Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage

Accepted Manuscript Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage Qi Tang, Peng Wu, Zhaoyang Hu, Yanhua Yang, Lipeng Qiu, Hanqing Liu, Shanying Zhu, Zhongjian Guo, Hengchuan Xia, Keping Chen, Guohui Li PII: DOI: Reference:

S0022-2011(17)30199-4 http://dx.doi.org/10.1016/j.jip.2017.08.004 YJIPA 6979

To appear in:

Journal of Invertebrate Pathology

Received Date: Revised Date: Accepted Date:

20 April 2017 31 July 2017 5 August 2017

Please cite this article as: Tang, Q., Wu, P., Hu, Z., Yang, Y., Qiu, L., Liu, H., Zhu, S., Guo, Z., Xia, H., Chen, K., Li, G., Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage, Journal of Invertebrate Pathology (2017), doi: http://dx.doi.org/10.1016/j.jip.2017.08.004

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Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage Qi Tang1, Peng Wu1, Zhaoyang Hu1,Yanhua Yang1, Lipeng Qiu1, Hanqing Liu2, Shanying Zhu3, Zhongjian Guo1, Hengchuan Xia1, Keping Chen1,*,Guohui Li1,* 1

Institute of Life Sciences, Jiangsu University, 301# Xuefu Road, Zhenjiang 212013, China.

2

Center for Nano Drug/ Gene Delivery and Tissue Engineering, Jiangsu University, Zhenjiang,

301# Xuefu Road, Zhenjiang 212013, China. 3

School of the Environment and Safety Engineering, Jiangsu University, 301# Xuefu Road,

Zhenjiang 212013, China. *Corresponding author. E-mail address: [email protected] (K. Chen), [email protected] Fax: +86 511 8791923.

1

Abstract: It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage.

The localization of Bm65 was studied using enhanced green

fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid.

We also showed that

after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light. Keywords：BmNPV, Bombyx mori, Bm65, UV radiation. 1. Introduction Baculoviruses are arthropod-specific, enveloped, double-stranded DNA viruses, and can be used as environmentally friendly biopesticides (Herniou et al. 2003; Jehle et al. 2006). UV radiation reduces the viability of baculoviruses (Black et al. 1997; David

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and Gardiner 1967). Exposure to UV radiation can cause significant DNA damage. UV radiation can be divided into three parts: UVA, UVB, and UVC. UVC can be completely absorbed by the ozone layer. UVA is the least harmful part of UV radiation. UVC and the short wave part of UVB can cause DNA crosslinking such as CPDs and 6-4PPs. UVC is often chosen to induce DNA damage, because UVC is very hazardous to organisms (Ananthaswamy et al. 1990; Hollosy, et al. 2002; Fotouhi, et al. 2015; Surjadinata, et al. 2017). Some viruses can repair UV-induced DNA damage using host enzymes or their own enzymes (Millhouse et al. 2012; Friedberg et al. 1995; Furuta et al. 1997). Nucleotide excision repair (NER) is a very important pathway for repair of UV-induced DNA damage and is responsible for the removal of bulky adducts such as CPDs caused by UV.

Amongst sequenced baculoviruses, genes encoding photolyase

homologs with possible or demonstrated DNA repair activity have been identified in Trichoplusia ni single nucleocapsid nucleopolyhedrovirus, Pseudoletia includens nucleopolyhedrovirus

(NC_026268.1),

Spodoptera

frugiperda

granulovirus

(NC_026511.1), Spodoptera litera granulovirus (NC_009503.1), Sucra jujuba nucleopolyhedrovirus

(NC_028636.1),

Lymantria

dispar

multiple

nucleopolyhedrovirus isolate BNP (NC_001973.1), and Chrysodeixis chalcites nucleopolyhedrovirus (NC_007151.1) (Van et al. 2004; Van et al. 2005; Biernat et al. 2012; Willis et al. 2005; Wang et al. 2008; Xu et al. 2008, Biernat et al. 2011, Rabalski et al. 2016). However, the mechanisms and pathways for the repair of UV-induced damage of baculovirus DNA are not understood. Knowledge of such

