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. 2001 Aug;75(16):7692-702.
doi: 10.1128/JVI.75.16.7692-7702.2001.

Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein

R J Hurrelbrink  1 , P C McMinn
Affiliations

Attenuation of Murray Valley encephalitis virus by site-directed mutagenesis of the hinge and putative receptor-binding regions of the envelope protein

R J Hurrelbrink et al. J Virol. 2001 Aug.

Abstract

Molecular determinants of virulence in flaviviruses cluster in two regions on the three-dimensional structure of the envelope (E) protein; the base of domain II, believed to serve as a hinge during pH-dependent conformational change in the endosome, and the lateral face of domain III, which contains an integrin-binding motif Arg-Gly-Asp (RGD) in mosquito-borne flaviviruses and is believed to form the receptor-binding site of the protein. In an effort to better understand the nature of attenuation caused by mutations in these two regions, a full-length infectious cDNA clone of Murray Valley encephalitis virus prototype strain 1-51 (MVE-1-51) was employed to produce a panel of site-directed mutants with substitutions at amino acid positions 277 (E-277; hinge region) or 390 (E-390; RGD motif). Viruses with mutations at E-277 (Ser-->Ile, Ser-->Asn, Ser-->Val, and Ser-->Pro) showed various levels of in vitro and in vivo attenuation dependent on the level of hydrophobicity of the substituted amino acid. Altered hemagglutination activity observed for these viruses suggests that mutations in the hinge region may indirectly disrupt the receptor-ligand interaction, possibly by causing premature release of the virion from the endosomal membrane prior to fusion. Similarly, viruses with mutations at E-390 (Asp-->Asn, Asp-->Glu, and Asp-->Tyr) were also attenuated in vitro and in vivo; however, the absorption and penetration rates of these viruses were similar to those of wild-type virus. This, coupled with the fact that E-390 mutant viruses were only moderately inhibited by soluble heparin, suggests that RGD-dependent integrin binding is not essential for entry of MVE and that multiple and/or alternate receptors may be involved in cell entry.

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Figures

FIG. 1
FIG. 1
Growth kinetics of hinge (A) and RGD (B) mutant viruses in Vero cells. Monolayers were infected at a multiplicity of infection of 5, and samples of cell culture supernatant were collected at the times indicated. Viral titers were determined by plaque assay on Vero cells. All assays were performed in duplicate.
FIG. 2
FIG. 2
Growth kinetics of hinge (A) and RGD (B) mutant viruses in infected mouse brain. Mice were inoculated i.c. with 103 PFU of virus, and brains were collected at the times indicated. Viral titers were determined by plaque assay of 10% homogenates on Vero cells. All assays were performed in duplicate.
FIG. 3
FIG. 3
Inhibition of virus binding by soluble heparin. Rates of inhibition were calculated using the following formula: (a − b)/a, where a is the number of plaques on cells incubated with untreated virus and b is the number of plaques on cells incubated with heparin-treated virus. CDV-1-51 is shown in black, E-277 mutant viruses are in white, and E-390 mutant viruses are in gray. Average results from two separate experiments are shown.
FIG. 4
FIG. 4
Plaque phenotype of CDV-1-51v5 (D→N) after multiple passage in Vero cells. A third passage C6/36 stock was progressively passaged to generate Vero passage 1 (VP1) through passage 3 (VP3) stocks. (A) Original C6/36 P3 stock; (B) CDV-1-51v5 (D→N) VP1; (C) CDV-1-51v5 (D→N) VP2; (D) CDV-1-51v5 (D→N) VP3.
FIG. 5
FIG. 5
The hinge region of the MVE E protein, based on the known three-dimensional structure of the TBE E protein (60). In MVE, the polar interface linking domains I and II includes residues E-52 to E-54 (hinge 1 [green]), E-129 to E-136 (hinge 2 [orange]), E-191 to E-200 (hinge 3 [red]), and E-266 to E-284 (hinge 4 [yellow]). Hinges 1 through 4 correspond to regions D0-a, e-E0, H0-f, and αB-I0, respectively, according to the nomenclature described by Rey et al. (60). Residue E-277, the residue selected for SDM, is highlighted in black.
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