Structural proteins in ZIKV consist of Capsid, prM and Envelope proteins. Capsid protein contains 102 residues and forms icosahedral capsid (30 nm in diameter) of the virus (Kuno and Chang, 2007), where the genomic RNA of ZIKV is encapsulated. Disordered regions of capsid protein include its N- and C-termini, with an overall predicted percent of intrinsic disorder (PPID) of 33.3% calculated from outputs of four predictors used in our study (Table 1 and Figure 4A). These observations are consistent with the results of previous studies on the disorder predisposition of proteins in flaviviruses (Goh et al., 2016). In vitro studies have implicated the role of intrinsic disorder for chaperone-like activities, such as viral genome packaging (Ivanyi-Nagy et al., 2008). In the case of Dengue virus, detailed functional study of capsid protein revealed that its N-terminal disordered region is responsible for carrying out multiple interactions necessary for mature virus particle formation (Martins et al., 2012). Furthermore, disordered N-terminal region interacts with phospholipids of lipid droplets (Martins et al., 2012). Similar studies have correlated the role of capsid disorder with the diverse functions in other flaviviruses, such as YFV and WNV. (Ivanyi-Nagy and Darlix, 2010) In other flaviviruses, it was shown that high virulence is correlated with the disorder levels of capsid protein (Goh et al., 2016). We assume that high abundance of disorder in capsid of ZIKV may provide an insight to uncover the mechanism of pathogenesis of this virus.

Next to capsid is the protein known as prM that acts as a chaperone for envelope protein. prM protein shows a central role in transition of immature virus particle to mature form, which is infectious, virulent, fusogenic, and can adhere to host cell membrane (Zhang et al., 2003). Immature viral particles of flaviviruses are noninfectious and are characterized by their “spiky” shape, possessing 60 trimeric E-prM heterodimer spikes (Zhang et al., 2003). The mature viral particle is smooth and contains 90 dimeric E:M heterodimers (Kuhn et al., 2002; Zhang et al., 2013). The low-pH environment of the trans-Golgi network is crucial for the maturation of viral particles, since it leads to the conformational changes of the surface glycoproteins needed for the cleavage of prM by the host protease furin to generate the pr peptide and mature protein M (Kuno and Chang, 2007). Subsequent removal of the pr peptide leads to the exposure of the ~12–amino acid-long fusion loop on the E protein, which, in the immature virus, is protected by the pr peptide (Yu et al., 2008). With exposed fusion loop, the virus is prepared for the low pH–mediated endosomal fusion (Yu et al., 2008). Therefore, both proteins eventually generated from the prM are functionally important, where Pr peptide protects the fusion loop of the E protein, and M protein acts as a transmembrane protein in the mature viral particle (Yu et al., 2008).

ZIKV prM contains 168 amino acids. In addition to the disordered N-and C-termini there is a long disordered central region in prM located between residues 80 and 120. This region contains the furin cleavage site (see Figure 4B and Figure 3). A multitool computational disorder analysis shows that the overall disorder of prM is 19.0% (see Table 1). Since the proteolytic cleavage of prM generates two functionally important proteins, the pr peptide and M protein (Yu et al., 2008). The analysis of both proteins separately revealed the PPID of 16.1% and 24% for pr and M proteins respectively (see Table 1). Despite high level of predicted disorder, M protein is able to form stable structure, being complexed with the E protein (see Figure 4D). Structurally, ZIKV M protein is characterized by N-terminal soluble loop (M loop) which contain two short α-helices (residues 6–10 and 21–39) and two transmembrane α-helices (residues 40–52 and 56–71) connected by very short loop that forms the stem and the transmembrane part of this protein embedded in the lipid bilayer. (Liu et al., 2004; Sirohi et al., 2016). Curiously, although the soluble M loop is predicted to be mostly disordered and contains little regular secondary structure in the E-M complex, it is crucial for stabilization of the E-M dimer, being intercalated into the E protein structure. Therefore, it is likely that this region of the M protein undergoes functional disorder-to-order transition.