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mechanisms and pathways may lead to ways to improve the baculovirus insecticides. Bombyx mori nucleopolyhedrovirus (BmNPV) specifically infects silkworms. Based on sequence comparisons, the BmNPV ORF Bm65 is a predicted member of the (GIY-YIG) nuclease superfamily (Tang et al. 2015) with 99% homology to Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) ORF79 (Ac79). Bm65 shares structural similarity with bacterial UvrC and intron-encoded endonucleases (Wu et al. 2012; Aravind et al. 1999). The bacterial UvrC family of nucleases was found to participate in the NER pathway for UV-induced photoproducts (Sancar et al. 1983). A previous study found that Bm65 improves the survival rate of Escherichia coli and BmNPV budded viruses (BVs) after UV radiation (Tang et al. 2015). In this study, additional lines of evidence are presented from a variety of assays that show that Bm65 may play a direct role in the repair of UV-induced DNA damage, or may directly affect the host’s ability to repair UV-induced damage. 2. Materials and methods 2.1 Plasmids, viruses and cells The plasmid pHTB-Pie-1-egfp was constructed by Dr. Guohui Li (Li et al. 2015) in our laboratory and was used to express the control protein, enhanced green fluorescent protein (EGFP). The plasmid pHTB-Pie-1-Bm65-egfp for expression of the fusion protein, Bm65-EGFP, was constructed according to methods described by Tang et al (Tang et al. 2015). Plasmids pPph-mCherry and pPie1-mCherry expressing the mCherry reporter gene were constructed previously (Guo et al. 2015). The mCherry encoding

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sequence was retrieved from pPph-mCherry by digestion with BamHI and HindIII, and then cloned into BamHI/HindIII sites of pPie-1 to generate the vector pPie1-mCherry. BmN cells were cultured at 27 °C in TC-100 insect medium supplemented with 10% fetal bovine serum (Gibco / Thermo Fisher Scientific, Waltham, MA, USA). 2.2 Subcellular localization of the Bm65 fusion protein during transient expression BmN cells were cultured on coverslips placed in a 60-mm dish and transfected with pHTB-Pie-1-Bm65-egfp (2 μg/well) using Cellfectin Reagent (Invitrogen, Carlsbad, CA, USA). As a control, BmN cells were transfected with pHTB-Pie-1-egfp to express EGFP. After culturing for 24 hours at 27 °C, the cells were washed with phosphate buffered saline (PBS, pH 7.2). Then, the cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized in 0.1% Triton X-100 for 15 min. After washing with PBS three times, the cells were stained with DAPI (Sigma, USA) for 10 min, washed with PBS. The coverslips with cells were removed from the dish and observed under a confocal microscope (Leica, Germany). 2.3 Host cell reactivation assay Host cell reactivation assay was performed according to methods described by Tan et al. (Tan et al. 2014) with some modifications. UV-induced DNA damage to a whole plasmid vector and its repair has been measured by reporter gene mCherry expression. BmN cells were seeded in 24-well plates at a density of 2.0×104 cells per well and incubated for 24 h at 27 °C. We exploited pHTB-Pie-1-mCherry, which can express mCherry under the control of the ie-1 promoter. The pHTB-mCherry construct was dissolved in 1 mL PBS buffer and irradiated with varying doses of UVC (254 nm

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wavelength) light generated by a hand-held wand. Unirradiated or irradiated pHTB-mCherry

