Dataset Information

Expression and antigenicity characterization for truncated capsid protein of porcine circovirus type 2.

ABSTRACT: Three pairs of specific primers were designed to amplify F2-1, F2-2, and XF2-2 truncated capsid protein genes of porcine circovirus type 2 (PCV-2). Amplified sequences were subcloned to pET-32a(+) vectors and expressed in Rosetta (DE3) Escherichia coli by induction of isopropy-?-D-thiogalactoside (IPTG). All of the fusion proteins had positive reactions to PCV-2 antiserum and His-XF2-2 showed the best reactivity. Proteins were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs), and 7 mAbs were selected. Capsid protein N-terminal parts 55 to 96 amino acid (aa), 97 to 141 aa, and 143 to 211 aa were confirmed as binding regions of the 7 mAbs. Reactivity between His-XF2-2 and the 7 mAbs was detected, FmAb-8 showed the best reactivity. The dominant B-cell epitope was located at 97 to 141 aa. The PEPSCAN indicated that the P122-136 peptide contained the dominant B-cell epitope.

SUBMITTER: Lou Z

PROVIDER: S-EPMC3003564 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Expression and antigenicity characterization for truncated capsid protein of porcine circovirus type 2.

Lou Zhongzi Z Li Xuerui X Li Zhiyong Z Yin Xiangping X Li Baoyu B Lan Xi X Yang Bin B Zhang Yun Y Liu Jixing J

Canadian journal of veterinary research = Revue canadienne de recherche veterinaire 20110101 1

Three pairs of specific primers were designed to amplify F2-1, F2-2, and XF2-2 truncated capsid protein genes of porcine circovirus type 2 (PCV-2). Amplified sequences were subcloned to pET-32a(+) vectors and expressed in Rosetta (DE3) Escherichia coli by induction of isopropy-β-D-thiogalactoside (IPTG). All of the fusion proteins had positive reactions to PCV-2 antiserum and His-XF2-2 showed the best reactivity. Proteins were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs), ...[more]

PMID: 21461197

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Project description:Porcine circovirus type 2 (PCV2) capsid protein (Cap) is a unique structure protein that plays pivotal roles in the process of viral replication and pathogenesis. Herein, we characterized a putative porcine Makorin RING finger protein 1 (pMKRN1) variant, an N-terminal-truncated variant of putative full-size porcine MKRN1 which has a unique expression pattern resulting from the porcine mkrn1 gene and which interacts with PCV2 Cap. A domain mapping assay showed that the C terminus of pMKRN1 and fragments (amino acids 108 to 198) of Cap are required for this interaction. PCV2 transiently upregulated pMKRN1 in PK-15 cells, but persistent viral infection downregulated pMKRN1 in major pathological tissues of PCV2-infected piglets. Overexpression of pMKRN1 significantly inhibited the generation of progeny PCV2 via ubiquitination and degradation of Cap, whereas knockout of pMKRN1 blocked Cap degradation and promoted progeny virus replication. pMKRN1 specifically targeted PCV2 Cap lysine residues 164, 179, and 191 to induce polyubiquitination and subsequent degradation. Mutation of either of the three lysine residues in the Cap protein or mutation of the histidine at residue 243 within the RING finger domain of pMKRN1 abrogated the E3 ligase activity of pMKRN1, rendering cells incapable of inducing Cap ubiquitination and degradation. Consistent with this finding, a Cap ubiquitination-deficient PCV2 strain showed enhanced virus replication and produced severe histological lesions in the lung and lymph node tissues compared with wild-type PCV2. Taken together, the results presented here suggest that PCV2 downregulates the pMKRN1 variant to avoid pMKRN1-mediated Cap ubiquitination and degradation, thus promoting viral replication and pathogenesis in its targeted tissues.IMPORTANCE Porcine circovirus type 2 is the pathogen to which pigs are the most susceptible, causing immense economic losses in the global swine industry, but whether host cells have developed some strategies to prevent viral replication is still unclear. Here, we found that porcine MKRN1 (pMKRN1) was upregulated in the early stage of PCV2 infection and mediated the polyubiquitination and degradation of Cap protein to block PCV2 replication, yet persistent PCV2 infection downregulated pMKRN1 levels to avoid degradation, promoting viral replication and pathogenesis in its targeted tissues. These data present new insight into the molecular mechanisms underlying the antiviral effects of pMKRN1 E3 ligase during PCV2 infection and also suggest potential new control measures for PCV2 outbreaks.

Dataset Information

Expression and antigenicity characterization for truncated capsid protein of porcine circovirus type 2.

Publications

Expression and antigenicity characterization for truncated capsid protein of porcine circovirus type 2.

Similar Datasets

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