At the next step, we analyzed the envelope protein E (500 residues) that participates in the membrane fusion between host late endosomes and virion. Protein E heterodimerizes with M, and this complex stabilizes E protein likely due to the chaperone-like activity of M (Hamel et al., 2015). The averaged PPID of 7.6% is observed in E protein (Table 1), which is rather low. However, several IDPRs of different length are predicted in the ZIKV E protein (Figure 4C). Viral capsid consists of 180 copies each of the E glycoprotein and the M protein anchored in a lipid membrane (Sirohi et al., 2016). Structurally, E protein consists of four domains; stem transmembrane domain and three ectodomains t external surface of the viral capsid and are mostly β-structural (see Figure 4D).

Previous studies on other flaviviruses (e.g., Dengue virus and West Nile virus) demonstrated that glycosylation of the E protein at specific sites provides the ability to attach to different cell types (Beasley et al., 2005; Pokidysheva et al., 2006; Miller et al., 2008). Cryo-EM structure of mature ZIKV viral particle revealed that the glycosylation site is located at the loop region nearby the fusion peptide (Sirohi et al., 2016). Besides being needed for pH-mediated endosomal fusion, the ZIKV fusion loop (residues 98-110) can act as an epitope and react with different antibodies. Figure 4C shows that although this region is predicted to be mostly ordered, it has some weak tendency for the presence of disorder-based binding sites as evidenced by the output of the ANCHOR algorithm. In fact, the ANCHOR-based analysis of protein E revealed the absence of disordered binding regions that function via undergoing a disorder-to-order transition upon binding to a globular protein partner. However, the fusion loop is located in close proximity to the fifth highest spike found within the ANCHOR profile of protein E (see Figure 4C). Furthermore, our disorder analysis revealed that the longest disorder region found in ZIKV E protein (located within the 120-180 region) contains the glycosylation site (Asn154) (Sirohi et al., 2016). High disorder propensity of the loop encompassing the glycosylation site is in line with the reported sequence variability of this region among ZIKV strains (Faye et al., 2014) and in other flaviviruses (Sirohi et al., 2016), suggesting that local structural dynamics and sequence variability could be of functional importance for this protein. In fact, sites of various enzymatically-catalyzed posttranslational modifications in proteins are commonly located within their IDPRs (Iakoucheva et al., 2004; Pejaver et al., 2014). Therefore, finding intrinsic disorder in functionally important regions of the ZIKV E protein signifies the potential therapeutic importance of its IDPRs.

The glycoprotein NS1 is considered as a key molecule in replication, immune evasion and pathogenesis of flaviviruses. NS1 is also secreted out into extracellular space as hexameric lipoprotein particles that are involved in multiple interactions with various components of immune system and host cell molecules (Suthar et al., 2013). In fact, it is believed that NS1 can be responsible for the diverse clinical consequences of infection caused by flaviviruses (Kuno et al., 1998; Cheng et al., 2009). Recently, the crystal structure of a C-terminal fragment of ZIKV NS1 (PDB ID: 5IY3) has been solved (Song et al., 2016). The sequence of this crystallized C-terminal domain (residues 176-351) along with the sequence of the full-length NS1 protein was used in our disorder analysis. This analysis revealed that the full-length NS1 is characterized by the PPID of 10.8% (see Figure 5A and Table 1), whereas the crystallizable C-terminal fragment (PDB ID: 5IY3) shows the PID of 14.1%. Structurally, NS1 is arranged in a rod-like head to head dimers. Each dimer has 20 β-strand showing ladder-like arrangement on one surface and a complex arrangement of several loops on an opposite surface (see Figure 5B) (Song et al., 2016). Typically, loops between the β-strands are short, with the exception to the spaghetti loop connecting strands β4 and β5 (residues 218–272), which lacks regular ordered structure and is predicted to contain significant amount of disorder (see Figure 5A). Furthermore, a potential glycosylation site is located within the short loop between the β3 and β4 strands (residues 207–209), which is also predicted to have high level of disorder. Therefore, the high abundance of disorder in NS1 is correlated with the complex arrangement of loops in 3D crystal structure (Song et al., 2016) (see Figure 5B) and has functional significance. A phylogenetic analysis based on the amino acid sequences of NS1 proteins from 10 flaviviruses revealed some unique sequence characteristics of ZIKV NS1 that positioned it in the individual phylogenetic group (Song et al., 2016). It was emphasized that there is a very large variability in positively and negatively charged surfaces in central loop regions of the NS1 proteins from DENV, WNV, and ZIKV (Song et al., 2016). This variability in loop regions of flaviviruses may be implicated in the diversity of their pathogenicity. In Dengue and WNV, these loops of NS1 have been implicated in immune pathogenesis, whereas in ZIKV, this mechanism needs to be explored (Suthar et al., 2013).

