A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation

Abstract

West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo. Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains. IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus. However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.

Molecular Determinants of West Nile Virus Virulence and Pathogenesis in Vertebrate and Invertebrate Hosts

“…The table details residues involved in virulence variation in vivo and in vitro and shared by at least two flaviviruses. TBEV: tick-borne encephalitis virus; DENV: dengue virus; ZIKV: zika virus; YFV yellow fever virus; JEV: Japanese encephalitis virus; MVEV: Murray valley encephalitis virus [92,97,99,102,[106][107][108]112,113,116,177,. [112,116,187,190] DENV G102S, F108A [191] Domain III (receptor binding site) JEV E306K…”

“…Some studies have shown that mutations in the protein M could produce attenuated flaviviruses, such as JEV [96]. A recent study of Basset et al [97] investigated the effect of protein M mutation in WNV. The authors generated WNV mutants by PCR from an IC of WNV Israel 98 (IS98) [3], which is genetically closely related to the NY99 strain.…”

“…influences binding to glycosaminoglycans (residues 325-326 and 380) or other cell receptors, modulates the efficacy of virus spread, neuroinvasiveness and neurovirulence -diminishes infection rates in Aedes aegypti mosquitoes (YFV), not studied in Culex mosquitoes [192] YFV, DENV S305F [181,[193][194][195] TBEV D308K [197] JEV A315V [198] YFV S325P, E326K/R [193,195,196] YFV, MVEV R380T [193,194,199,200] NS1 N-glycosylation site DENV N130A + N208A N130 + N207 ablation of the first glycosylation site (N130) decreases replication, viral production and neurovirulence and diminishes vector competence of Culex tarsalis mosquitoes for WNV [177,198] YFV N130A + N208A Decreased replication and neurovirulence (1st glycosylation site) [116,201,202] 3 It would also be a good starting point to compare the virulence and pathogenesis of the WNV with those of other flaviviruses of interest, such as the dengue and yellow fever viruses [97]. It is noteworthy that the study of flavivirus molecular determinants has been driven by the identification in the field or by artificial selection of mutants with properties of interest, such as attenuation, neuroadaptation, and escape from neutralization.…”

Neurotropic Viruses, Astrocytes, and COVID-19

“…Animal experiments have confirmed that a vaccine derived from a chimeric virus, which was constructed using the structural proteins (M and E) of the Kunjin WNV strain and the genome backbone of the insect-specific Binjari virus, offered efficient protection against virulent WNV strains, including the highly pathogenic WNV NY 99 strain ( Vet et al, 2020 ). In the WNV, the structural proteins E and M are responsible for binding to receptors, e.g., the dendritic cell- and liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrins (DC-SIGN and L-SIGN, respectively), and have been associated with flavivirus-induced pathogenesis ( Khoo et al, 2008 ; Basset et al, 2020 ). Hence, their function in the WNV is to some extent similar to the function of the spike (S) protein of SARS-CoV-2 ( Huang et al, 2020 ).…”

The key amino acids of E protein involved in early flavivirus infection: viral entry

“…A common strategy for obtaining a live vaccine is to pass the isolated wild strains in serial passages to generate attenuated strains with mutations in certain residues, and the attenuated strains are candidates for vaccines [ 71 , 72 ]. Usually, attenuated strains will differ from the wild type in many ways, for example, by influencing the secretion of the virus and decreasing viremia levels and the efficiency of replication in major target organs [ 60 , 73 ]. JEV attenuated strain SA14-14–2 (JEV SA14-14–2) is a vaccine strain with good protection effectiveness and safety [ 74 , 75 ].…”