Manuscript Click here to download Manuscript Manuscript-Wu-R1.doc Click here to view linked References 1 Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) 1 2 2 genome and its expression strategy in LD652 cells 3 4 3 Pengjun Xu1, 2, Robert I. Graham3, Kenneth Wilson4 and Kongming Wu1* 5 6 1 7 4 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of 8 9 5 Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, P.R. China, 10 2 11 6 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 12 13 7 P.R. China 14 3 15 8 Crop and Environment Sciences, Harper Adams University, Edgmond, Shropshire 16 17 9 TF10 8NB, UK 18 19 10 4Lancaster Environment Centre, Lancaster University, Lancaster, UK 20 21 11 Email address of all authors: Pengjun Xu: [email protected]; Robert I. Graham: 22 23 12 [email protected]; Kenneth Wilson: [email protected]; 24 25 13 Kongming Wu: [email protected] 26 27 14 *Corresponding author: Kongming Wu 28 29 15 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of 30 31 16 Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuan Ming 32 33 17 Yuan Road, Beijing 100193, P. R. China. E-mail: [email protected]. 34 35 18 36 37 19 38 39 40 20 41 42 21 43 44 22 45 46 23 47 48 24 49 50 25 51 52 26 53 54 27 55 56 28 57 58 29 59 60 30 61 62 1 63 64 65 31 Abstract 1 2 32 Backgroud: Densoviruses (DVs) are highly pathogenic to their hosts. However, we 3 4 33 previously reported a mutualistic DV (HaDV2). Very little was known about the 5 6 34 7 characteristics of this virus, so herein we undertook a series of experiments to 8 9 35 explore the molecular biology of HaDV2 further. 10 11 36 Results: Phylogenetic analysis showed that HaDV2 was similar to members of the 12 13 37 genus Iteradensovirus. However, compared to current members of the genus 14 15 38 Iteradensovirus, the sequence identity of HaDV2 is less than 44% at the 16 17 39 nucleotide-level, and lower than 36%, 28% and 19% at the amino-acid-level of VP, 18 19 40 NS1 and NS2 proteins, respectively. Moreover, NS1 and NS2 proteins from HaDV2 20 21 41 were smaller than those from other iteradensoviruses due to their shorter N-terminal 22 23 42 sequences. Two transcripts of about 2.2 kb coding for the NS proteins and the VP 24 25 43 proteins were identified by Northern Blot and RACE analysis. Using specific 26 27 44 anti-NS1 and anti-NS2 antibodies, Western Blot analysis revealed a 78 kDa and a 48 28 29 45 kDa protein, respectively. Finally, the localization of both NS1 and NS2 proteins 30 31 46 within the cell nucleus was determined by using Green Fluorescent Protein (GFP) 32 33 47 labelling. 34 35 48 Conclusion: The genome organization, terminal hairpin structure, transcription and 36 37 49 38 expression strategies as well as the mutualistic relationship with its host, suggested 39 40 50 that HaDV2 was a novel member of the genus Iteradensovirus within the subfamily 41 42 51 Densovirinae. 43 44 52 Keywords: densovirus, HaDV2, expression, transcription 45 46 53 47 48 54 49 50 55 51 52 56 53 54 57 55 56 58 57 58 59 59 60 60 61 62 2 63 64 65 61 Backgroud 1 2 62 The subfamily of Densovirinae within the family Parvoviridae is a group of small 3 4 63 (18–26 nm diameter), non-enveloped, icosahedral viruses containing a linear 5 6 64 7 single-stranded DNA genome ranging between 4–6 kb with characteristic terminal 8 9 65 hairpins [1-3]. Members of this subfamily typically produce “cellular dense 10 11 66 nucleosis” pathogenesis in their hosts, hence, they are commonly termed 12 13 67 densoviruses (DVs) [4-8]. Since the first identification of a densovirus in the greater 14 15 68 wax moth Galleria mellonella [9], DVs have been isolated from many arthropods, 16 17 69 including species from six insect orders (Lepidoptera, Diptera, Orthoptera, 18 19 70 Dictyoptera, Odonata and Hemiptera) and decapod crustaceans (shrimps and crabs) 20 21 71 [10-12]. 22 23 72 To date, many DVs have been identified and sequenced. Unlike vertebrate 24 25 73 parvoviruses, which all exhibit a monosense organization of their genome with 26 27 74 nonstructural protein (NS) and structural protein (VP) open reading frames (ORFs) 28 29 75 located on the same strand, arthropod DVs possess two types of genomes: 30 31 76 monosense and ambisense [13-18]. Previously, the taxonomy of DVs was ambiguous, 32 33 77 which was based on the organization of coding sequences, as well as genome size, 34 35 78 terminal hairpin structure, gene expression strategy and host range [19]. Under the 36 37 79 38 proposal of the International Committee on Taxonomy of Viruses (ICTV), Cotmore 39 40 80 et al. [2] reconstructed the taxonomy of the family Parvoviridae in which DVs were 41 42 81 classified into five distinct genera: Ambidensovirus, Brevidensovirus, 43 44 82 Iteradensovirus, Hepandensovirus and Penstyldensovirus according to phylogenetic 45 46 83 analysis and sequence homology. 