Circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection - PubMed (original) (raw)
Circulating clinical strains of human parainfluenza virus reveal viral entry requirements for in vivo infection
Samantha G Palmer et al. J Virol. 2014 Nov.
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 hemagglutinin-neuraminidase 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.
Importance: Acute 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.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Figures
FIG 1
The HPIV3 clinical isolate HN has the highest neuraminidase/avidity ratio, indicating the least engagement with receptor. (A) To measure relative avidity for receptor, cell monolayers transiently expressing reference strain HN (circles), HAE-adapted strain HN (squares), or CI-1 HN (open diamonds) were assayed by hemadsorption (HAD) (at 4°C) using a series of receptor-depleted RBCs, as described in Materials and Methods. The binding (y axis) at the indicated degree of receptor depletion, expressed as milliunits of neuraminidase treatment (x axis), is expressed as the percentage of binding obtained with undepleted RBCs. The results are means ± standard errors from triplicate experiments. (B) To measure relative neuraminidase activity, cell monolayers transiently expressing reference strain HN, HAE-adapted strain HN, CI-1 HN, or empty vector were assayed at 37°C at pH 5. These results are means ± standard errors from triplicate experiments and expressed as relative fluorescent units (RFU)/s (y axis).
FIG 2
The HN/F pairs of the lung fit variants are less efficient at promoting cell-cell fusion than the proteins of the laboratory reference virus, and the difference is mediated by both HN and F partners in the pair. 293T cells coexpressing the indicated HN and F pair were allowed to fuse with receptor-bearing cells at 37°C for 3 h. Cell fusion was measured as RLU (y axis; log scale) in the presence of each HN (as indicated on the x axis) coexpressed with reference strain F (dark bars), HAE-adapted strain F (gray bars), or CI-1 F (white bars). The bars represent means ± standard errors of results from three different wells in triplicate experiments. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01.
FIG 3
Activation of F over time at 45°C either alone or with HN (reference strain, HAE adapted, or CI-1). 293T cells transiently expressing influenza virus HA (A, B, C) or HN D216R (D, E, F) and either F from the reference strain, F from the HAE-adapted strain, or F from the clinical isolate (CI-1) were overlaid with RBCs at 45°C for 10, 30, or 60 min as indicated. The percentage of RBCs released (white bars), bound (gray bars), or fused (black bars) was quantified. The values are means ± standard deviations (SD) from triplicate samples from a representative experiment.
FIG 4
Inhibition of HPIV3 reference strain, HAE-adapted strain, and CI-1 infection by the cholesterol-tagged HPIV3 F HRC peptides in vitro. (A) Schematic of the monomeric cholesterol-tagged HPIV3 F HRC peptide, including its sequence derived from HPIV3 F amino acids 449 to 488, and the cholesterol tag attached to the C terminus by a maleimide (MAL) 4-unit polyethylene glycol (PEG 4) linker. (B) CV1 cell monolayers were infected with HPIV3 reference strain (circle), HAE-adapted strain (square), or CI-1 (open diamond) at a multiplicity of infection (MOI) of 6.7 in the presence of increasing concentrations of the PEG 4-cholesterol peptide. After a 90-min incubation at 37°C, cells were overlaid with Avicel, and plaques were stained and counted after 24 h. The percent inhibition of viral entry (compared to results for control cells infected in the absence of inhibitors) is shown as a function of the (log scale) concentration of peptide. Data points are means (±SD) from triplicate samples. These data are representative of results from three experiments.
FIG 5
Inhibition of CI-1 infection by lipid-tagged HPIV3 F HRC peptides in HAE cells and cotton rats. (A) HAE were infected with 4,000 PFU of HPIV3 CI-1 in the absence or presence of 0.1, 1, or 10 μM of PEG 4-cholesterol peptide. After a 90-min incubation at 37°C, the inoculum was removed and the HAE were incubated at 37°C. The virus released from the apical surface was collected at 1 day after infection. The viral titer (PFU/ml, log scale) on the y axis is shown as a function of the concentration of HPIV3 HRC peptide. Data points are means (±standard errors) of results from three separate experiments. (B) Groups of 4 cotton rats were infected with HPIV3 CI-1 and treated with 2 mg/kg/day of monomeric peptide and then sacrificed 3 days postinfection. Control animals were infected with the respective virus but not treated with peptide. Viral titer (PFU/g lung tissue) (y axis) was determined by plaque assay.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous