黑角舞蛾核多角體病毒基因體解序及一種類吉普賽舞蛾核多角體病毒特性之分析

Genomic Anlyses of LyxyMNPV and the Characterization of an LdMNPV-like Virus

乃育昕(Yu-Shin Nai)

指導教授 ： 王重雄

共同指導教授 ： 柯俊成(Chiun-Cheng Ko)

國立臺灣大學/生物資源暨農學院/昆蟲學研究所/博士(2010年)

https://doi.org/10.6342/NTU.2010.00481

摘要

黑角舞蛾 (Lymantria xylina Swinhoe) 為台灣林木及果樹類重要害蟲，本實驗室由野外罹病蟲體分離出兩種病毒病原體，皆屬桿狀病毒科，Alphabaculovirus 屬。在野外黑角舞蛾族群中以 LyxyMNPV 感染為主要，將 LyxyMNPV 基因體解序，並進行深入研究。LyxyMNPV 基因體大小為 156,344 bp，GC 百分比為 53.47%，其中包含 157 個開放譯讀區 (open reading frame, ORF)，13 個同源區域 (homologus regsions, hrs) 及 14 個桿狀病毒重複開放譯讀區 (baculovirus repeat orf, bro)。親緣關係分析上顯示 LyxyMNPV 屬於 group II NPV 與 LdMNPV 最為接近，唯基因排列上有所差異。LyxyMNPV 157 個 ORFs 中，包括 151 個 ORFs 與 LdMNPV 互為同源基因；2 個 ORFs 與其他桿狀病毒之基因同源；4 個 ORFs為特有基因，而其中一個為桿狀病毒中首次發現之特有基因，為 gag-like 的同源基因。LyxyMNPV 具有兩個非經由複製產生之 odv-e27 同源基因。LyxyMNPV 缺乏 host range factor-1 (hrf-1)，但仍可感染 NTU-LY-1 (LY) 及 IPLB-LD-652Y (LD) 細胞；進一步比較三種可感染 LD 細胞之病毒基因體 (LyxyMNPV、LdMNPV 及 OpMNPV)，結果顯示其共同擁有三個基因：inhibitor of apoptosis-3 (iap-3)、ribonucleotide reductase 2b (rr2b) 及 dutpase，其中 iap 基因在病毒感染細胞初期扮演非常重要的角色。
LyxyMNPV 具有兩個 iap 基因：iap-2 (687 bp) 與 iap-3 (450 bp)，經轉錄本分析結果顯示，iap-2 之轉譯起始點 (ATG) 上游 (upstream) 第15個鹼基對具有一個晚期及非常晚期基因轉錄起始位置 (TAAG)，而 iap-2 之轉錄起始點位於 ATG 上游第 16 個鹼基對；iap-3 之轉錄起始點位於 ATG 上游第 44 個鹼基對上之 CTTGT 保守區域，而保守區域下游第 25 個鹼基對接有 TCGT 序列；利用反轉錄-聚合酶鏈鎖反應得知此二基因 mRNA 表現量在 LyxyMNPV 感染寄主 LD 細胞後 6 小時開始上升至 72 小時，但於 72 小時後便開始下降直至 120 小時；而在 SF 細胞中，iap-2 之mRNA 表現量至感染後 120 小時仍相當低，而 iap-3 之mRNA 表現量在感染 48 小時後開始表現，且持續上升至感染後 120 小時。進一步在 SF 細胞株中利用共表現實驗 (co-expression) 進行蛋白質功能截斷分析，結果顯示 IAP-2、IAP-3 及 IAP-2-BIR 均可抑制由果蠅 RPR 蛋白所誘導之細胞凋亡反應 (P<0.05) 而 IAP-3-BIR、IAP-2-RZF 及 IAP-3-RZF在抑制由果蠅 RPR 蛋白所誘導之細胞凋亡反應則無顯著功效，推測 IAP-3 需要全長表現才具抑制由果蠅 RPR 蛋白所誘導之細胞凋亡效果。
另一株由黑角舞蛾分離出的病毒，經由 polyhedrin，lef-8 及 lef-9 基因序列分析，証實此病毒係異於 LyxyMNPV 而近於 LdMNPV的病毒，因此命名為 LdMNPV-like virus，其感染 LD 細胞呈現典型寡多角體 (Few polyhedra, FP) 病徵，經由選殖定發現其 fp25k 基因缺失 44 bp。此病毒是首次野外所發現呈現 FP 的病毒。

