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Duplicate US1 Genes of Duck Enteritis Virus Encode a Non-essential Immediate Early Protein Localized to the Nucleus.

ABSTRACT: The duplicate US1 genes of duck enteritis virus (DEV) encode a protein with a conserved Herpes_IE68 domain, which was found to be closely related to the herpes virus immediate early regulatory protein family and is highly conserved among counterparts encoded by Herpes_IE68 genes. Previous studies found the homologous proteins HSV-1 ICP22 and VZV ORF63/ORF70 to be critical for virus transcription and replication. However, little is known about the DEV ICP22 protein. In this paper, we describe the characteristics of this protein based on pharmacological experiments, real-time quantitative Polymerase Chain Reaction, Western blot, and immunofluorescence assays. We also investigate the role of the protein in DEV replication via mutation of US1. As a result, we found that the DEV ICP22 protein is a non-essential immediate early protein predominantly located in the nucleus of infected DEF cells and that DEV replication is impaired by US1 deletion. We also found that ICP22 contains a classical nuclear localization signal (NLS) at 305-312AA, and ICP22 cannot enter the nucleus by itself after mutating residue 309.

SUBMITTER: Li Y

PROVIDER: S-EPMC6979402 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Duplicate US1 Genes of Duck Enteritis Virus Encode a Non-essential Immediate Early Protein Localized to the Nucleus.

Li Yangguang Y Wu Ying Y Wang Mingshu M Ma YunChao Y Jia Renyong R Chen Shun S Zhu Dekang D Liu Mafeng M Yang Qiao Q Zhao Xinxin X Zhang Shaqiu S Huang Juan J Ou Xumin X Mao Sai S Zhang Ling L Liu Yunya Y Yu Yanling Y Pan Leichang L Tian Bin B Rehman Mujeeb Ur MU Chen Xiaoyue X Cheng Anchun A

Frontiers in cellular and infection microbiology 20200117

The duplicate US1 genes of duck enteritis virus (DEV) encode a protein with a conserved Herpes_IE68 domain, which was found to be closely related to the herpes virus immediate early regulatory protein family and is highly conserved among counterparts encoded by Herpes_IE68 genes. Previous studies found the homologous proteins HSV-1 ICP22 and VZV ORF63/ORF70 to be critical for virus transcription and replication. However, little is known about the DEV ICP22 protein. In this paper, we describe the ...[more]

PMID: 32010642

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Project description:BACKGROUND: At present, alphaherpesviruses gI gene and its encoding protein have been extensively studied. It is likely that gI protein and its homolog play similar roles in virions direct cell-to-cell spread of alphaherpesviruses. But, little is known about the characteristics of DEV gI gene. In this study, we expressed and presented the basic properties of the DEV gI protein. RESULTS: The special 1221-bp fragment containing complete open reading frame(ORF) of duck enteritis virus(DEV) gI gene was extracted from plasmid pMD18-T-gI, and then cloned into prokaryotic expression vector pET-32a(+), resulting in pET-32a(+)-gI. After being confirmed by PCR, restriction endonuclease digestion and sequencing, pET-32a(+)-gI was transformed into E.coli BL21(DE3) competent cells for overexpression. DEV gI gene was successfully expressed by the addition of isopropyl-?-D-thiogalactopyranoside(IPTG). SDS-PAGE showed that the recombinant protein His6-tagged gI molecular weight was about 61 kDa. Subsequently, the expressed product was applied to generate specific antibody against gI protein. The specificity of the rabbit immuneserum was confirmed by its ability to react with the recombinant protein His6-tagged gI. In addition, real time-PCR was used to determine the the levels of the mRNA transcripts of gI gene, the results showed that the DEV gI gene was transcribed most abundantly during the late phase of infection. Furthermore, indirect immunofluorescence(IIF) was established to study the gI protein expression and localization in DEV-infected duck embryo fibroblasts (DEFs), the results confirmed that the protein was expressed and located in the cytoplasm of the infected cells, intensively. CONCLUSIONS: The recombinant prokaryotic expression vector of DEV gI gene was constructed successfully. The gI protein was successfully expressed by E.coli BL21(DE3) and maintained its antigenicity very well. The basic information of the transcription and intracellular localization of gI gene were presented, that would be helpful to assess the possible role of DEV gI gene. The research will provide useful clues for further functional analysis of DEV gI gene.

Dataset Information

Duplicate US1 Genes of Duck Enteritis Virus Encode a Non-essential Immediate Early Protein Localized to the Nucleus.

Publications

Duplicate US1 Genes of Duck Enteritis Virus Encode a Non-essential Immediate Early Protein Localized to the Nucleus.

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