Novel Flavivirus Attenuation Markers Identified in the Envelope Protein of Alfuy Virus

Abstract

Alfuy (ALFV) is an attenuated flavivirus related to the Murray Valley encephalitis virus (MVEV). We previously identified markers of attenuation in the envelope (E) protein of the prototype strain (ALFV₃₉₂₉), including the hinge region (E273-277) and lack of glycosylation at E154-156. To further determine the mechanisms of attenuation we assessed ALFV₃₉₂₉ binding to glycosaminoglycans (GAG), a known mechanism of flaviviruses attenuation. Indeed, ALFV₃₉₂₉ exhibited reduced binding to GAG-rich cells in the presence of heparin; however, low-passage ALFV isolates were relatively unaffected. Sequence comparisons between ALFV strains and structural modelling incriminated a positively-charged residue (K327) in ALFV₃₉₂₉ as a GAG-binding motif. Substitution of this residue to the corresponding uncharged residue in MVEV (L), using a previously described chimeric virus containing the prM & E genes of ALFV₃₉₂₉ in the backbone of MVEV (MVEV/ALFV-prME), confirmed a role for K327 in enhanced GAG binding. When the wild type residues at E327, E273-277 and E154-156 of ALFV₃₉₂₉ were replaced with the corresponding residues from virulent MVEV, it revealed each motif contributed to attenuation of ALFV₃₉₂₉, with the E327/E273-277 combination most dominant. These data demonstrate that attenuation of ALFV₃₉₂₉ is multifactorial and provide new insights for the rational design of attenuated flavivirus vaccines.

Keywords: Alfuy virus (ALFV); Murray Valley encephali-tis virus (MVEV); attenuation; flavivirus; glycosaminoglycan (GAG).

Figure 1

Heparin inhibition by of ALFV₃₉₂₉ and other small plaque ALFV derivatives. Viruses were incubated with heparin (50 µg/mL or 200 µg/mL) for 15 min prior to their addition to SW13 (A,C) or C6/36 (B,D) cell monolayers. Error bars indicate the standard deviation values calculated for each triplicate. DM stands for the double mutant which contains the MVEV sequence in both the hinge and glycosylation sites. Percentage inhibition of titre caused by heparin was calculated as follows: 100 − (TCID₅₀ in the presence of heparin/TCID₅₀ of mock treated control) × 100%. MVE/ALFVstr-K327Q-DM = MVE/ALFstr-CHO+/H^MVEV/K327Q, MVE/ALFstr-K327Q-Hinge = MVE/ALFstr-H^MVEV/K327Q.

Figure 2

Computer generated map of the surface of E protein Domain III. Surface map was generated by Swiss-Pdb-Viewer Ver.4.0.4. Electrostatic potential was calculated using the Poisson-Boltzmann method. Blue indicates a positive charge, while red indicates a negative charge.

Figure 3

Growth kinetics of glycosaminoglycans GAG mutants. Growth kinetics of Murray Valley encephalitis virus (MVEV), ALFV3929 MVE/ALFVstr and associated mutants on PS-EK (A), Vero (B) and SW-13 cells (C). Cells infected at an m.o.i of 0.1, viral titre determined by TCID₅₀ on C6/36 cells.

Figure 4

Kaplan-Meier survival curves of Swiss mice inoculated i.p. with 10⁵ infectious units with MVEV/ALFVstr and associated mutants. (n = 10) p-Value is 0.0195. Curves produced using GraphPad Prism Ver.5.0.