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Baculoviral IAP2 and IAP3 encoded by Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) suppress insect cell apoptosis in a transient expression assay

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Abstract

Baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation stage of viral infection. While some iap genes in baculoviruses are not directly involved in anti-apoptotic activity, two iap genes (ly-iap2 and ly-iap3) were found and cloned from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) and used to investigate the roles in LyxyMNPV infection. From the transcriptional start site analysis, ly-iap2 consisted of a late promoter motif (TAAG) located upstream at nt −17 bp, and ly-iap3 had a potential baculoviral promoter consensus sequence (GCTTGTT) located upstream at nt −44 bp. Both the ly-iap2 and ly-iap3 genes were initially transcribed in the LyxyMNPV-infected Ld652Y cells (IPLB-LD652Y cell line) at 3 h post infection (h p.i.), increased dramatically at 12 h p.i. and reached the peak at 72 h p.i. according to a quantitative RT-PCR (qRT-PCR) assay. Functional assays of these iap genes were performed using an overexpression method in Sf9 cells. Full-length LY-IAP2, LY-IAP3 and LY-IAP2-BIR (baculoviral IAP repeat) could inhibit varying degrees of apoptosis induced by Drosophila RPR protein (DRPR). Interestingly, Ly-IAP2-BIR showed higher inhibitory activity than full-length Ly-IAP2, and the protein expression level was dramatically increased while the Ly-IAP2-RING domain was deleted. The effect of MG-132 on the overexpression of Ly-IAP2 was investigated. Under MG-132 treatments, high-molecular-weight signals were also detected. Taken together, this study suggested that both Ly-IAP2 and Ly-IAP3 were anti-apoptotic protein through the BIR functional domain. The Ly-IAP2-RING domain was possibly involved in ubiquitin E3 ligase activity, leading to the degradation of Ly-IAP2 protein, whereas Ly-IAP3-RING might be working as a “helper domain” to inhibit DRPR-induced apoptosis.

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Acknowledgments

We appreciate Dr. Kuo-Ping Chiu at the Genomics Research Center, Academia Sinica, Taiwan, for providing the experimental reagents and Mr. Chong-Yu Ko and Ms. Po-Ya Hsu for their assistance with this work. This study was supported by grant 103-2313-B-197-002-MY3 from the National Science Council of Taiwan and a grant from the Council of Agriculture, Executive Yuan, Republic of China.

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    Authors and Affiliations

    1. Department of Biotechnology and Animal Science, National Ilan University, No. 1, Sec. 1, Shennong Rd., Yilan City, Yilan County, 260, Taiwan, R.O.C

      Yu-Shin Nai, Yue-Wen Chen & Chung-Hsiung Wang

    2. Department of Agricultural Biology, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju, 561-756, Republic of Korea

      Yi-Ting Yang & Jae Su Kim

    3. Department of Entomology, National Taiwan University, Taipei, Taiwan, R.O.C

      Chih-Yu Wu & Chung-Hsiung Wang

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    1. Yu-Shin Nai
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    Yu-Shin Nai and Yi-Ting Yang contributed equally as the first author.

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    13355_2016_403_MOESM1_ESM.tiff

    Supplement Fig. 1. Ly-IAP3-RING showed ubiquitination activity under proteasome inhibitor MG-132 treatment. Ly-iap3-RING transfected Sf9 cells were treated with DMSO (negative control) and 10 μM MG-132 after heat shock. At 1 h post-heat shock, the cell lysates were harvested and subjected to SDS/PAGE, and the gels were stained with Coomassie blue (loading control) or used for Western blot analysis with α-V5 antibody. A slight increase in the protein expression level was detected in MG-132-treated cells. (TIFF 835 kb)

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    Nai, YS., Yang, YT., Kim, J.S. et al. Baculoviral IAP2 and IAP3 encoded by Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) suppress insect cell apoptosis in a transient expression assay. Appl Entomol Zool 51, 305–316 (2016). https://doi.org/10.1007/s13355-016-0403-x

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