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 (Ser3Ile, Ser3Asn, Ser3Val, and Ser3Pro) 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 (Asp3Asn, Asp3Glu, and Asp3Tyr) 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.
Murray Valley encephalitis virus (MVE) is a member of theFlavivirus genus (family Flaviviridae) and is a small, lipid-enveloped virus which contains a single-stranded positive-sense RNA genome. The genome is approximately 11 kb in length and contains a single open reading frame which is posttranslationally cleaved to generate three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. Viral genomic RNA also has a methylated cap at its 5Ј terminus and forms a highly conserved stem-loop structure at its 3Ј end (61). As for many flaviviruses, MVE causes clinically significant disease in humans and, together with Kunjin (KUN) virus, is responsible for almost all cases of flaviviral encephalitis in mainland Australia (41).In recent years, infectious cDNA clones have been produced for a number of flaviviruses, including MVE (31, 39), enabling manipulation of the genome at the nucleotide level. Such clones have been used to examine the glycosylation, cleavage, and function of the prM and E (4,20,28,33,55,57,68), NS1 (53, 55, 57), NS2B/NS3 (9, 10, 54), and NS5 (34, 35, 36) proteins, as well as to generate viruses with deletions in their 5Ј and 3Ј untranslated regions (6,38,43,48). More recent work has seen the generation of chimeric yellow fever viruses (YF)...