中山大学袁美妗组在杆状病毒功能基因研究方面取得新进展

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中山大学生命科学学院袁美妗组在杆状病毒功能基因研究方面取得重要进展，研究揭示了高度保守的ac75基因可影响核衣壳出核和核内微囊泡的产生。该成果在线发表于国际病毒学期刊Journal of Virology上。原文题为“Autographa californica Nucleopolyhedrovirus ac75 is Required for the Nuclear Egress of Nucleocapsids and Intranuclear Microvesicle Formation”。

杆状病毒是专一性感染节肢动物的病毒，其基因组为大型环状双链DNA，核衣壳呈杆状，外有囊膜包裹。苜蓿丫蚊夜蛾核多角体病毒（Autographa californica multiple nucleopolyhedrovirus，AcMNPV）是杆状病毒的代表种。杆状病毒的基因组复制和核衣壳装配均在宿主细胞的细胞核中完成。成熟的核衣壳运出细胞核，在细胞质膜处出芽，形成芽生型病毒粒子（budded virion，BV）；或者滞留在细胞核内，被病毒诱导产生的核内微囊泡包裹，形成包埋型病毒粒子（occlusion-derived virion，ODV）。已有的研究表明，核衣壳主要通过从核膜出芽进入细胞质，而核内微囊泡则是由核膜向核质方向出芽而成。因此，两种病毒粒子的形态发生都包括核膜出芽这一过程，但具体的机制尚不清楚。

AcMNPV的ac75基因在杆状病毒中高度保守，除了侵染蚊子的杆状病毒外，其他已测定全基因组序列的杆状病毒中均存在ac75的同源基因，但其在杆状病毒生活周期中的功能未知。研究人员构建了ac75基因缺失型重组病毒以及融合标签蛋白编码序列的补回型重组病毒，研究该基因的缺失对病毒复制的影响。研究结果显示缺失了ac75基因后，核衣壳滞留在细胞核内无法出核，核内微囊泡也无法形成，从而导致BV和ODV的产生均被阻断（图一）。

研究人员进一步检测了Ac75蛋白的表达特征、定位及其与其他病毒蛋白的互作，发现ac75是晚期基因，可表达分子量不同（18 kDa和15 kDa）的两种蛋白产物。有趣的是，18 kDa的蛋白分布在BV的核衣壳和囊膜上，而15 kDa的蛋白仅分布在BV和ODV的核衣壳上。免疫荧光分析结果显示Ac75在感染早期沿细胞核膜呈环状分布，在感染后期则主要分布于核内环带区。然而，相分离实验表明，Ac75不是整合膜蛋白，但其可与目前发现的唯一一个同样影响核内微囊泡产生的整合膜蛋白Ac76在细胞质、核膜和核内环带区发生相互作用（图二），暗示Ac75可能是通过与整合膜蛋白Ac76的相互作用结合到核膜上，并影响核膜的功能。

继ac93之后，本研究鉴定了第二个同时影响核衣壳出核和核内微囊泡产生的杆状病毒基因——ac75。该基因的功能鉴定和深入研究有助于揭示核衣壳出核和核内微囊泡产生的共同步骤的分子机制，进而促进我们对杆状病毒两种病毒粒子形态发生机制的理解。

中山大学生命科学学院袁美妗副教授为论文的通讯作者，石安琪博士研究生和胡朝阳博士为论文的并列第一作者。本研究由国家自然科学基金面上项目和广州市科技计划项目资助完成。

ABSTRACT

Autographa californica nucleopolyhedrovirus (AcMNPV) orf75 (ac75) is a highly conserved gene of unknown function. In this study, we constructed an ac75knockout AcMNPV bacmid and investigated the role of ac75 in the baculovirus life cycle. The expression and distribution of the Ac75 protein were characterized, and its interaction with another viral protein was analyzed to further understand its function. Our data indicated that ac75 was required for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent budded virion (BV) formation, as well as occlusion-derived virion (ODV) envelopment and embedding of ODVs into polyhedra. Western blot analyses showed that two forms of 18 and 15 kDa of FLAG-tagged Ac75 protein were detected. Ac75 was associated with both nucleocapsid and envelope fractions of BV, but only the nucleocapsid fraction of ODV; the 18-kDa form was associated with only BVs, whereas the 15-kDa form was associated with both types of virion. Ac75 was predominantly localized in the intranuclear ring zone during infection and exhibited a nuclear rim distribution during the early phase of infection. A phase-separation assay suggested that Ac75 was not an integral membrane protein. A coimmunoprecipitation assay revealed an interaction between Ac75 and the integral membrane protein Ac76, and bimolecular fluorescence complementation assays identified the sites of the interaction within the cytoplasm, at the nuclear membrane and ring zone in AcMNPV-infected cells. Our results have identifiedac75 as a second gene that is required for both the nuclear egress of nucleocapsids and the formation of intranuclear microvesicles.

IMPORTANCE

During the baculovirus life cycle, the morphogenesis of both budded virions (BVs) and occlusion-derived virions (ODVs) is proposed to involve a budding process at the nuclear membrane, which occurs while nucleocapsids egress from the nucleus or when intranuclear microvesicles are produced. However, the exact mechanism of virion morphogenesis remains unknown. In this study, we identified ac75 as a second gene, in addition to ac93, that is essential for the nuclear egress of nucleocapsids, intranuclear microvesicle formation, and subsequent BV formation, as well as ODV envelopment and embedding of ODVs into polyhedra. Ac75 is not an integral membrane protein. However, it interacts with an integral membrane protein (Ac76) and is associated with the nuclear membrane. These data enhance our understanding of the commonalities between nuclear egress of nucleocapsids and intranuclear microvesicle formation and may help to reveal insights into the mechanism of baculovirus virion morphogenesis.

来源：中国病毒学英文版

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