Influence of the Human Parainfluenza Virus 3 Attachment Protein's Neuraminidase Activity on Its Capacity To Activate the Fusion Protein

Abstract

In order to examine functions of the hemagglutinin-neuraminidase (HN) protein that quantitatively influence fusion promotion, human parainfluenza virus 3 (HPIV3) variants with alterations in HN were studied. The variant HNs have mutations that affect either receptor binding avidity, neuraminidase activity, or fusion protein (F) activation. Neuraminidase activity was regulated by manipulation of temperature and pH. To dissect the specific contribution of neuraminidase to triggering, two variant HNs that are triggering-defective due to a mutation in the HN stalk were evaluated. One of these variants has, in addition, a mutation in the globular head that renders it neuraminidase dead, while the HN with the stalk mutation alone has 30% of wt neuraminidase. While the variant without neuraminidase activity triggered F effectively at 37°C irrespective of pH, the variant possessing effective neuraminidase activity completely failed to activate F at pH 5.7 and was capable of only minimal triggering activity even at pH 8.0. These results demonstrate that neuraminidase activity impacts the extent of HPIV3-mediated fusion by releasing HN from contact with receptor. Any particular HNs competence to promote F-mediated fusion depends on the balance between its inherent F-triggering efficacy and its receptor-attachment regulatory functions (binding and receptor cleavage).Human parainfluenza virus 3 (HPIV3), like other paramyxoviruses, possesses two envelope proteins directly involved in viral entry and cytopathology. Hemagglutinin-neuraminidase (HN), by binding to sialic acid-containing cellular surface receptors, brings the viral envelope in proximity to the plasma membrane. Fusion protein (F) is the active mediator of fusion between the viral and cell membranes, which leads to release of the viral nucleocapsid into the cytoplasm. In order to mediate fusion, F must be present in its cleaved state and then undergo a second activation step to assume its fusion-ready conformation. It has been shown that HN plays an essential role in the fusion process, and one proposed role of HN is to drive the final conformational change in F that renders it fusion active (30). Fusion of the virus with the target cell, which permits synthesis of viral macromolecules and expression of viral envelope proteins on the host cell's surface, is followed by two processes that lead to the spread of infection and HPIV3 cytopathogenesis. One is the assembly and budding of new virions that then infect distant as well as neighboring cells. Second, by virtue of HN and F expressed on its surface, an infected cell can fuse with adjacent uninfected cells, forming syncytia. In the spread of infection via budding, HNЈs receptordestroying neuraminidase activity plays the essential role of preventing progeny virions from remaining aggregated on the infected cell's surface rather than spreading to additional cells (29). For syncytium formation, no direct role has yet been found for HNЈs neuraminidase activity, although the magnitude of the receptor-cleaving activity...

Premature Activation of the Paramyxovirus Fusion Protein before Target Cell Attachment with Corruption of the Viral Fusion Machinery

“…The timing of F-activation is critical for paramyxovirus entry (15,25,28,57,58). In the case of PIV5, a mutated F protein with a hyperactive fusion phenotype releases the fusion peptide before reaching the target cell, thus preventing fusion with the target cell (58).…”

“…Although F-activation is key for viral entry (19), triggering must occur only when F is close enough to then make contact with the target cell membrane. We suggest that this timing of activation represents a potential target for intervention with paramyxovirus infection (15,25,27).…”

Entry of parainfluenza virus into cells as a target for interrupting childhood respiratory disease

“…Viruses with alterations in the HN protein that led to either an increased affinity of the protein for its receptor or decreased neuraminidase activity are more potent at inducing membrane fusion, which leads to the conclusion that it is the interaction of the HN protein with its receptor that is required for activation of F protein (29). HN proteins with either increased receptor affinity or decreased enzymatic (receptor-cleaving) activity stay engaged with their receptor longer than WT HN proteins (29).…”

“…HN proteins with either increased receptor affinity or decreased enzymatic (receptor-cleaving) activity stay engaged with their receptor longer than WT HN proteins (29). Both cell surface receptor-binding and neuraminidase activities of the HN protein, by impacting the duration of HN-receptor engagement, regulate F protein activation and fusion promotion.…”

“…While receptor binding is an important component of this process, attachment is not sufficient (23,25,28); many F proteins demonstrate a requirement for the presence of an HN protein from the same type of virus (the homotypic HN protein) in order to mediate fusion (25,26). One proposed explanation for this requirement is that the interaction between the HN and F proteins may be type specific and/or that a specific relationship between the structures and/or activities of the 2 proteins is required in order to maintain function (29). This final key function of the HN protein - promotion of fusion - has become amenable to mechanistic study only recently; upon binding to its receptor, parainfluenza HN protein plays a critical role in activating or "triggering" the F protein to assume its fusion-ready conformation (30,31).…”