Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

Abstract

Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo.IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by specific interactions between the receptor binding molecule (hemagglutinin-neuraminidase [HN]) and the fusion protein (F). Here we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion and directly impact infection. The crystallographic and biochemical data point to a structural explanation for the HN’s altered ability to activate F for fusion and reveal properties that are critical for infection by this important lung virus in vivo.

Circulating Clinical Strains of Human Parainfluenza Virus Reveal Viral Entry Requirements for In Vivo Infection

“…The HPIV3 HAEadapted strain and the CI-1 strain attain viral titers of up to 10 7 and 10 8 PFU/ml, respectively, in HAE, and greater than 10 5 PFU/g of lung tissue in cotton rats compared to those of the HPIV3 laboratory reference strain, which we previously found to peak at 10 5 PFU/ml in HAE and 10 4 PFU/g of lung tissue in cotton rats (20). Viruses collected from HAE infected with the HAE-adapted HPIV3 produced small plaques (average diameter of 0.1 mm) relative to those of the reference strain (average diameter of 0.6 mm) (19), and the CI-1 virus produced plaques similar in size to those of the HAE-adapted strain (data not shown).…”

“…None of the strains with proficient fusion activities that grow efficiently in cultured monolayer cells succeed in vivo. We observed that the HPIV3 CI-1 collected directly from a patient grew efficiently in cotton rats, producing 2 to 3 logs more virus than the laboratory reference strain at the peak of infection, similarly to the HAE-adapted strain, which we had previously found to be viable in vivo (19,20).…”

“…In contrast, viruses that grow well in monolayer cell cultures have a higher binding avidity than their neuraminidase activity, allowing for more receptor contact, and they have more fusogenic HN/F fusion machineries and thus would be at a disadvantage in vivo (20).…”

“…The laboratory reference strain and the HN Q552 strain, with an HN that interacts with F to trigger fusion more efficiently, are fusogenic in monolayer cell culture. However, these strains grow poorly or not at all in human airway or in vivo (18)(19)(20). The HAE-adapted strain (HN Q552-R559/F D396) has reduced receptor binding, interaction with F, and fusion promotion and grows poorly in monolayer cell culture but well in vivo (20), indicating that the optimal balance of binding and fusion properties is different in vivo and in cultured cell monolayers and that virus in HAE reflects infection in vivo.…”

“…The efficiency of a virus' fusion machinery correlated inversely to sensitivity to fusion peptide inhibition and growth in vivo (16)(17)(18)20). We hypothesized that the HPIV3 CI's fusion characteristics would resemble viruses derived from adaptation to growth in HAE, rendering it sensitive to peptide inhibitors.…”