黑角舞蛾核多角體病毒融合蛋白基因之選殖及其在吉普賽舞蛾細胞之表現

Abstract

以黑角舞蛾核多角體病毒 (Lymantria xylina multiple nucleopolyhedrovirus; LyxyMNPV) 感染吉普賽舞蛾細胞株 (IPLB-LD652Y-5d) 後，發生細胞融合形成一大的多核細胞，此細胞融合現象可能與病毒套膜融合蛋白有關。核多角體病毒的套膜蛋白質包括 gp64 與 ld130 基因，此兩個蛋白質在低 pH 值的誘導下，會導致感染的細胞融合。第一群核多角體病毒的套膜融合蛋白為 GP64；第二群病毒則為 LD130。黑角舞蛾核多角體病毒之 ld130 基因全長為 2,025 個鹼基對。黑角舞蛾核多角體病毒與吉普賽舞蛾核多角體病毒的 ld130 之核苷酸與胺基酸序列，相似度分別為 93 與 96 %。電腦分析黑角舞蛾核多角體病毒的 LD130 蛋白質序列，發現具有一訊息肽 (signal peptide) 和穿膜區域 (transmembrane domain)，預測其為一膜蛋白。黑角舞蛾核多角體病毒 ld130 基因轉錄本 (transcript) 之分析顯示，其 5’ 端序列具有早期和晚期轉錄起始點。而在 3’ 端的聚腺嘌呤序列位於加聚腺嘌呤訊號 (ATTAAA, poly A signal) 下游第 17 個核苷酸的位置。利用 ld130 基因序列進行親緣關係的分析，黑角舞蛾核多角體病毒屬於第二群病毒。以西方墨漬法偵測 LyxyMNPV 的LD130 蛋白，發現 LD130 為一結構蛋白。以桿狀病毒表現系統 (baculovirus transient expression vector) 極早期基因啟動子所構築之重組 ld130 進行暫時性表現，顯示 LD130 蛋白質的表現和細胞融合有關，並且在酸的誘導下會促使細胞融合。此結果確定了黑角舞蛾核多角體病毒具有 LD130 之套膜融合蛋白，且功能上可能與核多角體病毒第二群之 LD130 相同，為酸誘導後促進細胞融合的重要蛋白質。

IPLB LD-652Y-5d cells infected with Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) were found that the infection cells were fused together and formed syncytial giant cells. This phenomenon is probably caused by the viral envelope fusion protein in the cell membrane of the infected cells. Recently, two viral genes encoding the envelope fusion proteins, GP64 and LD130, had been identified. Both GP64 and LD130 envelope fusion proteins mediate the cell fusion at low pH value. The envelope fusion protein of NPV Group I is GP64 while Group II is LD130. We cloned and sequenced ld130 from LyxyMNPV. The LyxyNPV ld130 consists of 2,025 bp. Comparison of the nucleotide and amino acid sequences of LyxyMNPV ld130 with those of LdMNPV ld130 showed 93 and 96% identities, respectively. The predicted peptide sequence of LyxyMNPV LD130 contains signal and transmembrane domains that are significant homology to membrane proteins. Analysis of LyxyNPV ld130 transcript revealed both early and late transcriptional initiation sites located at upstream of the 5’ ATG initiation codon. In addition, a poly A sequence located at 17 nt downstream of the 3’ polyadenylation signal (ATTAAA). Based on the sequences of ld130 gene, the NPV species can be divided into two Groups, I and II, in phylogenetic analysis, LyxyMNPV belongs to Group II. The LD130 of budded virus detected by Western blot revealed that LD130 is a viral structural protein. The LD130 were constructed with the promoter of immediately early gene and expressed by baculovirus transient expression vector in uninfected insect cells showed that LD130 could mediate the cell-to-cell fusion at low pH. These results suggest that a functional homolog of LD130 envelope fusion protein in group II NPVs was found in LyxyMNPV.