**1. Introduction**

Viral hemorrhagic fever is a global problem, with most cases due to dengue virus (DENV), which originates over 390 million infections per year worldwide, being a major socio-economic burden, mainly for tropical and subtropical developing countries [1]. A working vaccine was registered in Mexico in December 2015, approved for official use in some endemic regions of Latin America and Asia and, as of October 2018, also in Europe [2–4]. However, this vaccine is not 100% effective against all DENV serotypes. Thus, research into new prophylactics is still ongoing, with a new vaccine proposed

recently being now in phase 3 clinical trials [5]. In spite of these recent developments, fully effective prophylactics approaches are lacking and there are no effective therapies. This is in part, due to a poor understanding of key steps of the viral life cycle.

There are four dengue serotypes occurring: DENV-1, DENV-2, DENV-3 and DENV-4 [6]. Here, if not otherwise indicated, DENV refers to DENV-2. DENV is a member of the *Flavivirus* genus, part of the Flaviviridae family, a genus which comprises 53 viral species [6]. Many of these are important human pathogens as well, such as hepatitis C (HCV), tick-borne encephalitis (TBEV), yellow fever (YFV), West Nile (WNV) and Zika (ZIKV) viruses [6–9]. Flaviviridae are single-stranded positive-sense RNA viruses with approximately 11 kb, containing a single open reading frame [10]. Using the host cell translation machinery, the *Flavivirus* RNA genome is translated into a polyprotein that is co- and post-translationally cleaved by cellular and viral proteases into three structural proteins and seven non-structural proteins [10]. Structural proteins are named as such since they are present in the mature virion structure [11]. Nevertheless, they may also have non-structural roles, such as the capsid (C) protein. This is a structural protein that also mediates viral assembly and encapsidation, crucial steps of the viral life cycle. Given the C protein key roles, it is the focus of this work and will be described in detail below.

DENV C contains 100 amino acid residues, which form an homodimer with an intrinsically disordered protein (IDP) region in the N-terminal followed by four α-helices, α1 to α4, per monomer [12]. Overall, the main structural/dynamics regions consist of the disordered N-terminal, a short flexible intermediate fold and, finally, a large conserved fold region, which greatly stabilizes the protein homodimer structure [12–16]. The C protein has an asymmetric charge distribution: one side of the dimer contains a hydrophobic pocket (α2–α2 interface), responsible for, alongside the disordered N-terminal, the binding to host lipid droplets (LDs) [12–16]. The other is the positively charged C-terminal side (α4–α4 interface), proposed to mediate the C protein binding to the viral RNA [12]. It is noteworthy that several transient conformations for DENV C N-terminal were proposed, which may help modulate DENV C interaction with host lipid systems, via an autoinhibition mechanism [15].

DENV infection affects the host lipid metabolism, increasing host intracellular LDs and unbalancing plasma lipoprotein levels and composition [17–19]. Importantly, DENV C binds LDs, an interaction essential for viral replication [18,20]. DENV C-LDs binding requires potassium ions, the LDs surface protein perilipin 3 (PLIN3) and involves specific amino acid residues of DENV C α2–α2 helical hydrophobic core and of the N-terminal [14,20]. This knowledge led us to design pep14-23, a patented peptide, based on a *Flavivirus* C protein conserved N-terminal motif. We then established that pep14-23 inhibits DENV C-LDs binding [14], acquiring α-helical structure in the presence of anionic phospholipids [15]. Moreover, we also found that DENV C binds specifically to very low-density lipoproteins (VLDL), requiring K<sup>+</sup> ions and a specific VLDL surface protein, apolipoprotein E (APOE), being also inhibited by pep14-23 [21]. This is analogous to DENV C-LDs interaction. The similarities between APOE and PLIN3 further reinforce this, suggesting a common mechanism [22]. The role of LDs in *Flavivirus* infection is well known and has been recently reviewed [14,18,20,23–25]. Given that, pep14-23 is an excellent drug development lead. Further developments require a better understanding of the function of the C protein of dengue and of *Flavivirus* in general.

Therefore, here, we seek to contribute to understand the C proteins biological activity, with a special focus on DENV C. Briefly, we studied DENV C structure-activity relationship in the context of similar and highly homologous mosquito-borne *Flavivirus* C proteins. Our findings shed light into the structure-function relationship behind the C protein biological roles, which may contribute to future therapeutic approaches against DENV and closely related *Flavivirus*.
