Zika virus (ZIKV) harbours a single-stranded positive-sense RNA genome, encoding a polyprotein cleaved into three structural (C, PrM/M, E) and seven non-structural proteins (NS1, NS2A-B, NS3, NS4A-B, and NS5), forming the replication complex on the endoplasmic reticulum membrane.1–9
In the investigated NS1, NS3, and NS5 proteins, regions with high probability scores in epitope prediction coincide with observed responses in the membrane (Fig. 5). NS1, the initial protein in membrane synthesis, is noteworthy for its multifactorial virulence function in ZIKV and conservation across flaviviruses. It contains 12 cysteines and undergoes glycosylation similarly to other NS proteins 20–24
Protein NS1, a primary antigenic marker of viral infection, is implicated as a key genetic determinant influencing diverse clinical outcomes among Flaviviruses, which consist of over 70 members. Despite this, our understanding of NS1 in Zika virus (ZIKV) remains limited due to its unique pathogenesis.20,24–27
NS1 interacts with both innate and adaptive immune components, contributing to immune evasion and pathogenesis. Its antigenic significance positions it as a potential early biomarker for ZIKV detection. NS1's molecular mechanisms resemble those of Dengue virus (DENV) and West Nile virus (WNV), hinting at its coding sequence as a potential determinant of Flavivirus clinical variability.20–25
Intracellular glycosylated NS1 associates with lipids, forming homodimers crucial for late-stage ZIKV replication. Furthermore, NS1 undergoes secretion as a hexameric lipoprotein particle, actively contributing to immune evasion and pathogenesis by interacting with host factors involved in innate and adaptive immune responses.20,25
NS1, a major antigenic marker, is proposed, along with other markers, as a biomarker for early viral infection detection. While NS1's molecular mechanisms are well-established for Dengue virus (DENV) and West Nile virus (WNV), its coding sequence is suspected to be a significant genetic determinant contributing to diverse clinical outcomes observed in flaviviruses.20
The Flavivirus NS1 protein comprises three distinct domains: a hydrophobic β-roll N-terminal chain, an alpha/beta wing, and a C-terminal central β-pleated portion. A flat cross-shaped dimer forms the fundamental unit, featuring hydrophobic and glycosylated polar faces.26 Spot-synthesis experiments in this study revealed regions of NS1 with a correlation between epitope prediction scores and membrane spot intensity (Pep 01), displaying significant immunogenicity.
Comparing the relative intensities in mothers and newborns, a similar recognition pattern is observed for domain two, albeit with lower signal intensity. This likely stems from antibody transmission from mother to baby, primarily through transplacental and breastfeeding routes, where IgA antibodies predominate, with a lesser presence of IgG and IgM.26,28,29
NS3 protein exhibits notable immunogenic potential in present experiments, characterized by numerous regions with high solvent accessibility and elevated scores in both secondary structure and epitope predictions. Several membrane-reactive regions in NS3 coincide with the predicted epitopes, highlighting their correspondence. Additionally, distinct regions with high membrane reactivity were identified, yet did not align with the predicted epitopes.
The NS3 protein plays a pivotal role in Zika virus (ZIKV) replication, possessing enzymatic functions critical for viral polyprotein processing. While the rationale for housing these diverse catalytic activities within the NS3 protein remains enigmatic, their evolutionary conservation across the Flaviviridae family hints at a crucial functional significance.25
Flaviviral NS3 harbours a dedicated protease domain responsible for cleaving the 375 kDa polyprotein precursor at specific junctions. Notably, cleavage events at the C-prM, prM-E, E-NS1, NS4A-NS4B, and NS1-NS2A sites are mediated by host cell peptidases within the endoplasmic reticulum.25,30 In stark contrast, NS3 cleaves the peptide bonds between NS2A-NS2B, NS2B-NS3, NS3-NS4A, and NS4B-NS5. This underscores the criticality of NS3 protease activity for viral replication, making it a prime target for therapeutic intervention against Flavivirus infections.25,30
Furthermore, NS3 activation is contingent upon a 40-amino acid region in the NS2B protein, which functions as a crucial cofactor. This interaction forms a heterodimeric complex resembling a chaperone, stabilizing NS3 and facilitating its enzymatic function.25,30,31
Specific regions within NS3 have been demonstrated to exhibit activity in association with the membrane, based on sequence motifs observed in secondary structure predictions. These motifs are exposed on the highly accessible surface of the full-length protein, which adopts a mixed alpha-helix and beta-sheet architecture. These predictions suggest a domain of high immunogenic potential, corroborated by both the summation of observed experimental responses and the protein illustrations presented in Fig. 5.
