Functional analysis of a small basic accessory protein encoded by paramyxovirus

麻疹病毒(MV)C蛋白是一种小而基本的非结构蛋白，但其功能尚不清楚。首先，我们利用反向遗传学系统产生了一个缺乏C蛋白的重组野生型MV，称为WTMV(C-)。WTMV(C-)在B95a细胞中生长正常，但在293/hSLAM细胞中生长不正常。表达EGFP的WTMV(C-)持续诱导后期细胞圆形和细胞死亡，提示C蛋白可以防止细胞死亡，是长期感染MV所必需的。当食蟹猴感染WTMV(C-)或WTMV时，WTMV(C-)感染的胸腺细胞数比WTMV(C-)少1000倍。免疫组织化学分析显示，在感染WTMV(C-)的食蟹猴的脾、淋巴结、扁桃体和喉部有MV抗原的强烈表达。这些结果表明，无论是在体外还是在食蟹猴体内，MV C蛋白都是MV高效复制所必需的。其次，我们发现，从cDNA表达的标记野生型MV C蛋白仅在细胞核中积累。突变分析表明，经典的核定位信号^&lt；41&gt；PPARKRRQ^&lt；48&gt；对核定位具有重要作用。C蛋白的氨基酸序列分析表明，它具有核输出信号(NES)样序列，^&lt；76&gt；LEKAMTTLKL^&lt；85&gt；将假定的NES加入到EGFP中，导致EGFP移位到细胞质中。REV(1.4)-EGFP核出口分析表明，该NES具有依赖CRM1的NES活性。异核体分析表明，c-EGFP在HeLa核中可转移到NIH3T3核中。在MV感染的细胞中，C-EGFP在早期积聚在细胞核内，而在晚期积聚在细胞质中。这些结果表明，NES具有一定的功能，C蛋白具有穿梭于胞核和胞质之间的能力。

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.