Adaptation of Human Parainfluenza Virus to Airway Epithelium Reveals Fusion Properties Required for Growth in Host Tissue

Abstract

Paramyxoviruses, a family of RNA enveloped viruses that includes human parainfluenza virus type 3 (HPIV3), cause the majority of childhood croup, bronchiolitis, and pneumonia worldwide. Infection starts with host cell receptor binding and fusion of the viral envelope with the cell membrane at the cell surface. The fusion process requires interaction of the two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). We have previously shown that viruses with an HN/F pair that is highly fusogenic in monolayers of immortalized cells due to mutations in HN’s secondary sialic acid binding site are growth impaired in differentiated human airway epithelium (HAE) cultures and in vivo. Here we have shown that adaptation of HPIV3 to growth in the lung is determined by specific features of HN and F that are different from those required for growth in cultured immortalized cells. An HPIV3 virus bearing a mutated HN (H552Q), which is fit and fusogenic in immortalized cells but unfit for growth in the lung, evolved into a less-fusogenic but viable virus in differentiated human airway epithelium. Stepwise evolution led to a progressive decrease in efficiency of fusion activation by the HN/F pair, with a mutation in F first decreasing the activation of F by HN and a mutation in HN’s secondary sialic acid binding site decreasing fusion activation further and producing a stable virus. Adaptation of HPIV3 to successful growth in HAE is determined by specific features of HN and F that lead to a less easily activated fusion mechanism.

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). CI-1 HN/F, like the HAE-adapted strain HN/F, fuses cultured cells little or not at all.…”

“…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).…”

“…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.…”

“…Infection and growth in vivo correlate with the HAE-adapted/CI constellation of fusion properties. In fact, the growth of the HPIV3 strains in vivo ranked in the opposite order of their fusogenicity in monolayer culture cells (19) and in the opposite order of the activation readiness of their fusion machineries. None of the strains with proficient fusion activities that grow efficiently in cultured monolayer cells succeed in vivo.…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…These cells express basal cell markers including cytokeratin 5 and the transcription factor p63 (Hackett et al, 2011a) and have been used as models of basal cells. al., 2010), mucociliary transport (Seagrave et al, 2012), airway toxicology (Mathis et al, 2013;Watson et al, 2010), electrophysiology (Hirsh et al, 2008), and bacterial and viral infection studies (Deng et al, 2014;Mitchell et al, 2011;Palmer et al, 2012;Ren and Daines, 2011;Ren et al, 2012). The MucilAir cultures have been characterized electrophysiologically and are fully functional: the activity of the main epithelial ionic channels, such as CFTR, EnaC, Na/K ATPase, etc.…”