Human Parainfluenza Virus Infection of the Airway Epithelium: Viral Hemagglutinin-Neuraminidase Regulates Fusion Protein Activation and Modulates Infectivity

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN′s receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN′s F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN′s F-triggering activity may have promise in vivo.Paramyxoviruses are enveloped viruses that enter cells by fusing directly with the cell membrane. During entry, the viral surface glycoproteins hemagglutinin-neuraminidase (HN) (the receptor-binding molecule) and F (the fusion protein) cooperate in a highly specific way to mediate fusion upon receptor binding. To understand these mechanisms, elucidate how paramyxoviruses enter cells, and develop strategies to prevent or treat infection, we study human parainfluenza virus (HPIV), an important cause of croup and bronchiolitis in children. Our results have uncovered fundamental roles of the receptor-binding protein in paramyxovirus fusion and principles of coordinated interaction between the glycoproteins during the viral life cycle.To understand how the diverse functions of the viral glycoproteins are regulated during the viral life cycle, we have used viruses bearing variant HN molecules with mutations at the binding/F-triggering site (and/or the primary receptor-binding site) to study how this molecule works to trigger F (2, 3, 7, 10, 15, 18, 20). The correct timing of F activation (triggering) by HN is essential for entry. For infection to occur, triggering must occur only when F is in proximity to the target cell membrane, and we propose that the regulation of F triggering is essential for the survival of the virus. The outcome of infection is determined by the target cell''s properties and its receptors, and specific mechanisms that are relevant to pathogenesis need to be tested using tissues that reflect the natural host. We therefore tested the hypothesis that a dysregulation of F triggering precludes successful infection in both a cotton rat model and the natural host airway epithelium.For the cotton rat model, previous studies suggested that altered pathogenesis in HPIV infection might be caused by specific HN mutations (24). The present detailed studies of the cotton rat using HN viral variants suggest that the extent of lung infection correlates with the ability of each variant to grow in vivo. The most striking finding is that the ability of the HN variants to grow in vivo is inversely related to their ability to fuse a monolayer of cultured cells. In order to understand the determinants of infection in the natural host, we therefore turned to a model that closely reflects the natural human host tissue, the human airway epithelium (HAE). This model utilizes a recently developed method for culturing primary HAE cells at an air-liquid interface, generating a differentiated, pseudostratified, mucociliary epithelium that faithfully represents the HAE (16). The HAE model was previously used to characterize the polarity and cell specificity of respiratory syncytial virus (26) and HPIV type 3 (HPIV3) (25), confirming that it is suited to studying paramyxovirus-HAE interactions that reflect those in the human lung.We used viruses bearing HNs that are altered in receptor binding or F triggering to reveal the functional relevance of these properties in the HAE and to establish the key role of HN binding site II in infection in the natural host. We propose that an enhanced triggering of F by HN may be a disadvantage in vivo and that the function and timing of F triggering are critical in the target tissue. The correct balance between the three functions of HN (receptor binding, receptor cleaving, and F triggering) resides in the functions of the two binding sites (18), binding and release in site I and F triggering in site II, and both sites of HN play key roles in the natural host.