Effect of Hemagglutinin Glycosylation on Influenza Virus Susceptibility to Neuraminidase Inhibitors

Abstract

The two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), of influenza A virus interact with cellular receptors containing terminal neuraminic acid (NeuAc) moieties. HA initiates infection by binding to cellular receptors, whereas NA destroys the receptors by cleaving off NeuAc moieties (49). In addition, HA binds to complementary, as-yetunidentified, cellular receptors that lack NeuAc moieties (5, 46). The functional balance between HA and NA in influenza virus infections has been intensively studied (4,10,22,23,33,49,50). The HA precursor (HA0) is proteolytically cleaved into two subunits, HA1 and HA2; the HA1 subunit carries the NeuAc-binding site, and the HA2 subunit is responsible for fusion of viral and cellular membranes (51). NA is not essential for influenza A virus assembly or budding (25), but its enzymatic activity facilitates the progeny virions' release from infected cells (6). The anti-influenza drugs oseltamivir and zanamivir inhibit NA activity by targeting the enzyme active site formed by highly conserved residues (6, 24). Substitutions at those residues confer virus resistance to NA inhibitors (28). However, drug resistance can also be conferred via an NAindependent mechanism. Amino acid substitutions in HA can lessen viral dependence on NA activity for release from infected cells and thus decrease susceptibility to NA inhibitors by reducing efficiency of virus binding to cellular receptors (11,29,41,49).Propagation of virus in the presence of an NA inhibitor should result in retention of NeuAc moieties by the complex glycans attached to HA (3, 39). Of note, negatively charged NeuAc moieties situated near the HA receptor-binding site have a potential to impair the virus binding to cellular receptors (38). Therefore, viruses containing complex glycans at the receptor-binding site would exhibit lower susceptibility to NA inhibitors than those lacking such glycans. To investigate this possibility, we utilized the influenza A viruses that belong to the H1N1 antigenic subtype as a model. Viruses of this subtype produced the devastating "Spanish flu" pandemic in 1918 (42), and antigenic drift variants of this virus remained in circulation in the human population until mid-1950s. In 1977, the virus reemerged in the human population, causing the "Russian flu" epidemic; since that time, antigenic drift variants have been in circulation in the human population. There is a considerable variance in the glycosylation patterns near the HA receptorbinding site among the human influenza A/H1N1 viruses, due to antigenic drift and host adaptation (20). For example, the so-called early laboratory-passaged virus A/WSN/33 (WSN) contains a single glycosylation site at Asn129, whereas the contemporary virus, A/Charlottesville/31/95 (CH/95), contains two additional glycosylation sites at Asn 94a and Asn 163) ( Fig. 1) (numbering according to reference 52). In our previous studies, we demonstrated that both viruses were equally susceptible to NA inhibitors by the enzyme inhibition assay, whereas in ce...

Thiazolides, a New Class of Anti-influenza Molecules Targeting Viral Hemagglutinin at the Post-translational Level

“…By using different biochemical approaches, we show that TIZ blocks HA terminal glycosylation at a stage preceding resistance to endoglycosidase H digestion, which is a marker for transport into the cis and middle Golgi compartments (29). Immunomicroscopy studies and analysis of viral particles produced by infected cells confirm that the TIZ-induced alterations impair HA0 trafficking between the endoplasmic reticulum and the Golgi complex, preventing its transport and insertion into the host cell plasma membrane and blocking the exit of mature virions from host cells.…”

“…The mannose-rich sugar component is processed in the Golgi apparatus during the transport to the cell surface, and terminal glycosylation occurs in trans cisternae of the Golgi apparatus (27). To investigate whether TIZ could affect HA0 passage through the Golgi, we subjected aliquots of radiolabeled proteins and HA0 immunoprecipitated samples to digestion with Endo-H, an enzyme that removes N-linked carbohydrate chains that have not been terminally glycosylated (28) or with PNGase-F, an enzyme that removes all N-glycans (29). As expected, both forms of the protein were sensitive to PNGase-F digestion; however, whereas HA0 from control cells was terminally glysosylated becoming Endo-H-resistant, HA0 from TIZ-treated cells remained sensitive to digestion with the protease up to 4 h after synthesis (Fig.…”

Inhibition of Influenza Virus Infection by a Novel Antiviral Peptide That Targets Viral Attachment to Cells

“…Based on the ability of EB to inhibit viral attachment, we hypothesized that the peptide interacted with either NA or HA since changes to either surface glycoprotein can alter fitness of the virus (22). To determine if EB inhibited NA enzymatic activity, untreated or peptide-treated Tk/Ont was tested for enzymatic activity by MUNANA [2Ј-(4-methylumbelliferyl)-␣-D-N-acetylneuraminic acid].…”

Evolutionary Characterization of Human H1N1 Influenza Virus Hemagglutinin Genes Isolated from 1947 to 2009 in China

“…Although glycosylation sites were not necessary for the survival of influenza virus, they played an important role in determining some properties of these viruses. Vasiliy [ see 27,28] reported that a negatively charged glycosylation site near HA RB sites affect HA receptor binding, leading to reduced influenza virus dependence on NA which was the target of antiinfluenza drug failure. Furthermore, the 3 RB sites underwent considerable changes with time.…”