(2

μg/well)

were

then

co-transfected

with

either

pHTB-Pie-1-Bm65-egfp (1 μg/well) or pHTB-Pie-1-egfp in BmN cells. pHTB-Pie-1-egfp plasmid was used as a control. EGFP intensity was observed under a fluorescent microscope to assess transfection efficiency. At 24, 48 and 72 hpt, the extent of UV-induced DNA damage was then quantitatively assessed by monitoring the expression of mCherry fluorescent protein simultaneously. The mCherry quantity expressed from unirradiated pHTB-mCherry in cells transfected with pHTB-Pie-1-egfp was set as 100%, with mCherry levels in other treatments expressed as quantities relative to the unirradiated pHTB-mCherry. Each sample was assayed in triplicate and experiments were performed three times. Statistical analysis was performed using single factor analysis of variance. 2.4 UV-induced DNA damage assays UV-induced DNA damage was measured using an OxiSelect Cellular UV-Induced DNA Damage ELISA Kit (CPDs; Cell Biolabs Inc, San Diego, CA, USA) according to the manufacturer’s instructions. The Kit is an enzyme immunoassay developed for rapid detection of CPDs in genomic DNA of cultured cells. Cells were seeded in 96-well culture plates at a density of 2×104 cells/well and incubated for 24 h at 27 °C. Cells were then transfected with either pHTB-Pie-1-Bm65-egfp or pHTB-Pie-1-egfp. pHTB-Pie-1-egfp plasmid was used as a control. The cells were observed under a fluorescence microscope and decide that there were no visually obvious differences in EGFP expression. Subsequently, cells were irradiated with an incident UVC dose of

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30 J/m2. After culturing for 0, 3, 12 or 24 h, the medium was removed and cells were treated according to the manufacturer’s instructions. Finally, CPDs were quantified by measuring the absorbance of each well at 450 nm using a microplate reader. CPD levels in each treatment were expressed as a percentage of the value obtained from the egfp-transfected cells at 0 h post UV irradiation. Each sample was assayed in triplicate and experiments were performed three times. Statistical analysis was performed using single factor analysis of variance. 2.5 Cell viability analysis Cell viability was measured by the Cell Counting Kit-8 assay (CCK8, Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. Cells were seeded in 96-well culture plates at a density of 5×103 cells/well and incubated for 24 h at 27 °C. These cells were then transfected with either pHTB-Pie-1-Bm65-egfp or pHTB-Pie-1-egfp. The egfp-transfected cells were used as controls. EGFP intensity was observed under a fluorescent microscope to assess transfection efficiency. Subsequently, these cells were irradiated with varying doses of UVC. After 24 h, the cell monolayer was rinsed with PBS three times and then 10 µl of CCK8 solution was added. Cells were then incubated for 2 h at 27 °C. The absorbance was measured by a microplate reader set to a wavelength of 450 nm. Cell viability was expressed as a percentage of the value obtained from the egfp-transfected cells without UV radiation. Each sample was assayed in triplicate and experiments were performed three times. Statistical analysis was performed using single factor analysis of variance.

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3. Results 3.1 Subcellular localization of the Bm65 fusion protein during transient expression The intracellular localization of Bm65 was studied using EGFP fusion constructs transiently expressed in insect cells. In order to detect the subcellular localization of the Bm65-EGFP fusion protein, BmN cells were transfected with either pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp, and cells observed by confocal fluorescent microscopy. Transient expression assays in insect cells showed that Bm65-EGFP mainly accumulated within the nucleus at 24 h post-transfection. Cells transfected with the control plasmid pHTB-Pie-1-egfp were found to display uniformly distributed EGFP intensity within the cytoplasm and nucleus (Fig. 1). These results suggest that Bm65 can enter the nucleus in the absence of other BmNPV proteins.

Figure. 1 Subcellular localization of the Bm65 fusion protein in cells during transient expression. BmN cells were transfected with either pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp and observed by confocal fluorescent microscopy at 24 h post-transfection.

3.2 Host cell reactivation assays Using a host cell reactivation assay, we observed that BmN cells expressing Bm65 had an increased capacity to repair a UV-damaged reporter gene. UV-irradiated plasmids containing the mCherry reporter gene were co-transfected with either plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp in BmN cells. The results showed there was no difference in the expression yield of unirradiated mCherry between Bm65-transfected cells and egfp-transfected cells. The quantity of mCherry in each