關鍵字

黑角舞蛾核多角體病毒 ； 基因體解序 ； 抑制細胞凋亡基因 ； 基因表現 ； 功能分析 ； 類吉普賽舞蛾核多角體病毒

並列摘要

The casuarina moth, Lymantria xylina Swinhoe (Lepidoptera: Lymantriidae), is an important forest pest in Taiwan. Two nucleopolyhedrovirus (NPV) strains were isolated from L. xylina larvae with an epizootic nucleopolyhedrosis. In the field, LyxyMNPV is the most prevalent vius strain, therefore, the nucleotide sequence was determined and analysed. The genome of LyxyMNPV consists of 156,344 bases with a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The LyxyMNPV genome encoded 14 bro genes. 13 homologous regions (hrs) were identified. In a phylogenetic analysis, LyxyMNPV is a member of Group II NPV and closely related to LdMNPV but with highly distinct genomic organization. The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified homologous to those reported in the LdMNPV genome. Two genes were homologous to other baculoviruses and four unique ORFs were identified in LyxyMNPV genome, including a gag-like gene which was not reported in baculoviruses. Two putative odv-e27 homologues were identified in LyxyMNPV, each of which has been acquired independently and not by gene duplication. LyxyMNPV lacks host range factor-1 (hrf-1). However, in vitro host range assay indicated that LyxyMNPV could infect both NTU-LY and IPLB-LD-652Y cell lines with the absence of hrf-1. Therefore, we compared LyxyMNPV gene content to those of LdMNPV and OpMNPV, and found that these three NPVs shared three genes, iap-3, rr2b, and dutpase, that are absent in AcMNPV genome, of these three genes, iap-3 is one of the baculovirus genes that affect viral host range and prevent apoptosis in baculovirus-infected cells.
Two groups of anti-apoptotic genes, p35 and iap (inhibitor of apoptosis) family, have been identified in baculovirus. There are two iap genes, iap-2 (687 bp) and iap-3 (450 bp), in LyxyMNPV genome. The 5’UTR of iap-2 contains a transcriptional initiation site, TAAG, of late and very late gene motif, but 5’UTR of iap-3 contains CTTGT promoter motif plus TCGT in 25 bp spacing context. The mRNA expression profiles of these two genes in permissive cell line LD were evaluated using RT-PCR. The expression levels of iap-2 and iap-3 in the LD cells infected with LyxyMNPV raised from 3 to 72 hours post-infection (p.i.), but declined after 72 to 120 hours. Interestingly, the expression level of iap-2 and iap-3 of the infected SF cells showed significant difference. The mRNA of iap-2 was not detected; in contrary, iap-3 was detectable. During the process of infecting SF cells with LyxyMNPV, iap-3 presented a delayed expression pattern, which was detected at 48 hours p.i. and continued to rise for 120 hours. Functional assay of two iap genes was performed using over-expression method. In SF cells, full length IAP-2, IAP-3 and IAP-2-BIR domain inhibited the apoptosis induced by Drosophila RPR protein (P<0.05), however, IAP-3-BIR domain could not rescue the apoptosis induced by D-RPR.
LdMNPV-like virus was nominaded base on the sequences analyses of polyhedrin, lef-8 and lef-9. This virus was closely related to LdMNPV but far from LyxyMNPV. LdMNPV-like virus showed a few polyhedra (occlusion bodies) CPE in the infected LD cells. A significant deletion of a 44 bp sequence found in LdMNPV-like virus was noted in the fp25k sequences of LdMNPV and LyxyMNPV and may play an important role in the few polyhedra CPE. This is the first FP virus isolated from the field-collected larvae infected with NPV.

並列關鍵字

LyxyMNPV ； genomic sequence ； antiapoptosis genes ； gene expression ； functional assay ； LdMNPV-like virus

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