Functional analysis of a small basic accessory protein encoded by paramyxovirus

副粘病毒编码的小碱性辅助蛋白的功能分析

• 批准号: 17590414
• 负责人: TAKEUCHI Kaoru
• 金额: $ 2.24万
• 依托单位: University of Tsukuba
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590414/
• 关键词: paramyxovirus; measles virus; C protein; subcellular localization; nuclear localization signal; nuclear export signal; pathogenicity; Magnetofection; EGFP; CRM-1; ウイルス増殖機構; カニクイザル; 免疫組織染色; インターフェロン

Measles virus (MV) C protein is a small and basic nonstructural protein, but its function is not well understood. First, we generated a recombinant wild-type MV lacking the C protein, called wtMV(C-), by using a reverse genetics system. wtMV(C-) grew normally in B95a cells, but not in 293/hSLAM cells. EGFP-expressing wtMV(C-) consistently induced late-stage cell rounding and cell death, suggesting that the C protein can prevent cell death and is required for long-term MV infection. When cynomolgus monkeys were infected with wtMV(C-) or wtMV, the number of MV-infected cells in the thymus was >1,000-fold smaller for wtMV(C-) than for wtMV. Immunohistochemical analyses showed strong expression of an MV antigen in the spleen, lymph nodes, tonsils, and larynx of a cynomolgus monkey infected with wtMV(C-). These data indicate that the MV C protein is necessary for efficient MV replication both in vitro and in cynomolgus monkeys.Second, we have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. Mutation analysis revealed that ^<41>PPARKRRQ^<48>, belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, ^<76>LEKAMTTLKL^<85>. Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1. 4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicated that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.

麻疹病毒（MV）C蛋白是一种小的碱性非结构蛋白，但其功能尚不清楚。首先，我们通过使用反向遗传学系统产生了缺乏C蛋白的重组野生型MV，称为wtMV（C-）。wtMV（C-）在B 95 a细胞中生长正常，而在293/hSLAM细胞中生长不正常。表达EGFP的wtMV（C-）持续诱导晚期细胞变圆和细胞死亡，表明C蛋白可以防止细胞死亡，并且是MV长期感染所必需的。当食蟹猴感染wtMV（C-）或wtMV时，胸腺中MV感染细胞的数量wtMV（C-）比wtMV小> 1，000倍。免疫组织化学分析显示，在感染wtMV的食蟹猴（C-）的脾脏、淋巴结、扁桃体和喉中MV抗原的强表达。这些数据表明，MV C蛋白是必要的有效MV复制在体外和在食蟹猴。第二，我们已经发现，FLAG标记的野生型MV C蛋白的cDNA表达的积累只在细胞核中。突变分析表明，^<41>PPARKRRQ^<48>是经典的核定位信号，对核定位有重要作用。C蛋白氨基酸序列分析表明，它具有核输出信号（内斯）样序列^<76>LEKAMTTLKL^<85>。将推定的内斯添加到EGFP中导致EGFP易位到细胞质中。《The Rev.》（1）4)-EGFP核输出实验表明该内斯具有CRM 1依赖的内斯活性。在异核体实验中，HeLa细胞核中积累的C-EGFP可转位到NIH 3 T3细胞核中。在MV感染的细胞中，C-EGFP在早期聚集在细胞核中，而在晚期聚集在细胞质中。这些结果表明，推测的内斯是功能性的，C蛋白具有在细胞核和细胞质之间穿梭的能力。

项目成果

Stringent Requirement for the C Protein of Wild-Type Measles Virus for Growth both In Vitro and in Macaques

• DOI: 10.1128/jvi.79.12.7838-7844.2005
• 发表时间: 2005-06
• 期刊: Journal of Virology
• 影响因子: 5.4
• 作者: K. Takeuchi;M. Takeda;N. Miyajima;Y. Ami;N. Nagata;Y. Suzaki;Jamila Shahnewaz;Shin-ichi Kadota;K. Nagata

Stringent requirement for the C protein of wild-type measles virus for growth both in vitro and in macapues.

野生型麻疹病毒在体外和猕猴体内生长对 C 蛋白的严格要求。

• 发表时间: 2005
• 期刊: Journal of Virology 79
• 作者: Kaoru Takeuchi;Makoto Takeda;Naoko Miyajima;Yasushi Ami;Noriyo Nagata;Yuriko Suzaki;Jamila Shahnewaz;Shin-ich Kadoda;Kyosuke Nagata.
• 通讯作者: Kyosuke Nagata.

The C protein of wild-type measles virus has the ability to shuttle between the nucleus and the cytoplasm.

野生型麻疹病毒的C蛋白具有在细胞核和细胞质之间穿梭的能力。

• 发表时间: 2007
• 期刊: Microbes and Infection 9
• 作者: Nishie;T.;Nagata;K.;Takeuchi;K.
• 通讯作者: K.

Enhancing of measles virus infection by magnetofectiion

磁转染增强麻疹病毒感染

• 发表时间: 2005
• 期刊: Journal of Virological Methods 128
• 作者: Chandy S;Mitra S;Sathish N;Vijayakumar TS;Abraham OC;Jesudason MV;Abraham P;Yoshimatsu K;Arikawa J;Sridharan G;Kadota S et al.
• 通讯作者: Kadota S et al.

Efficient rescue of measles virus from cloned cDNA using SLAM-expressing Chinese hamster ovary cells

使用表达 SLAM 的中国仓鼠卵巢细胞从克隆的 cDNA 中有效拯救麻疹病毒

• 发表时间: 2005
• 期刊: Virus Res. 108
• 作者: Takeda M;Ohno S;Seki F;Hashimoto K;Miyajima N;Takeuchi K;Yanagi Y.
• 通讯作者: Yanagi Y.