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treatment was expressed as a percentage of the value obtained from the cells which were co-transfected with pHTB-Pie-1-egfp plasmid and unirradiated pHTB-mCherry. At 24, 48 and 72 hours post transfection (hpt), the extent of UV-induced DNA damage was quantitatively observed by monitoring cells expressing mCherry. The fluorescent images showed that mCherry expression decreased markedly with increasing UVC dose from 500 to 1500 J/m2 (Fig. 2 A). Since UV damage reduces mCherry expression from the pHTB-Pie-1-mCherry plasmid, the level of normalized mCherry is an indirect measure of the degree of DNA repair. Furthermore, the ratio of mCherry expression was analyzed at 72 hours post-transfection (Fig. 2 B). After UV radiation, the ratio of mCherry expression in Bm65-transfected cells was higher than egfp-transfected cells. The difference was highly significant when the UV dose was 1000 or 1500 J/m2. The higher expression yield of reporter proteins in cells co-transfected with Bm65 indirectly indicated greater reporter gene repair, suggesting Bm65 may be involved in the dark repair of UV-induced DNA damage.

Figure. 2 Host cell reactivation assay. A). Fluorescent images of BmN cells. The plasmid pHTB-Pie-1-mCherry was UV-irradiated or unirradiated and co-transfected into BmN cells with 1 µg of plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp. Triplicate dishes received the same transfection mixture. At 24, 48 and 72 hpt, cells were observed under a fluorescence microscope. B). mCherry expression analysis. At 72 hpt, the level of mCherry was expressed as a percentage of the value obtained from the cells which were co-transfected with pHTB-Pie-1-egfp plasmid and

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unirradiated pHTB-mCherry. Error bars indicate the mean ± SD from three independent experiments. ** indicates a statistically significant difference (P < 0.01).

3.3 UV-induced DNA damage assays The Cellular UV-induced DNA Damage ELISA Kit was used to measure the levels of CPDs in the presence and absence of Bm65 expression. There was no difference in the

absorbance

representing

CPDs

between

Bm65-transfected

cells

and

egfp-transfected cells at 0 h after UV irradiation. CPD levels in each treatment were expressed as a percentage of the value obtained from the egfp-transfected cells at 0 h post UV irradiation. The results showed that the CPD levels in Bm65-transfected cells were lower than in egfp-transfected cells at 3 h or 12 h post UV irradiation (Fig. 3). These results suggest that Bm65 may assist the repair of UV-induced DNA damage.

Figure. 3 DNA damage induced by UV light in BmN cells. After transfection with the plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp, cells were exposed to 30 J/m2 UVC light. At 0, 3, 12 or 24 h post UV irradiation, the absorbance of each microwell was read at 450 nm. CPD levels were expressed as a percentage of the value obtained from the egfp-transfected cells at 0 h post UV irradiation. Error bars indicate the mean ± SD from three independent experiments. ** indicates a statistically significant difference (P < 0.01).

3.4 Cell viability analysis In order to determine the UVC radiation sensitivity of BmN cells expressing Bm65, cell proliferation assays were conducted. After 10 or 50 J/m2 UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells (Fig.

10

4). However, the viability of cells treated with 100 J/m2 UVC was low, and there was no significant difference between the two groups. Therefore, these results demonstrate a correlation between Bm65 expression and reduced sensitivity of host cells to low-dose UVC radiation.

Figure. 4 Effect of Bm65 on BmN cell viability after UV treatment. After being transfected with the plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp, cells were exposed to an increase in UVC dose from 10 to 100 J/m2. At 24 h post UV irradiation, cell viability analysis was performed. Error bars indicate the mean ± SD from three independent experiments. * indicates a statistically significant difference (P < 0.05).

