need to consider additional ways to prevent and control A(H7N9) infection.…”