**1. Introduction**

Mosquito-borne Zika (ZIKV), dengue (DENV), Yellow fever (YFV) and Japanese encephalitis (JEV) viruses belonging to flavivirus genus (*Flaviviridae* family), are four enveloped RNA viruses of significant public health concern worldwide [1–4]. Recent ZIKV global outbreaks, with Brazil at the epicentre, highlighted how a previously neglected flavivirus can turn into a severe threat for human health. While human ZIKV infections remained only sporadic and with a limited impact for decades [5–8], recent outbreaks revealed that ZIKV caused clusters of severe congenital and neurological abnormalities in infants and peripheral nervous system impairments in adults [9–12]. Considering the dramatic increase of severe human cases, strategies to e fficiently control this virus, either in terms of antiviral therapies or vaccines, are urgently needed and a granted requirement for more extensive studies.

Flaviviruses contain a genomic single-stranded positive RNA encoding a single large polyprotein that is subsequently cleaved by cellular and viral proteases into three structural proteins (C, prM/M and E) and seven nonstructural proteins (NS1 to NS5). The latter are responsible for virus replication, assembly and escape from host immune system, while structural proteins form the viral particle surrounding genomic viral RNA. Among structural proteins, the E protein is responsible for viral entry into host cells. Viral E protein first binds to cellular attachment factors and receptors, leading to virion internalisation primarily through a clathrin-mediated endocytic pathway [13]. In endosomes, fusion of viral and cellular membranes occurs after E protein conformational changes triggered by low pH [14]. The E protein peptide chain folds into three distinct domains: a central ß-barrel (domain EDI), an elongated dimerization region (domain EDII), which includes the fusion loop, and a C-terminal, immunoglobulin-like module (domain EDIII) [15]. Most flavivirus E proteins are post-translationally modified by addition of a single N-linked oligosaccharide on residue N-154 located within the EDI-loop [16]. Flavivirus E proteins represent one of the key determinants for viral pathogenesis. Flavivirus envelope supports virus tropism and single amino-acid changes can redirect virus tropism [17]. Flavivirus E proteins also represent a major target for neutralizing antibodies but, at the same time, can be involved in enhancement/cross-reactivity of reactive antibodies [18–21].

Recently, our studies on chimeric ZIKV clones between an epidemic Brazilian strain of ZIKV BeH819015 (hereafter called BR15) and a historical African strain MR766 highlighted an important role of two structural proteins prM/M, and E in ZIKV ability to infect human cells [16,22–24]. We further showed that they contribute to the initiation of viral infection. Analysis of chimeric viruses indicated that C-prM region plays a role in triggering cell death by ZIKV and E protein is associated with viral attachment to host cells during early infection [23,24]. Flavivirus E proteins usually contain two *<sup>N</sup>*-glycosylation sites at position E-56 and E-154. The first site is lacking in ZIKV E protein and contribution of the second site in ZIKV viral cycle, including in the mosquito vector, has been recently highlighted [25]. N-linked glycosylation of the E protein was shown to be an important determinant of ZIKV virulence in a mouse model of viral encephalitis [22,26]. In invertebrate vectors, ZIKV bearing an unglycosylated E protein was attenuated in its capacity to replicate in *Aedes aegypti* [27]. Although a *<sup>N</sup>*-glycosylation site is highly conserved among flaviviruses, which suggests of its biological importance, E proteins could remain unglycosylated as it has been observed in some ZIKV strains. To date, the exact mechanism by which the *<sup>N</sup>*-glycosylation motif region of the E protein contributes to ZIKV infectivity still remains elusive. In the present study, we further characterised structural protein contribution in ZIKV infectivity by focusing on the ZIKV E protein. Our goal was to determine whether three E residues—Ile152, Thr156, and His158 (hereafter called as E-152/156/158 residues)—which surround the Asn154 composing the *<sup>N</sup>*-glycosylation site NDT, may have any e ffect on ZIKV's ability to infect human cells.