Non-structural proteins from NS2 to NS5 are involved in the formation of replication complex which is located on the endoplasmic reticulum membrane. NS2A possesses several important functions, such as involvement in viral RNA synthesis, virus-induced membrane formation, and inhibition of interferon α/β response (Xie et al., 2015). Table 1 shows that, being the most ordered of the ZIKV proteins, NS2A has the PPID of 5.4%. However, NS2B is on the other side of spectrum, being characterized by a high abundance of disordered residues with PPID of 16.2 % (Figures 5C,D). Figure 5D shows that NS2B contains a central long disordered region of 37 residues (residues 62-98). This region is responsible for interaction with the NS3 protease (Murray et al., 2008). In the case of DENV it was shown that a central hydrophilic region of NS2B binds to NS3 and is needed for the formation of an active NS2B-NS3 protease complex. NS2B also stabilizes NS3 by acting as a chaperone. In previous studies on other members of the Flaviviridae family (Dengue virus and WNV), the structure of NS2B in a complex with NS3 was determined (Erbel et al., 2006). Curiously, recent crystallographic analysis of the hybrid protein constituting a central of the ZIKV NS2B (residues 49–95) covalently linked via the Gly₄-Ser-Gly₄ artificial linker to the N-terminal protease domain of NS3 (residues 1–170) revealed that the NS3-binding region of NS2B wraps around the globular NS2B domain (see Figure 5E; PDB ID: 5LC0) (Lei et al., 2016). This structure clearly indicates that this NS2 region is disordered in its unbound form and folds upon binding to the globular protease domain of ZIKV NS3 protein. In the perspective of folding upon binding found for many IDPs, this seems to be a similar mechanism where protein acquires active conformation and function only by binding disordered partner as also evidenced in case of the model IDP systems such as KIX and cMyb (Gianni et al., 2012).

