Touris-Otero Fernando, Martínez-Costas José, Vakharia Vikram N, Benavente Javier
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Virology. 2004 Feb 5;319(1):94-106. doi: 10.1016/j.virol.2003.10.034.
The M3 genome segment of avian reovirus 1733, which encodes the nonstructural protein microNS, is 1996 nucleotides long and contains a long open reading frame that is predicted to encode a polypeptide of 635 amino acid residues. Examination of the deduced amino acid sequence of microNS revealed the presence of two regions near its carboxyl terminus with a high probability of forming alpha-helical coiled coils. Expression of the M3 gene in both infected and transfected cells revealed that this gene specifies two protein isoforms that are recognized by a microNS-specific antiserum. Only the larger microNS isoform, but not the smaller one, interacts with the nonstructural protein sigmaNS in infected cells, suggesting that the two isoforms play different roles during avian reovirus infection. In the second part of this study, we show that microNS and the nonstructural protein sigmaNS colocalize throughout the viral life cycle in large and small phase-dense globular cytoplasmic inclusions, which are believed to be the sites of viral replication and assembly. Individual expression of these proteins in transfected cells of avian and mammalian origin revealed that while microNS is able to form inclusions in the absence of other viral proteins, sigmaNS distributes diffusely throughout the cytoplasm in the absence of microNS. These data suggest that microNS is the minimal viral factor required for inclusion formation during avian reovirus infection. On the other hand, our findings that sigmaNS associates with microNS in infected cells, and that sigmaNS colocalizes with microNS in viroplasm-like inclusions when the two proteins are coexpressed in transfected cells, suggest that microNS mediates the association of sigmaNS to inclusions in avian reovirus-infected cells.
禽呼肠孤病毒1733的M3基因组片段编码非结构蛋白microNS，长度为1996个核苷酸，包含一个长开放阅读框，预计编码一个由635个氨基酸残基组成的多肽。对推导的microNS氨基酸序列的检查显示，在其羧基末端附近存在两个区域，极有可能形成α-螺旋卷曲螺旋。M3基因在感染细胞和转染细胞中的表达表明，该基因指定了两种蛋白异构体，它们可被microNS特异性抗血清识别。在感染细胞中，只有较大的microNS异构体与非结构蛋白sigmaNS相互作用，而较小的异构体则不相互作用，这表明这两种异构体在禽呼肠孤病毒感染过程中发挥不同作用。在本研究的第二部分，我们表明，在整个病毒生命周期中，microNS和非结构蛋白sigmaNS共定位于大小不同的相致密球状细胞质内含物中，这些内含物被认为是病毒复制和组装的场所。在禽源和哺乳动物源的转染细胞中单独表达这些蛋白表明，虽然microNS在没有其他病毒蛋白的情况下能够形成内含物，但在没有microNS的情况下，sigmaNS在整个细胞质中呈弥散分布。这些数据表明，microNS是禽呼肠孤病毒感染期间形成内含物所需的最小病毒因子。另一方面，我们的研究结果表明，在感染细胞中sigmaNS与microNS相关联，并且当这两种蛋白在转染细胞中共表达时，sigmaNS与microNS在类病毒质体的内含物中共定位，这表明microNS介导了sigmaNS与禽呼肠孤病毒感染细胞中内含物的关联。