4. Discussion In this study, transient expression assays in BmN cells showed that the Bm65-EGFP fusion protein was localized in the nucleus. Using a host cell reactivation assay, cells expressing Bm65 were found to display an enhanced repair capacity based on rescue of the UV-damaged reporter gene. These results indicate that Bm65 might enter the nucleus and play a role in repairing UV-induced DNA damage in the absence of other BmNPV proteins. Furthermore, CPD levels in Bm65-transfected cells was lower than in egfp-transfected cells at 3 h or 12 h post UV irradiation. The nucleotide excision repair also found in the silkworm (Xu et al. 2009). After low doses of UV radiation, the host cells will also repair the damage. Therefore, there is little difference between Bm65-transfected cells and egfp-transfected cells in the late time after UV irradiation. It was speculated that Bm65 might help host cells to repair UV-induced DNA damage

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more quickly in the early time after UV irradiation. Viruses can interact with host cells to direct activation and deactivation of DNA damage response pathways (Hollingworth et al. 2015). Previous findings showed that viral infection often inhibited NER, and some viral proteins were found to interfere with the NER pathway. Human hepatitis B virus X protein and hepatitis C virus core protein were found to decrease the repair capacity of their host to UV-induced lesions (Jia et al. 1999; O'Dowd et al. 2012; van et al. 2004). Only a few viral proteins are reported to have a positive effect on the host cell response to DNA damage. The human immunodeficiency virus type 1 Tat protein was found to increase cell survival in response to cisplatin (Galina et al. 2004). In this study, the results showed that Bm65 expression in uninfected host cells could improve their viability after UV treatment. The mechanism of Bm65 in uninfected and virus-infected host cells requires further investigation. Acknowledgements: We would like to thank Dr. Zhongjian Guo of Institute of Life Sciences, Jiangsu University, China for kindly providing pPie1-mCherry. The research were supported by the National Natural Science Foundation of China (Nos. 31402016, 81402840, 31372259, 81402145 and 31300138), China Postdoctoral Science Foundation funded project (No. 2015M571675), Start-Up Research Funding of Jiangsu University (No. 14JDG026) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Natural Science Foundation of Jiangsu Province of China (No. BK20130495 and BK20140572). Reference Ananthaswamy, H.N., Pierceall, W.E., 1990. Molecular mechanisms of ultraviolet

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Figure. 1 Subcellular localization of the Bm65 fusion protein in cells during transient expression. BmN cells were transfected with either pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp and observed by confocal fluorescent microscopy at 24 h post-transfection.

Figure. 2 Host cell reactivation assay. A). Fluorescent images of BmN cells. The plasmid pHTB-Pie-1-mCherry was UV-irradiated or unirradiated and co-transfected into BmN cells with 1 µg of plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp. Triplicate dishes received the same 18

transfection mixture. At 24, 48 and 72 hpt, cells were observed under a fluorescence microscope. B). mCherry expression analysis. At 72 hpt, the level of mCherry was expressed as a percentage of the value obtained from the cells which were co-transfected with pHTB-Pie-1-egfp plasmid and unirradiated pHTB-mCherry. Error bars indicate the mean ± SD from three independent experiments. ** indicates a statistically significant difference (P < 0.01).

Figure. 3 DNA damage induced by UV light in BmN cells. After transfection with the plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp, cells were exposed to 30 J/m2 UVC light. At 0, 3, 12 or 24 h post UV irradiation, the absorbance of each microwell was read at 450 nm. CPD levels were expressed as a percentage of the value obtained from the egfp-transfected cells at 0 h post UV irradiation. Error bars indicate the mean ± SD from three independent experiments. ** indicates a statistically significant difference (P < 0.01).

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Figure. 4 Effect of Bm65 on BmN cell viability after UV treatment. After being transfected with the plasmid pHTB-Pie-1-egfp or pHTB-Pie-1-Bm65-egfp, cells were exposed to an increase in UVC dose from 10 to 100 J/m2. At 24 h post UV irradiation, cell viability analysis was performed. Error bars indicate the mean ± SD from three independent experiments. * indicates a statistically significant difference (P < 0.05).

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Graphical abstract: BmNPV Bm65 protein might help to repair UV-induced DNA damage (CPDs) and increase the viability of host cells after ultraviolet radiation.

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Highlights: 1: Bm65 significantly increased the expression of mCherry reporter gene damaged by UV. 2: The DNA damage induced by UV light in Bm65-transfected BmN cells decreased faster than in control cells. 3: The viability of UV-induced BmN cells which were transiently expressing Bm65-EGFP were higher than control cells.

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