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Duck enteritis virus (DEV) UL54 protein, a novel partner, interacts with DEV UL24 protein.

ABSTRACT: UL24 is a multifunctional protein that is conserved among alphaherpesviruses and is believed to play an important role in viral infection and replication.In this paper, to investigate putative UL24-binding proteins and to explore the functional mechanisms of DEV UL24, yeast two-hybrid (Y2H) was carried out, and further verified the interaction between UL24 and partners by co-immunoprecipitation and fluorescence microscopy experiments. Interaction partners of UL24 protein were screened by yeast two-hybrid (Y2H) with the cDNA library of DEV-CHv strain post-infection DEF cells. A novel partner, DEV UL54 protein, was discovered by Y2H screening and bioinformatic. Co-immunoprecipitation experiments suggested that DEV UL24 interacted with UL54 proteins. And distribution of a part of UL54 protein was changed from nucleus to cytoplasm in DF-1 cells of co-subcellular localization experiments which also showed that DEV UL24 interacted with UL54 proteins.The interaction between the DEV UL24 and UL54 proteins was discovered for the first time. Thus, DEV UL54 protein as a novel partner interacted with DEV UL24 protein.

SUBMITTER: Gao X

PROVIDER: S-EPMC5575879 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Duck enteritis virus (DEV) UL54 protein, a novel partner, interacts with DEV UL24 protein.

Gao Xinghong X Jia Renyong R Wang Mingshu M Yang Qiao Q Chen Shun S Liu Mafeng M Yin Zhongqiong Z Cheng Anchun A

Virology journal 20170829 1

<h4>Background</h4>UL24 is a multifunctional protein that is conserved among alphaherpesviruses and is believed to play an important role in viral infection and replication.<h4>Results</h4>In this paper, to investigate putative UL24-binding proteins and to explore the functional mechanisms of DEV UL24, yeast two-hybrid (Y2H) was carried out, and further verified the interaction between UL24 and partners by co-immunoprecipitation and fluorescence microscopy experiments. Interaction partners of UL ...[more]

PMID: 28851454

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Project description:BackgroundHighly pathogenic avian influenza virus (AIV) subtype H5N1 remains a threat to poultry. Duck enteritis virus (DEV)-vectored vaccines expressing AIV H5N1 hemagglutinin (HA) may be viable AIV and DEV vaccine candidates.MethodsTo facilitate the generation and further improvement of DEV-vectored HA(H5) vaccines, we first constructed an infectious clone of DEV Chinese vaccine strain C-KCE (DEV(C-KCE)). Then, we generated a DEV-vectored HA(H5) vaccine (DEV-H5(UL55)) based on the bacterial artificial chromosome (BAC) by inserting a synthesized HA(H5) expression cassette with a pMCMV IE promoter and a consensus HA sequence into the noncoding area between UL55 and LORF11. The immunogenicity and protective efficacy of the resulting recombinant vaccine against DEV and AIV H5N1 were evaluated in both ducks and chickens.ResultsThe successful construction of DEV BAC and DEV-H5(UL55) was verified by restriction fragment length polymorphism analysis. Recovered virus from the BAC or mutants showed similar growth kinetics to their parental viruses. The robust expression of HA in chicken embryo fibroblasts infected with the DEV-vectored vaccine was confirmed by indirect immunofluorescence and western blotting analyses. A single dose of 10(6) TCID50 DEV-vectored vaccine provided 100 % protection against duck viral enteritis in ducks, and the hemagglutination inhibition (HI) antibody titer of AIV H5N1 with a peak of 8.2 log2 was detected in 3-week-old layer chickens. In contrast, only very weak HI titers were observed in ducks immunized with 10(7) TCID50 DEV-vectored vaccine. A mortality rate of 60 % (6/10) was observed in 1-week-old specific pathogen free chickens inoculated with 10(6) TCID50 DEV-vectored vaccine.ConclusionsWe demonstrate the following in this study. (i) The constructed BAC is a whole genome clone of DEV(C-KCE). (ii) The insertion of an HA expression cassette sequence into the noncoding area between UL55 and LORF11 of DEV(C-KCE) affects neither the growth kinetics of the virus nor its protection against DEV. (iii) DEV-H5(UL55) can generate a strong humoral immune response in 3-week-old chickens, despite the virulence of this virus observed in 1-week-old chickens. (iv) DEV-H5(UL55) induces a weak HI titer in ducks. An increase in the HI titers induced by DEV-vectored HA(H5) will be required prior to its wide application.

Project description:BACKGROUND: Knowledge of the subcellular localization of a protein can provide useful insights about its function. While the subcellular localization of many alphaherpesvirus UL51 proteins has been well characterized, little is known about where duck enteritis virus (DEV) UL51 protein (pUL51) is targeted to. Thus, in this study, we investigated the subcellular localization and distribution of DEV pUL51 by computer aided analysis, as well as indirect immunofluorescence (IIF) and transmission immunoelectron microscopy (TIEM) approaches in DEV-infected cells. RESULTS: The DEV UL51 gene product was identified as an approximate 34 kDa protein in DEV-infected cells analyzed by western blotting. Computer aided analysis suggested that DEV pUL51 is not targeted to the mitochondrial, extra-cellular or nucleus, but be targeted to the cytoplasmic in host cells, more specifically, palmitoylation of the pUL51 through the N-terminal cysteine at position 9 makes membrane association and Golgi localization possible. Using IIF analysis, we found that DEV pUL51 was first detected in a juxtanuclear region of DEV-infected cells at 9 h postinfection (p.i.), and then was detected widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region from 12 to 60 h p.i. TIEM analysis revealed that DEV pUL51 was mainly associated with cytoplasmic virions and also with some membranous structure near the pUL51-specific immuno-labeling intracellular virion in the cytoplasmic vesicles; moreover, the pUL51 efficiently accumulated in the Golgi apparatus at first, and then was sent to the plasma membrane from the Golgi by some unknown mechanism. CONCLUSION: In this work, we described the basic characteristics of pUL51 subcellular localization and distribution for the first time. From these results, we concluded that palmitoylation at the N-terminal cysteine, which is conserved in all alphaherpesvirus UL51 homologs, is required for its membrane association and Golgi localization, and the pUL51 mainly localized to the juxtanuclear region of DEV-infected cells, as well seemed to be incorporated into mature virions as a component of the tegument. The research will provide useful clues for DEV pUL51 functional analysis, and will be usefull for further understanding the localization properties of alphaherpesvirus UL51 homologs.

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Duck enteritis virus (DEV) UL54 protein, a novel partner, interacts with DEV UL24 protein.

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Duck enteritis virus (DEV) UL54 protein, a novel partner, interacts with DEV UL24 protein.

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