Muscovy duck reovirus p10.8 protein localizes to the nucleus via a nonconventional nuclear localization signal

Guo, Dongchun; Qiu, Na; Shaozhou, Wulin; Bai, Xiaofei; He, Yilong; Zhang, Qingshan; Zhao, Jian; Liu, Ming; Zhang, Yun

doi:10.1186/1743-422x-11-37

Cited by 10 publications

(10 citation statements)

References 25 publications

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“…IFA and DAPI staining showed that the VP3 protein was located both in the cytoplasm and nucleus of DHAV-1-infected DEF cells, which is consistent with the location of VP3 in FMDV infected cells 20 . Most proteins of RNA viruses enter the nucleus via nuclear localization signals (NLSs), which contain a continuous of basic amino acid residues 21 – 23 ; when we scanned the VP3 protein sequence, we noticed multiple basic amino acids ( 1 G KRK PC RR PI HK P K N 15 ) at the N-terminal of the VP3 protein. Whether this basic motif serves NLS function needs to be proved.…”

Section: Discussionmentioning

confidence: 99%

“…Blocking was carried out in 10% FBS solution at room temperature for 1 h. After washing for three times, the cells were incubated with fluorescent-conjugated Goat anti-Mouse IgG antibody (KPL, MD, USA) (500 × dilution). To confirm the location of the VP1 protein, the DEF cells were then stained with DAPI as described previously 21 . Bound antibodies and stained cells were visualized under a fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

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Mapping a Type-specific Epitope by Monoclonal Antibody against VP3 Protein of Duck Hepatitis A Type 1 Virus

Zhang

Meng

et al. 2017

Sci Rep

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Duck hepatitis A subtype 1 virus (DHAV-1) infection causes high mortality in ducklings, resulting in significant losses to duck industries. VP3 is a structural protein of DHAV-1. However, B-cell epitopes on VP3 have not been investigated. To stimulate VP3 antibody response, eukaryotic expression plasmid pCI-neo-VP3 was constructed and used as DNA immunogen to prepare mAbs. Western blot showed that 25.5 kDa VP3 could be detected by mAbs in duck embryo fibroblast (DEF) cells transfected with pCI-neo-VP3. Immunofluorescence assay showed that mAbs could specifically bind to DEF cells infected with DHAV-1. DAPI staining indicated that VP3 localizes to the cytoplasm and nucleus of DHAV-1 infected DEF. With neutralizing mAb 3B7, minimal epitope PSNI was mapped. Sequence alignment indicated that 205PSNI208 is highly conserved among DHAV-1, but different from those of DHAV-2 and DHAV-3. Epitope peptide reacted specifically with DHAV-1-positive duck sera by dot blotting, revealing PSNI is DHAV-1 type-specific epitope and the importance of these amino acids in antibody-epitope binding reactivity. These findings provided useful information for understanding the antigenicity of VP3 and might be valuable in the development of epitope-based vaccine or diagnostic kit for DHAV-1 infection and provide insights for understanding the pathogenesis of DHAV-1.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mapping a Type-specific Epitope by Monoclonal Antibody against VP3 Protein of Duck Hepatitis A Type 1 Virus

Zhang

Meng

et al. 2017

Sci Rep

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“…Most proteins enter the nucleus via nuclear localization signals (NLSs). NLSs have been shown to contain a continuous motif of basic amino acid residues [ 20 , 27 , 28 ]; When we scanned the VP1 protein sequence, we noticed a cluster of basic amino acids ( 189 K M KKR W K P R 197 ) (basic amino acids underlined) ( S2 Table ) at the C-terminal of the VP1 protein, which may represent an NLS. Whether this basic motif serves some nuclear import function should be evaluated.…”

Section: Discussionmentioning

confidence: 99%

“…Then, 50 μL/well of FITC-conjugated goat anti-mouse IgG (KPL, MD, USA) at a 1:400 dilution was added and the cells were incubated for 1 h at 37°C. To confirm the location of the VP1 protein, the cells were then stained with DAPI as described previously [ 20 ]. The stained cells were viewed by using a Zeiss Axioplan-2 or a confocal LSM 700 (Carl Zeiss) fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

Zhang

et al. 2015

PLoS ONE

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BackgroundThe VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized.Methods and ResultsTo characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1–positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope.Conclusions and SignificanceWe identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

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“…The cells were then incubated with different anti-E mAbs (1:500 dilutions) and normal mouse serum (as a negative control, 1:500 dilutions) for 1 h at 37°C and then with fluorescein-labeled antibodies against mouse IgG (KPL, MD, USA) (1:500 dilutions). To determine the location of the native E protein, the cells were stained with DAPI as described previously [6] and viewed using a Zeiss Axioplan-2 or a confocal LSM 700 (Carl Zeiss) fluorescence microscope.…”

Section: Isotyping and Serological Screeningmentioning

confidence: 99%

Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection

et al. 2015

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The E protein of flaviviruses is the primary antigen that induces protective immunity, but a monoclonal antibody (mAb) against the E protein of duck Tembusu virus (DTMUV) has never been characterized. Six hybridoma cell lines secreting DTMUV anti-E mAbs were prepared and designated 2A5, 1F3, 1G2, 1B11, 3B6, and 4F9, respectively. An immunofluorescence assay indicated that the mAbs could specifically bind to duck embryo fibroblast (DEF) cells infected with DTMUV and that the E protein was distributed in the cytoplasm of the infected cells. Immunoglobulin isotyping differentiated the mAbs as IgG1 (1G2, 1B11, 4F9, 1F3, and 2A5) and IgG2b (3B6). The mAbs were used to identify three epitopes, A (2A5, 1F3, and 1G2), B (1B11 and 4F9), and C (3B6) on the E protein on the basis of a competitive binding assay. By using mAbs 1F3 and 3B6, we developed an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect E antigen from clinical samples. The AC-ELISA did not react with other known pathogens, indicating that the mAbs are specific for DTMUV. Compared to RT-PCR, the specificity and sensitivity of the AC-ELISA was 94.1 % and 98.0 %, respectively. This AC-ELISA thus represents a sensitive and rapid method for detecting DTMUV infection in birds.

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Muscovy duck reovirus p10.8 protein localizes to the nucleus via a nonconventional nuclear localization signal

Cited by 10 publications

References 25 publications

Mapping a Type-specific Epitope by Monoclonal Antibody against VP3 Protein of Duck Hepatitis A Type 1 Virus

Mapping a Type-specific Epitope by Monoclonal Antibody against VP3 Protein of Duck Hepatitis A Type 1 Virus

Identification of a Conserved B-Cell Epitope on Duck Hepatitis A Type 1 Virus VP1 Protein

Characterization of monoclonal antibodies against duck Tembusu virus E protein: an antigen-capture ELISA for the detection of Tembusu virus infection

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