The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. unrelated to inhibition of neuraminidase activity. The order-of-magnitude lower 50% inhibitory concentrations of 4-GU-DANA (and also DANA CDDO-EA and 4-AM-DANA) for plaque area reduction and for inhibition in the fusion assay than for reducing plaque quantity or obstructing hemadsorption indicate the particular efficacy of these sialic acid analogs in interfering with cell-cell fusion. In cell lines expressing influenza computer virus hemagglutinin (HA) as the only viral protein, we found that 4-GU-DANA experienced no effect on hemadsorption but did inhibit HA2b-red blood cell fusion, as judged by both lipid combining and content material combining. Therefore, 4-GU-DANA can interfere with both influenza computer virus- and HPF3-mediated fusion. The results indicate that (i) in HPF3, 4-GU-DANA and its analogs have an affinity not only for the neuraminidase active site of HN but also for sites important for receptor binding and cell fusion and (ii) sialic acid-based inhibitors of influenza computer virus neuraminidase can also exert a direct, negative effect on the fusogenic function of the additional envelope protein, HA. Sialic acid is the receptor determinant for the human being parainfluenza computer virus type 3 (HPF3) hemagglutinin-neuraminidase (HN) glycoprotein, the molecule responsible for binding of the computer virus to cell surfaces. In addition to binding receptor and contributing to fusion promotion, the HPF3 HN molecule consists of receptor-destroying (sialidase) activity (11). The putative active sites are in the extracellular website of this type II integral membrane protein; however, since the crystal structure of HPF3 HN is not available, the locations of these sites, as well as the structural requirements for binding to the cellular receptor(s), are unfamiliar. In the case of influenza computer virus, studies of the neuraminidase molecule like a potential target of antiviral therapy led to the synthesis of potent inhibitors of this enzyme (31). One of these unsaturated sialic acid analogs, 4-GU-DANA (4-guanidino-Neu5Ac2en; zanamivir), has recently been demonstrated to be a clinically BSPI effective anti-influenza computer virus agent (6, 19). The common mechanism whereby such transition state analogs of sialic acid are thought to block the spread of infection is usually inferred from extensive information about the functions of the two influenza computer virus envelope CDDO-EA proteins, hemagglutinin (HA) and neuraminidase (NA). HA, which recognizes the sialic acid moiety CDDO-EA around the cell surface receptor, mediates both binding of the computer virus to the cell surface and fusion of the viral envelope with the endosomal membrane; NA is not involved in these processes but is necessary for promoting the release of newly formed virions from the cell surface by removing receptors for the computer virus. Thus, restriction by neuraminidase inhibitors of the number of virions available for infecting neighboring uninfected cells is usually believed to underlie the decrease in plaque size and, in the case of even more severe restriction, reduction of plaque number in the CDDO-EA presence of an inhibitor (14, 30, 33). Our interest in examining the possible effect of 4-GU-DANA on HPF3 stems from our observations for another unsaturated sialic acid analog, DANA (Neu5Ac2en) (13). After demonstrating the inhibitory potential of DANA on HPF3 neuraminidase, we showed that this analog also interferes with viral attachment and fusion. Notably, DANA blocked hemadsorption of HPF3-infected cells at a heat where neuraminidase is usually inactive. In addition, in our assay system for quantitating HN-receptor conversation, DANA inhibited the fusion of persistently infected cells with uninfected cells (13). As you possibly can interpretations of these results, we proposed that (i) binding of DANA to the neuraminidase active site of HN induces an inactivating change in the protein at the site(s) with receptor-binding and fusion-promoting function; (ii) there is one site responsible for both neuraminidase and receptor binding; or (iii) alternatively, DANA may bind independently to both the neuraminidase- and receptor-binding sites. In this study, aimed primarily at separating HN’s binding and enzymatic properties, we showed that 4-GU-DANA is usually a more effective, and 4-AM-DANA (4-amino-Neu5Ac2en) is usually a less effective, HPF3 neuraminidase inhibitor than DANA. We compared their effects on HN-mediated processes in several test systems and extended the inquiry to a newly isolated neuraminidase-deficient HPF3 variant (M. Porotto and.

The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation