The cytoplasmic terminus of Kaposi's sarcoma-associated herpesvirus glycoprotein B is not essential for virion egress and infectivity.
The cytoplasmic terminus of Kaposi's sarcoma-associated herpesvirus glycoprotein B is not essential for virion egress and infectivity.
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卡波西肉瘤相关疱疹病毒糖蛋白 B 的细胞质末端对于病毒颗粒的排出和感染性并不是必需的。
DOI：
10.1128/jvi.00617-08
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发表时间：
2008
影响因子：
5.4
通讯作者：
Kousoulas,KG
中科院分区：
文献类型：
--
作者：
Subramanian,R;D'Auvergne,O;Kong,Haixia;Kousoulas,KG
卡波西肉瘤相关疱疹病毒 (KSHV) 编码的糖蛋白 B (gB) 是病毒感染性和病毒颗粒排出的重要决定因素。设计了一种基于小干扰 RNA (siRNA) 的策略来抑制 KSHV gB 基因表达。在293细胞中瞬时共转染组成型表达gB和抗gB siRNA的质粒显着抑制了gB mRNA水平和蛋白质产生。类似地，siRNA瞬时表达到原代渗出性淋巴瘤细胞系BCBL-1中导致gB转录物和蛋白质合成大幅减少。采用针对裂解性 KSHV 基因 ORF59 的 TaqMan 实时 PCR 测定和对 293 细胞的感染性测定，分别评估抑制 gB 合成对 BCBL-1 细胞病毒体排出的影响和对 293 细胞的感染性的影响。这些实验表明 gB 对于病毒颗粒的排出和感染性至关重要。转染密码子优化的 gB 基因，其中前 540 个核苷酸发生改变，因此不会被针对天然序列而非密码子优化序列的抗 gB siRNA 识别，在存在抑制野生型 gB 表达的 siRNA 的情况下，有效地挽救了 BCBL-1 细胞中的病毒粒子流出和感染性。为了评估 gB 胞质结构域在病毒颗粒排出中的作用，生成了突变的 gB 基因，该基因指定了 25 和 58 个氨基酸的羧基末端截断，破坏了与病毒诱导的细胞融合相关的两个突出的预测 α 螺旋结构域。第三次截断去除了整个预测的 84 个氨基酸的细胞质末端，而第四次截断去除了 110 个氨基酸，包括末端最疏水的膜内锚定序列。病毒粒子流出实验表明，所有截短的 gB 都有利于病毒粒子从 BCBL-1 细胞中流出，但最大的 110 个氨基酸截短除外，它去除了 gB 锚定序列。重要的是，去除整个预测的细胞质结构域的 gB 截短增加了病毒粒子的出口，表明位于 gB 细胞质结构域内膜内序列附近的出口调节结构域的存在。所有上清病毒粒子对 293 细胞都具有感染性，这表明 gB 的羧基末端对于病毒粒子排出或病毒感染性都不是必需的。
Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded glycoprotein B (gB) is an important determinant of viral infectivity and virion egress. A small interfering RNA (siRNA)-based strategy was devised to inhibit KSHV gB gene expression. Transient cotransfection of plasmids constitutively expressing gB and anti-gB siRNAs in 293 cells substantially inhibited gB mRNA levels and protein production. Similarly, transient expression of siRNAs into the primary effusion lymphoma cell line BCBL-1 caused a substantial reduction of gB transcripts and protein synthesis. TaqMan real-time PCR assays against the lytic KSHV gene ORF59 and infectivity assays on 293 cells were employed to assess the effect of inhibiting gB synthesis on virion egress from BCBL-1 cells and infectivity on 293 cells, respectively. These experiments showed that gB was essential for virion egress and infectivity. Transfection of a codon-optimized gB gene with the first 540 nucleotides altered, and therefore not recognized by anti-gB siRNAs that target the native but not the codon-optimized sequence, efficiently rescued virion egress and infectivity in BCBL-1 cells in the presence of siRNAs inhibiting wild-type gB expression. To assess the role of the cytoplasmic domain of gB in virion egress, mutant gB genes were generated specifying carboxyl terminal truncations of 25 and 58 amino acids disrupting two prominent predicted α-helical domains associated with virus-induced cell fusion. A third truncation removed the entire predicted cytoplasmic terminus of 84 amino acids, while a fourth truncation removed 110 amino acids, including the terminal most hydrophobic, intramembrane anchoring sequence. Virion egress experiments revealed that all truncated gBs facilitated virion egress from BCBL-1 cells, with the exception of the largest 110-amino-acid truncation, which removed the gB anchoring sequence. Importantly, the gB truncation that removed the entire predicted cytoplasmic domain increased virion egress, suggesting the presence of a egress regulation domain located proximal to the intramembrane sequence within the cytoplasmic domain of gB. All supernatant virions were infectious on 293 cells, indicating that the carboxyl terminus of gB is not essential for either virion egress or virus infectivity.