Looking at the NS5 protein, which has been highly reactive in the membrane, and more reactive peptides with high probability score of epitopes, four peptides had good epitope potential in this protein, and shows a differential spot intensity with more reactivity in Newborn group.
NS5 protein is the largest and most conserved among Flavivirus and has three enzymatic activities. In its N-terminal region, it has activity of guanyliltranferase and S-adenosylmethionine methyltransferase, comprised in 320 residues of this region. Guanylyltransferase is responsible for adding a GTP (guanosine triphosphate) molecule to the 5 'end of viral RNA after RTP (remove a phosphate group) activity from another NS virus protein, NS3.32,33
The activity of NS3 allows NS5 to form a triphosphate bridge over RNA. The enzyme S-adenosylmethionine methyltransferase then transfers a methyl group of S-adenosylmethionine to the seventh guanine nitrogen, finally completing the capping of genomic RNA.32,33
In the C-terminal region (between residues 420 and 900), it has RNA-dependent RNA polymerase activity and is responsible for the polymerization of intermediate RNA (negative sense), which serves as a template for the synthesis of other RNA molecules. positive sense that will be encapsidated in the virion.32,33
Among the methyltransferase and polymerase domains, the protein has an interdomain amino acid sequence that functions as a signal for nuclear localization and has already been found in the nucleus of infected mammalian cells. This sequence is recognized with high specificity by the import subunits (β-importina, mainly) recognizing nuclear localization sequence, although all replications of Flaviviruses occur in the cytosol.32,33
In this interdomain region, NS5 also has a CK2 (Casein Kinase II) phosphorylation region. More precisely, the threonine-395 residue. It is believed that both the nuclear localization signal and the CK2 phosphorylation site are ways of regulating the presence of NS5 in the cytoplasm of the infected cell, indicating that NS5 activity is only required at specific times of infection, such as during replication of genomic RNA.32,33
In all Flaviviruses, proteins with RNA polymerase activity have a specific amino acid sequence: the Gly-Asp-Asp motif, typical of RNA polymerases and the catalytic site.32,33
In the experimental model used in this work the NS5 protein showed great immunogenic potential, with some regions reactive in the membrane and many regions with high probability score in the prediction of epitopes. Membrane reactive regions were shown in the prediction of secondary structure, as structures exposed in the protein with high absolute accessibility to the solvent and constituted mainly by alpha-helices and in some regions are constituted by spirals in the protein.34–36
The potential for immunological recognition of NS5 protein has already been observed in other studies.34–36
Certain regions within NS1, 3, and 5 exhibit high membrane reactivity and epitope recognition potential. NS1 displays significant immunogenicity with solvent-accessible regions overlapping predicted epitopes as evidenced literature. NS5 reveals four highly reactive peptides with good epitope potential, showcasing differential activity in newborns.37–41
The ZIKV and DENV comparison, shows a different response pattern as expected in NS zika peptides, with a strong recognition of NS5 peptides. Recent literature references some different aspects between zika/dengue immune responses.42–44
Further studies will be carried out following this work, to better elucidate the immunogenic mechanisms of ZIKV proteins and to describe potential epitopes in structural proteins of ZIKV, ELISA-SPOT will be used to validate the found peptides in this work, as showed in Chikungunya virus envelope protein.45