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      Duck enteritis virus UL54 is an IE protein primarily located in the nucleus

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          Abstract

          Background

          The UL54 protein of Duck Enteritis Virus (DEV) is a homolog of herpes simplex virus-1 (HSV-1) immediate-early infectious cell protein 27 (ICP27), a multifunctional protein essential for viral infection. Nonetheless, there is little information on the UL54 protein of DEV.

          Methods

          The UL54 gene was cloned into the pPAL7 vector, and the recombinant protein, expressed in the E. coli Rosetta, was used to produce a specific antibody. Using this antibody, Western blotting and indirect immunofluorescence analysis (IFA) were used to analyze the expression level and intracellular localization, respectively, of UL54 in DEV-infected cells at different times. Real-time quantitative reverse transcription PCR (RT-PCR) and the pharmacological inhibition test were utilized to ascertain the kinetic class of the UL54 gene.

          Results

          UL54 was expressed as a fusion protein of approximately 66.0 kDa using the prokaryotic expression system, and this protein was used to generate the specific anti-UL54 antibody. The UL54 protein was initially diffusely distributed throughout the cytoplasmic region; then, after 2 h, it gradually distributed into the nucleus, peaking at 24 h, and complete localization to the nucleus was observed thereafter. The UL54 transcript was detected as early as 0.5 h, and peak expression was observed at 24 h. The UL54 gene was insensitive to the DNA polymerase inhibitor Ganciclovir (GCV) and the protein synthesis inhibitor Cycloheximide (CHX), both of which confirmed that UL54 was an immediate early gene.

          Conclusions

          The DEV UL54 gene was expressed in a prokaryotic expression system and characterized for expression level, intracellular localization and gene kinetic class. We propose that these results will provide the foundation for further functional analyses of this gene.

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          Most cited references37

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          Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling.

          Host cells respond to viral infection by many mechanisms, including the production of type I interferons which act in a paracrine and autocrine manner to induce the expression of antiviral interferon-stimulated genes (ISGs). Viruses have evolved means to inhibit interferon signaling to avoid induction of the innate immune response. Herpes simplex virus 1 (HSV-1) has several mechanisms to inhibit type I interferon production, the activities of ISGs, and the interferon signaling pathway itself. We report that the inhibition of the Jak/STAT pathway by HSV-1 requires viral gene expression and that viral immediate-early protein ICP27 plays a role in downregulating STAT-1 phosphorylation and in preventing the accumulation of STAT-1 in the nucleus. We also show that expression of ICP27 by transfection causes an inhibition of IFN-induced STAT-1 nuclear accumulation. Therefore, ICP27 is necessary and sufficient for at least some of the effects of HSV infection on STAT-1.
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            ICP27 interacts with the C-terminal domain of RNA polymerase II and facilitates its recruitment to herpes simplex virus 1 transcription sites, where it undergoes proteasomal degradation during infection.

            Herpes simplex virus 1 (HSV-1) ICP27 has been shown to interact with RNA polymerase II (RNAP II) holoenzyme. Here, we show that ICP27 interacts with the C-terminal domain (CTD) of RNAP II and that ICP27 mutants that cannot interact fail to relocalize RNAP II to viral transcription sites, suggesting a role for ICP27 in RNAP II recruitment. Using monoclonal antibodies specific for different phosphorylated forms of the RNAP II CTD, we found that the serine-2 phosphorylated form, which is found predominantly in elongating complexes, was not recruited to viral transcription sites. Further, there was an overall reduction in phosphoserine-2 staining. Western blot analysis revealed that there was a pronounced decrease in the phosphoserine-2 form and in overall RNAP II levels in lysates from cells infected with wild-type HSV-1. There was no appreciable difference in cdk9 levels, suggesting that protein degradation rather than dephosphorylation was occurring. Treatment of infected cells with proteasome inhibitors MG-132 and lactacystin prevented the decrease in the phosphoserine-2 form and in overall RNAP II levels; however, there was a concomitant decrease in the levels of several HSV-1 late proteins and in virus yield. Proteasomal degradation has been shown to resolve stalled RNAP II complexes at sites of DNA damage to allow 3' processing of transcripts. Thus, we propose that at later times of infection when robust transcription and DNA replication are occurring, elongating complexes may collide and proteasomal degradation may be required for resolution.
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              The histone acetyltransferase CLOCK is an essential component of the herpes simplex virus 1 transcriptome that includes TFIID, ICP4, ICP27, and ICP22.

              Studies published elsewhere have shown that the herpes simplex virus regulatory protein ICP0 interacts with BMAL1, a partner and regulator of circadian histone acetyltransferase CLOCK, that both proteins localize at ND10 bodies and are stabilized by viral proteins, that enzymatically active CLOCK partially complements ΔICP0 mutants, and that silencing of CLOCK suppresses the expression of viral genes. Here we report that CLOCK is a component of the transcriptional complex that includes TFIID, ICP4, ICP27, and ICP22. The results suggest that the CLOCK histone acetyltransferase is a component of the viral transcriptional machinery throughout the replicative cycle of the virus and that ICP27 and ICP22 initiate their involvement in viral gene expression as components of viral transcriptome.
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                Author and article information

                Contributors
                +86 28 82691776 , chenganchun@vip.163.com
                +86 28 82691776 , mshwang@163.com
                Journal
                Virol J
                Virol. J
                Virology Journal
                BioMed Central (London )
                1743-422X
                25 November 2015
                25 November 2015
                2015
                : 12
                : 198
                Affiliations
                [ ]Avian Diseases Research Center, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan 611130 P.R. China
                [ ]Key Laboratory of Animal Diseases and Human Health of Sichuan Province, Wenjiang, Chengdu City, Sichuan 611130 P.R. China
                [ ]Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Wenjiang, Chengdu City, Sichuan 611130 P.R. China
                Author information
                http://orcid.org/0000-0002-4230-9159
                Article
                424
                10.1186/s12985-015-0424-z
                4658773
                26606920
                d2c53211-7586-4373-b2ad-6c3b74b41823
                © Liu et al. 2015

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 25 August 2015
                : 15 November 2015
                Categories
                Research
                Custom metadata
                © The Author(s) 2015

                Microbiology & Virology
                duck enteritis virus,ul54,expression,ie,intracellular localization
                Microbiology & Virology
                duck enteritis virus, ul54, expression, ie, intracellular localization

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