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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.

SUBMITTER: Hou W

PROVIDER: S-EPMC5729740 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Hou Wangheng W Cruz-Cosme Ruth R Armstrong Najealicka N Obwolo Lilian Akello LA Wen Fayuan F Hu Wenhui W Luo Min-Hua MH Tang Qiyi Q

Gene 20170715

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 m ...[more]

PMID: 28720531

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Project description:Snake venom metalloproteinases (SVMPs) are a superfamily of zinc-dependent proteases and participate in a number of important biological, physiological and pathophysiological processes. In this work, we simultaneously amplified nine cDNAs encoding different classes of metalloproteinases from glands of four different snake species (Agkistrodon contortrix laticinctus, Crotalus atrox, Crotalus viridis viridis and Agkistrodon piscivorus leucostoma) by RT-PCR with a pair of primers. Among the encoded metalloproteinases, two enzymes (AclVMP-I and AplVMP-I), three enzymes (CaVMP-II, CvvVMP-II and AplVMP-II) and four enzymes (AclVMP-III, CaVMP-III, CvvVMP-III and AplVMP-III) with the characteristic motif (HEXXHXXGXXH) of metalloproteinase belong to type P-I, P-II and P-III enzymes, respectively. Disintegrin domains of CaVMP-II and CvvVMP-II from two Crotatus snakes contain RGD-motif whereas AplVMP-II from Agkistrodon snake has KGD-motif. Instead of R/KGD-motif within disintegrin domain of SVMP-II enzyme, CaVMP-III, CvvVMP-III and AplVMP-III enzymes contain SECD-motif, while AclVMP-III has DDCD-motif in their corresponding position of disintegrin-like domains. There are 12 Cys amino acids in cysterin-rich domains of each P-III enzyme. Moreover, a disintegrin precursor (AplDis) with RGD-motif also simultaneously amplified from the glands of A.p. leucostoma while amplifying AplVMP-II and AplVMP-III, which indicated that different types of SVMPs and related genes are present in a single species of snake and share a consensus sequence at the 3' and 5' untranslated regions. RT-PCR result also showed that P-III is highly expressed in Crotalus snakes than in Agkistrodon snakes. Aligning the deduced amino acid sequence of these enzymes with other SVMPs from GenBank database indicated that this is the first report on the isolation of cDNAs encoding P-II and P-III enzymes from C.v. viridis and A.p. leucostoma snakes. The availability of these SVMP sequences directly facilitated further studies of structure characterization and diversified function analysis.

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

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

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