NS3 is a bifunctional enzyme that consists of two domains, such as the N-terminal protease domain (residues 1–167) and the C-terminal helicase domain (residues 168–617), which are essential for the polyprotein processing and the viral replication, respectively (Luo et al., 2015). NS3 consists of 617 residues, 12.6% of which are predicted to promote disorder. The longest disordered region found in this protein consists of 57 residues (residues 191–247) and is located within the helicase domain (Figure 6A). According to a recent report, Dengue virus NS3 contains the N-terminal proline-rich disordered region that plays a critical role in replication and virus particle formation (Gebhard et al., 2016). As it was already mentioned, the ZIKV NS3 protease domain (residues 1–170) was recently crystallized as a part of the hybrid protein containing the NS3-binding region of the ZIKV NS2B covalently linked by the Gly₄-Ser-Gly₄ peptide to N-terminus of the NS3 protein. No structural information was obtained for the 31 residue-long region connecting NS2B and NS3 and containing C-terminal tail of the NS2B, the Gly₄-Ser-Gly₄ linker, and the N-terminal 14 residues of the ZIKV NS3 containing the aforementioned proline-rich region (see Figure 5E; PDB ID: 5LC0) (Lei et al., 2016). Remaining part of the NS3 protease domain (except to its last 3 residues) was well-resolved and represents two β-barrels with strand orders AI-BI-CI-αI-DI-EIa-EIb-FI and AII-BIIa-BIIb-CII-DII-EIIa-EIIb-FII (Lei et al., 2016). Recently, a crystal structure of the NS3 helicase domain of ZIKV has been solved (PDB ID: 5JMT) (Tian et al., 2016) (see Figure 6B). In contrast to the DENV NS3 helicase, ZIKV has a monomeric helicase molecule. NS3 helicase is characterized by mostly ordered tertiary structure, with loops serving as connectors between the three domains of this protein (Tian et al., 2016). It was pointed out that the cleft between Domain I and II contains the NTPase active site and includes Walker A (or motif I or P-loop, residues 193–204) and B motifs (or motif II, residues 285–292) that play an important role in recognizing NTP and Mn²⁺ or Mg²⁺ cations (Caruthers and McKay, 2002; Tian et al., 2016). Figure 6A shows that both of these motifs are located within IDPRs. Although these loops are characterized by the considerable intrinsic flexibility, it was pointed out that they are highly conserved among flaviviruses and play an important role in binding and catalysis of NTP (Tian et al., 2016). Curiously, NS3 is one of the two ZIKV proteins that are predicted to have some disorder-based binding sites identified by ANCHOR. In fact, Figure 6A shows that there are 6 such sites in ZIKV NS3 (residues 1–4, 79–81, 147–150, 261–264, 311–314, and 374–376) which although were predicted by ANCHOR but then were filtered out by the algorithm because of their small size (they are ranging in length between 3 and 4 residues and ANCHOR uses the length threshold of 6 residues). Nevertheless, our analysis shows that ZIKV NS3 has several functionally important IDPRs.

Although non-structural proteins NS4A and NS4B from flaviviruses have not been crystallized as of yet, their biological roles in Dengue virus have been investigated experimentally. (Zou et al., 2015) NS4A functions by introducing rearrangements in membrane of host endoplasmic reticulum. These changes lead to the formation of virus-induced membranous vesicles. NS4A also control the ATPase activity of the NS3 helicase, whereas NS4B inhibits the interferon-induced host STAT1 phosphorylation and nuclear translocation (Kuno and Chang, 2007). Table 1 and Figures 6C,D show that although NS4A is predicted to be rather disordered, having the PPID of 16.5%, the level of intrinsic disorder in the NS4B protein is noticeably lower (its PPID is of 6.4%).

The NS5 protein is the largest ZIKV protein (903 residues) and has high degree of sequence similarity with the Dengue virus NS5 protein. In DENV, crystal structure of the full-length NS5 has been solved recently (PDB ID: 4V0Q) (Zhao et al., 2015), and structures of the methyltransferase domain (PDB ID: 1L9K) (Egloff et al., 2002), and the RNA-dependent RNA polymerase (RdRp) domain (PDB ID: 2J7W) of the NS5 (Yap et al., 2007) are also available. The NS5 is the most conserved protein amongst flaviviruses, exhibiting enzymatic activities that play vital roles in virus replication. In fact, it has the N-terminal domain (residues 1–262 in DENV3) that belongs to the S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase) superfamily (Egloff et al., 2002) and the C-terminal domain (residues 273–900) which serves as the RNA-dependent RNA polymerase (RdRp) that synthesizes the anti-genome and progeny genome (Yap et al., 2007). Since NS5 contains two functional domains with several key enzymatic activities crucial for the viral RNA replication in the host cell, this protein represents one of the major targets for the design of antiviral inhibitors. Our analysis revealed that despite being the multifunctional enzyme, ZIKV NS5 contains numerous IDPRs (in a range of 20) and is characterized by the PPID of 8.6%. Furthermore, ANCHOR analysis revealed the presence of 4 disorder-based binding sites (residues 22–27, 179–186, 324–329, and 377–383), with two more disorder-based binding sites that were filtered out by the algorithm (residues 476–484 and 846–849). Our analysis also revealed that most of the NS5 disordered regions are located in the central part of the protein, with the longest disordered region containing 40 amino acid residues (see Figure 6E). These findings of NS5 are also correlating well with the presence of long disordered region in DENV and JEV NS5 proteins.