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In vitro expression and development of indirect ELISA for Capsid protein of duck circovirus without nuclear localization signal.

ABSTRACT: Duck circovirus was a newly discovered pathogen that causing ducks immunosuppression in recent years, but it can not been cultured in vitro that limited its depth study. In the present study, the Cap gene that defect the nuclear localization signal (NLS) of DuCV was amplified and connected to the express vector pET-32a (+), to express the recombinant Cap protein in the bacterium Escherichia coli Rosetta. The recombinant Cap protein was purified and an indirect ELISA method was developed based on the recombinant Cap protein. The results showed that the truncated Cap gene was 567 bp and cloned into pET-32a (+) vector successfully. The recombinant Cap protein was expressed as inclusion bodies. The results of optimization for indirect ELISA revealed that the optimal antigen and serum dilutions were selected to be 4 ?g/well and 1:40, respectively; the coating condition was 37°C for 1 h and 4°C overnight; the blocking time in 1% BSA was 1 h at 37°C and the second antibody dilution was 1:800, the cut-off value was 0.352. Known anti-sera samples of other duck common pathogens were tested by the developed ELISA and the results showed it was specific for DuCV anti-sera detection. The sensitivity of indirect ELISA reached 1:2560, and the coefficient of variation between intra-assay and inter-assay were less than 10%. Compared the PCR and indirect ELISA methods, the positive compliance rate was 95.6% for detected 59 duck samples.

SUBMITTER: Lu Y

PROVIDER: S-EPMC4152055 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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In vitro expression and development of indirect ELISA for Capsid protein of duck circovirus without nuclear localization signal.

Lu Yanyan Y Jia Renyong R Zhang Zhilong Z Wang Mingshu M Xu Yu Y Zhu Dekang D Chen Shun S Liu Mafeng M Yin Zhongqiong Z Chen Xiaoyue X Cheng Anchun A

International journal of clinical and experimental pathology 20140715 8

Duck circovirus was a newly discovered pathogen that causing ducks immunosuppression in recent years, but it can not been cultured in vitro that limited its depth study. In the present study, the Cap gene that defect the nuclear localization signal (NLS) of DuCV was amplified and connected to the express vector pET-32a (+), to express the recombinant Cap protein in the bacterium Escherichia coli Rosetta. The recombinant Cap protein was purified and an indirect ELISA method was developed based on ...[more]

PMID: 25197365

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Project description:BackgroundPorcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication.ResultsThe PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2.ConclusionsRecovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.

Project description:A relatively stable and flexible capsid is critical to the viral life cycle. However, the capsid dynamics and cytosol trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation flexibility of PCV2 virions by genome labeling and the use of three monoclonal antibodies (MAbs) against the native capsid of PCV2. Genome labeling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65°C, causing binding incompetence for all antibodies, and the viral genome was released without capsid disassembly upon heating at 60°C. Antibody binding experiments with PCV2 showed that residues 186 to 192 were concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment and then exposed in PK-15 cytosol and the 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking were not significantly affected by microtubule depolymerization in monocytic 3D4/31 cells treated with nocodazole. These findings demonstrated that nuclear targeting of viral capsids involved conformational changes, the PCV2 genome was released from the assembled capsid, and the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.IMPORTANCE Circovirus is the smallest virus known to replicate autonomously. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of porcine circovirus type 2 (PCV2). Here, we report the release of the PCV2 genome from assembled capsid and the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.

Project description:Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses. Importance: Most DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.

Dataset Information

In vitro expression and development of indirect ELISA for Capsid protein of duck circovirus without nuclear localization signal.

Publications

In vitro expression and development of indirect ELISA for Capsid protein of duck circovirus without nuclear localization signal.

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