The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three distinct activities contributing to the ability of HN to promote viral fusion and entry: receptor binding, receptor cleavage (neuraminidase), and activation of the fusion protein. The relationship between receptor binding and fusion triggering functions of HN are not fully understood. For Newcastle disease virus (NDV), one bifunctional site (site I) on HN′s globular head can mediate both receptor binding and neuraminidase activities, and a second site (site II) in the globular head is also capable of mediating receptor binding. The receptor analog, zanamivir, blocks receptor binding and cleavage activities of NDV HN′s site I while activating receptor binding by site II. Comparison of chimeric proteins in which the globular head of NDV HN is connected to the stalk region of either human parainfluenza virus type 3 (HPIV3) or Nipah virus receptor binding proteins indicates that receptor binding to NDV HN site II not only can activate its own fusion (F) protein but can also activate the heterotypic fusion proteins. We suggest a general model for paramyxovirus fusion activation in which receptor engagement at site II plays an active role in F activation.

The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect.F usion between enveloped viruses and the host cell is a key step in viral infectivity, and interfering with this process can lead to highly effective antivirals. Viral fusion is driven by specialized proteins that undergo an ordered series of conformational changes resulting in the close apposition of the viral and host membranes and the formation of a fusion pore (reviewed in reference 14). The first step of infection with most paramyxoviruses is binding of the receptor binding protein to cell surface receptors. In the case of human parainfluenza virus (HPIV), the receptor binding protein, hemagglutinin (HA)-neuraminidase (HN), binds to sialic acidcontaining molecules. HN activates the viral fusion protein (F) to undergo the series of structural rearrangements that ultimately lead to direct fusion of the viral envelope with the plasma membrane of the cell (27,29,35,63). For F activation to initiate, the F protein must have been properly cleaved from its F0 precursor to its active processed form (F1ϩF2) during synthesis in the infected cell. For HPIV (36, 37) and for most other paramyxoviruses (4,8,17,20,26), interaction of HN with its receptor is essential in order for F to promote membrane fusion during infection although there are a few exceptions, e.g., respiratory syncytial virus (RSV) (7, 59) and human metapneumovirus (hMPV) (60, 61). HN also plays a key role after the assembly of progeny virions since its sialic acid-cleaving (neuraminidase) activity facilitates the release of newly budded virions and thus the spread of infection.HN is a tetrameric type II membrane protein consisting of a cytoplasmic domain, a membrane-spanning region, a stalk region, and a globular head. The stalk confers s...

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