Molecular cloning and characterization of the genes encoding the proteins of Zika virus

Abstract

Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells.

Endoplasmic reticulum: a focal point of Zika virus infection

“…ZIKV capsid interacts with multiple nucleolar proteins, whereas NS5 protein targets and disrupts the Cajal bodies in the nucleus [20]. This observation is consistent with the aforementioned experimental study that showed nuclear localization of ZIKV capsid and NS5 protein [19]. The study also identified numerous interactions between viral proteins (prM, Env, NS2A, NS2B, NS4A, and NS4B) with various ER proteins, which highlights the importance of the ER in ZIKV life cycle [20].…”

“…Three ZIKV proteins namely Env, prM, and NS2A are distributed to the ER as indicated by their co-localization with calreticulin expression. NS5, which contains nuclear localization signals, forms punctate distribution in the nucleus [19]. However, these data should be interpreted with caution as the subcellular localization of these cloned viral proteins may differ in ZIKV-infected cells.…”

“…ZIKV utilizes host translational machinery to produce a single polyprotein that is further cleaved by viral NS2B-NS3 serine protease and host cell protease into functional viral proteins [18]. These viral proteins are then distributed to cellular compartments for various functions [19].…”

“…Molecular cloning and expression of individual ZIKV proteins reveal distinct and specific organellar localization. ZIKV capsid localizes to several compartments including the nucleus and the Golgi [19]. In addition to capsid, NS2B and NS4A also localize to the Golgi.…”

Zika virus nonstructural protein 5 residue R681 is critical for dimer formation and enzymatic activity

“…Differential subcellular localization of NS5 has been reported for DENV where it is predominantly nuclear for DENV‐2, ‐3, and ‐4, and mostly cytoplasmic for DENV‐1 . More recently, it was demonstrated that the closely related flavivirus, ZIKV NS5 formed supramolecular nuclear bodies that were distinct from DENV‐2 NS5 despite being nuclear localized . Interestingly, previous works showed that ZIKV NS5 formed higher order oligomers in solution in which in part residues Y25, K28, and K29 have been proposed to be involved in .…”