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

Abstract

Human parainfluenza viruses (HPIVs) cause widespread respiratory infections, with no vaccines or effective treatments. We show that the molecular determinants for HPIV3 growth in vitro are fundamentally different from those required in vivo and that these differences impact inhibitor susceptibility. HPIV infects its target cells by coordinated action of the hemagglutininneuraminidase receptor-binding protein (HN) and the fusion envelope glycoprotein (F), which together comprise the molecular fusion machinery; upon receptor engagement by HN, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. Peptides derived from key regions of F can potently inhibit HPIV infection at the entry stage, by interfering with the structural transition of F. We show that clinically circulating viruses have fusion machinery that is more stable and less readily activated than viruses adapted to growth in culture. Fusion machinery that is advantageous for growth in human airway epithelia and in vivo confers susceptibility to peptide fusion inhibitors in the host lung tissue or animal, but the same fusion inhibitors have no effect on viruses whose fusion glycoproteins are suited for growth in vitro. We propose that for potential clinical efficacy, antivirals should be evaluated using clinical isolates in natural host tissue rather than lab strains of virus in cultured cells. The unique susceptibility of clinical strains in human tissues reflects viral inhibition in vivo. IMPORTANCEAcute respiratory infection is the leading cause of mortality in young children under 5 years of age, causing nearly 20% of childhood deaths worldwide each year. The paramyxoviruses, including human parainfluenza viruses (HPIVs), cause a large share of these illnesses. There are no vaccines or drugs for the HPIVs. Inhibiting entry of viruses into the human cell is a promising drug strategy that blocks the first step in infection. To develop antivirals that inhibit entry, it is critical to understand the first steps of infection. We found that clinical viruses isolated from patients have very different entry properties from those of the viruses generally studied in laboratories. The viral entry mechanism is less active and more sensitive to fusion inhibitory molecules. We propose that to interfere with viral infection, we test clinically circulating viruses in natural tissues, to develop antivirals against respiratory disease caused by HPIVs.

Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures

“…CX3CR1 is expressed in epithelial cells, smooth muscle cells, microglia, neurons, T cells, monocytes, dendritic cells, and NK cells [47][48][49][50][51][52][53]. RSV is able to infect nearly all immortalized cell lines and infects primary epithelial cells, smooth muscle cells, neuronal cells, eosinophils, and dendritic cells [19,40,54,55]. Here we found that CX3CR1 is detectable on the cilia of ciliated cells in HAE cultures, the cell targeted by RSV [19].…”

“…Differences in measles virus (MV) receptor usage between clinical isolates and laboratory strains have been well documented to be due to selection during growth in immortalized cells. SLAM (CD150) is used as a receptor by clinical isolates [40], but not by the laboratory adapted Edmonston strain of MV which uses CD46 [56]. Because mAb 131-2g blocks G protein binding to CX3CR1, our finding that mAb 131-2g neutralizes infection of clinical isolates for HAE cultures (Fig 1D) indicates that the use of CX3CR1 is not an artifact of using a laboratory strain of RSV.…”

“…For other paramyxoviruses, receptor binding differs between laboratory adapted strains and clinical isolates [39,40]. To confirm that mAb 131-2g neutralization of rgRSV (subgroup A) in HAE cultures is not an artifact of using a laboratory adapted virus, we examined the neutralizing ability of this mAb for a low-passage subgroup A clinical isolate, RSV A2001/2-20 (2-20) [41].…”

On the Stability of Parainfluenza Virus 5 F Proteins

“…The attachment protein's F-activating region then interacts with an exposed hydrophobic loop in the IgG-like domain of F to provide the energy needed to promote F refolding and membrane fusion (4,5). This energy barrier to the activation of F can also be overcome by increased temperature (6)(7)(8)(9)(10) or by the introduction of destabilizing point mutations (11)(12)(13). The paramyxovirus parainfluenza virus 5 (PIV5) WR isolate differs in sequence from the W3A isolate by only three residues: the L22, P443, and A516 in WR are P22, S443, and V516 in W3A (14).…”

Hijacking the Fusion Complex of Human Parainfluenza Virus as an Antiviral Strategy

“…F itself influences the extent and timing of fusion through its prefusion stability, kinetics of activation, and precursor cleavability. Fusion is moderated through a balance of these functions of HN and F. While viruses bearing the fusion complex of clinical strains grow efficiently in both the human airway and in vivo models, they fail to grow on immortalized cells, demonstrating the profound specificity of the HN/F complex for the authentic host (28,30).…”