Ionic Strength-Dependent, Reversible Pleomorphism of Recombinant Newcastle Disease Virus

Abstract

A genetically modified, recombinant form of Newcastle disease virus (rNDV) undergoes ionic strength-dependent changes in morphology, as observed by cryo-electron microscopy (cEM). In hypotonic solutions with ionic strengths ranging from < 0.01 to 0.02 M, rNDV virions are spherical or predominantly spherical. In isotonic and hypertonic solutions, rNDV displays pleomorphism and contains a mixed population of spherical and elongated particles, indicating that a change from spherical to elongated shape is induced with increasing salt concentration. This ionic strength-dependent transition is largely reversible, as determined by cEM. Concomitantly, we measured infectious titers of these same rNDV samples at different ionic strengths using a fluorescent focus assay (FFA). The infectivity of oncolytic rNDV was found to be independent of ionic strength, ranging from 0.01 M to approximately 0.5 M. These structural and functional observations, in combination, suggest that infectivity (and, by inference, oncolytic activity) of rNDV virions is fully maintained in their pleomorphic forms.IMPORTANCE Oncolytic viruses are being developed for cancer therapy, as they selectively target, infect, and kill cancer cells. NDV is particularly attractive because while it is pathogenic to avians (e.g., chickens), it does not cause significant viremia in humans. We have developed a genetically modified recombinant NDV (rNDV) that has much reduced pathogenicity in chickens but is highly oncolytic. The morphology of rNDV transitions from spherical at very low salt concentrations to a heterogeneous population of spherical and elongated virions in isotonic (physiologic salt concentration) and hypertonic solutions. The infectivity (cell-killing activity by infecting cells) of rNDV is unaltered by changes in salt concentration despite morphological changes. These observations are significant for purification and formulation of rNDV, as exposure to different salt concentrations may be needed. Importantly, at physiological salt concentration, relevant to clinical testing, infectivity and, therefore, oncolytic activity will not be compromised despite morphological heterogeneity.

Keywords: Newcastle disease virus; cryo-electron microscopy; fluorescent focus assay; genetically modified; infectious titer; ionic strength; oncolytic activity; pleomorphism; recombinant; recombinant Newcastle disease virus; reversible; virion morphology.

FIG 1

Cryo-EM images (×52,000 magnification) of rNDV samples incubated in different salt concentrations and ionic strengths (μ) while maintaining the pH at 7.0. (A) 6.5 mM sodium phosphate, 150 mM NaCl, μ = 0.160 M; (B) 5.3 mM sodium phosphate, 3.0 mM NaCl, 0.2% sucrose, μ = 0.011 M; (C) after incubation at μ = 0.011 M. As in panel B, the rNDV sample was incubated in 5.8 mM sodium phosphate, 258 mM NaCl, 0.06% sucrose, μ = 0.275 M. The purpose was to test if the ionic strength-dependent change in morphology was reversible between spherical and elongated shapes.