47 48 84 DVs are highly pathogenic viruses to their hosts, and have been documented as 49 50 85 being transmitted both horizontally and vertically [7, 9, 16]. Traditionally, these 51 52 86 properties have captured the interest of many researchers investigating the potential 53 54 87 application of DVs as biopesticides for biological control of insect pests or vectors 55 56 88 for transgenic insects [20-26]. However, we previously reported a novel DV 57 58 89 displaying a mutualistic interaction with its host (Helicoverpa armigera), and named 59 60 90 this virus HaDV2 (previously named HaDNV-1) to distinguish it from the HaDV1 61 62 3 63 64 65 91 reported by El-Far et al. [27-29]. In this current study, we report the genome 1 2 92 organization, transcription and expression strategies of the virus HaDV2. 3 4 93 Methods 5 6 7 94 Insect cell culture and transfection 8 9 95 Lymantria dispar LD652 cells, a gift from Central China Normal University in 10 11 96 Wuhan (China) [30], were cultured in Grace’s insect medium containing 10% fetal 12 13 97 bovine serum (FBS) and 1% Penicillin-Streptomycin (Invitrogen, Grand Island, NY, 14 o 15 98 USA) at 28 C. Purified plasmid (100 ng) containing the ORFs of NS1 and NS2 with 16 17 99 TIANpure Mini Plasmid Kit (TIANGEN, Beijing, China) was transfected into cells 18 19 100 using Cellfectin® II Reagent as recommended (Invitrogen). The luciferase activities 20 21 101 were determined using Luciferase Assay System (Promega, Madison, WI, USA). 22 23 102 Sequence analysis 24 25 103 Identity and alignment of the nucleotide and amino acid sequences was calculated 26 27 104 using CLUSTAL W software [31]. The ORFs were identified using the ORF Finder 28 29 105 (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Neighbor-joining trees with 30 31 106 Poisson-corrected distances for the DV nucleotide sequences and the amino acid 32 33 107 sequences of (NS1, NS2 and VP ORFs) were constructed using CLUSTAL W 34 35 108 software and MEGA6.0 software [32]. 36 37 38 109 Amplification of the stem-loop structure of HaDV2 genome by inverse PCR 39 40 110 The viral DNA was extracted from purified virus particles using TIANamp Genomic 41 42 111 DNA Kit (TIANGEN). According to the reported genome sequence of HaDV2 43 44 112 (GenBank accsession No.: HQ613271), three forward primers near the 3’ end (DVF1 45 46 113 [nt 4576-4595], DVF2 [nt 3891-3910], DVF3 [nt 4343-4362]), and two reverse 47 48 114 primers near the 5’ end (DVR1 [nt 1038-1057] and DVR2 [nt 832-889]) were 49 50 115 designed according to the genome sequence of HaDV2 (Table S1). PCR reactions 51 52 116 were performed using TransTaq DNA Polymerase High Fidelity (TransGen, Beijing, 53 54 117 China) and extracted viral DNA as a template. The PCR program was as follows: 30 55 56 118 s at 94 oC, 30 s at 57 oC, and 60 s at 72 oC for 40 cycles. 57 58 119 Mapping of the transcripts by 5’/3’ RACE and Northern blot 59 60 120 The 5’ and 3’ ends of the HaDV2 transcripts were amplified using the SMART 61 62 4 63 64 65 121 RACE cDNA Amplification Kit (Clontech, CA, USA), according to the 1 2 122 manufacturer’s instructions. cDNA was synthesized by RT-PCR from the total RNA 3 4 123 of migrating cotton bollworms infected by HaDV2 using primers NS3F1/UPM for 5 6 124 7 the 3’ end, NS5R1/UPM and NS5R2/UPM for the 5’ end of the NS genes, 3F1/UPM 8 9 125 for the 3’ end, VP5R1/UPM and VP5R2/UPM for the 5’ end of the VP genes, 10 11 126 respectively (Table S1). RNA (30 µg total) from insects infected by HaDV2 were 12 13 127 separated on 1.1% formaldehyde agarose gels using MOPS buffer and blotted onto a 14 15 128 positively charged nylon membrane (Roche, USA). Northern blot hybridization was 16 17 129 performed using DIG-labeled probes (DIG DNA Labeling and Detection Kit, Roche, 18 19 130 USA), according to the manufacturer’s instruction. A 543 bp NS probe (1073-1615 20 21 131 nt) and a 420 bp VP probe (4081-4500 nt) were amplified by PCR with primers pairs 22 23 132 NSF/NSR and VPF/VPR (Table S1) using 30 cycles on a thermocycler as follows: 24 25 133 30 s at 95 oC, 30 s at 50 oC, and 30 s at 72 oC.