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Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene.

ABSTRACT: Thus far, there have been no reports on the molecular characterization and antiapoptotic function of the DPV Us5 gene. To perform molecular characterization of DPV Us5, RT-PCR and pharmacological inhibition tests were used to ascertain the kinetic class of the Us5 gene. Western blotting and an indirect immunofluorescence assay (IFA) were used to analyze the expression level and subcellular localization of Us5 in infected cells at different time points. Us5 in purified DPV virions was identified by mass spectrometry. The results of RT-PCR, Western blotting, and pharmacological inhibition tests revealed that Us5 is transcribed mainly in the late stage of viral replication. The IFA results revealed that Us5 was localized throughout DPV-infected cells but was localized only to the cytoplasm of transfected cells. Mass spectrometry and Western blot analysis showed that Us5 was a virion component. Next, to study the antiapoptotic function of DPV Us5, we found that DPV CHv without gJ could induce more apoptosis cells than DPV-CHv BAC and rescue virus. we constructed a model of apoptosis in duck embryo fibroblasts (DEFs) induced by hydrogen peroxide (H2O2). Transfected cells expressing the Us5 gene were protected from apoptosis induced by H2O2, as measured by a TUNEL assay, a caspase activation assay and Flow Cytometry assay. The TUNEL assay and Flow Cytometry assay results showed that the recombinant plasmid pCAGGS-Us5 could inhibit apoptosis induced by H2O2 in DEF cells. However, caspase-3/7 and caspase-9 protein activity upregulated by H2O2 was significantly reduced in cells expressing the recombinant plasmid pCAGGS-Us5. Overall, these results show that the DPV Us5 gene is a late gene and that the Us5 protein is a component of the virion, is localized in the cytoplasm, and can inhibit apoptosis induced by H2O2 in DEF cells.

SUBMITTER: Zhao C

PROVIDER: S-EPMC6425025 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene.

Zhao Chuankuo C He Tianqiong T Xu Yang Y Wang Mingshu M Cheng Anchun A Zhao XinXin X Zhu Dekang D Chen Shun S Liu Mafeng M Yang Qiao Q Jia Renyong R Chen Xiaoyue X Wu Ying Y Zhang Shaqiu S Liu Yunya Y Yu Yanling Y Zhang Ling L

Scientific reports 20190319 1

Thus far, there have been no reports on the molecular characterization and antiapoptotic function of the DPV Us5 gene. To perform molecular characterization of DPV Us5, RT-PCR and pharmacological inhibition tests were used to ascertain the kinetic class of the Us5 gene. Western blotting and an indirect immunofluorescence assay (IFA) were used to analyze the expression level and subcellular localization of Us5 in infected cells at different time points. Us5 in purified DPV virions was identified ...[more]

PMID: 30890748

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Project description:BackgroundDuck viral enteritis, commonly known as duck plague (DP), is an acute and contagious fatal disease in ducks, geese, and swans caused by the DP virus (DPV). It poses a serious threat to the growth of duck farming in the Haor (wetland) areas of Bangladesh.AimThis study aimed to detect the circulating DPV by molecular characterization, followed by phylogenetic analysis, targeting the UL30 gene in infected ducks from five Haor districts in Bangladesh and to observe the variation in the genome sequence between the field virus and vaccine strain of DPV.MethodsA total of 150 samples (liver, 50; intestine, 50; and oropharyngeal tissue, 50) were collected from DP-suspected sick/dead ducks from 50 affected farms in Kishoreganj, Netrokona, B. Baria, Habiganj, and Sunamganj districts in Bangladesh. For the identification of DPV in collected samples, polymerase chain reaction (PCR) was utilized. Nucleotide sequences of the amplified UL30 gene were compared with those of other DPV strains available in GenBank.ResultsOf the 150 samples, 90 (60%) were found to be positive for DPV, as confirmed by PCR. Organ-wise prevalence was higher in the liver (72%), followed by the intestine (64%) and oropharyngeal tissue (44%). Regarding areas, the highest and lowest prevalence in the liver and intestine was observed in Habiganj and B. Baria, respectively, whereas the highest and lowest prevalence in the oropharyngeal tissue was observed in B. Baria and Habiganj, respectively. Two isolates, BAU/KA/DPV(B1)/2014 from Kishoreganj and BAU/KA/DPV(B4)/2014 from Sunamganj were sequenced, and phylogenetic analysis revealed that these isolates are evolutionarily closely related to Chinese isolates of DPV. Additionally, the isolates of DPV BAU/KA/DPV(B1)/2014 and BAU/KA/DPV(B4)/2014 showed the highest (98%) similarity to each other. The nucleotide sequence of the isolate BAU/KA/DPV(B1)/2014 exhibited higher nucleotide variability (246 nucleotides) than that of the vaccine strain (accession no. EU082088), which may affect protein function and additional drug sensitivity.ConclusionBased on the findings of the molecular study, it can be assumed that the Bangladeshi isolates and all Chinese isolates of DPV may have a common ancestry.

Project description:Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1-60 at the N-terminal, and amino acids 1-20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40-50 and 768-777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

Dataset Information

Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene.

Publications

Molecular characterization and antiapoptotic function analysis of the duck plague virus Us5 gene.

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