Hua J, Patton J T
Department of Microbiology and Immunology, University of Miami School of Medicine, Florida 33101.
Virology. 1994 Feb;198(2):567-76. doi: 10.1006/viro.1994.1068.
The rotavirus nonstructural protein NS53 (NSVP1), the product of genome segment 5, possesses RNA-binding activity and contains a highly conserved cysteine-rich motif located in the amino-terminal half of the protein. The genome of the bovine rotavirus variant, brvA, lacks a normal segment 5 but includes a novel dsRNA (gene A) of approximately 2600 basepairs (bp) that contains segment 5-specific sequences (F. Hundley, B. Biryahwaho, M. Gow, and U. Desselberger, Virology 143, 88-103, 1985). To gain information about the nature of the rearrangement in gene A and its capacity to encode a protein product, we prepared and sequenced complementary (c)DNA of the gene A RNA. The results showed that gene A is 2693 bp in size and contains a head-to-tail duplication of 1112 bp that originates from the open reading frame (ORF) of gene 5. The duplication begins at nucleotide (nt) 1454, which is 53 nt upstream from the end of the ORF for NS53. Gene A contains a point mutation at nt 808 which results in the presence of a nonsense codon near the middle of the ORF for NS53. Thus the predicted product of gene A is a truncated NS53 of 258 amino acids (aa) (31 kDa), approximately one-half the size of the authentic 491-aa NS53 (58 kDa). Examination of lysates from brvA-infected cells by Western blot assay using an NS53-specific antibody confirmed that the variant encodes only a truncated gene 5 product. Despite the truncation, analysis of the gene A product suggested that it, like full-length NS53, accumulated in association with the cytoskeleton of the infected cell, thus providing evidence that the subcellular localization signal in NS53 resides in the amino terminal half of the protein. Given that brvA is a viable, nondefective mutant, these results demonstrate that the carboxyl-terminal 233 aa of NS53 are not required for rotavirus replication in vitro.
轮状病毒非结构蛋白NS53（NSVP1）是基因组第5节段的产物，具有RNA结合活性，并且在该蛋白的氨基末端一半区域含有一个高度保守的富含半胱氨酸的基序。牛轮状病毒变种brvA的基因组缺少正常的第5节段，但包含一个约2600个碱基对（bp）的新型双链RNA（基因A），该双链RNA含有第5节段特异性序列（F. Hundley、B. Biryahwaho、M. Gow和U. Desselberger，《病毒学》143，88 - 103，1985）。为了获取有关基因A中重排性质及其编码蛋白质产物能力的信息，我们制备了基因A RNA的互补（c）DNA并进行了测序。结果表明，基因A大小为2693 bp，包含一个1112 bp的头对头重复序列，该重复序列源自基因5的开放阅读框（ORF）。重复从核苷酸（nt）1454开始，此位置距离NS53的ORF末端上游53 nt。基因A在nt 808处有一个点突变，这导致在NS53的ORF中部附近出现一个无义密码子。因此，基因A的预测产物是一个258个氨基酸（aa）（31 kDa）的截短型NS53，大约是真实的491个氨基酸（58 kDa）的NS53大小的一半。使用NS53特异性抗体通过蛋白质印迹分析检测brvA感染细胞的裂解物，证实该变种仅编码一种截短的基因5产物。尽管存在截短，但对基因A产物的分析表明，它与全长NS53一样，与感染细胞的细胞骨架相关联积累，从而提供了证据表明NS53中的亚细胞定位信号位于该蛋白的氨基末端一半区域。鉴于brvA是一个有活力的、无缺陷的突变体，这些结果表明NS53的羧基末端233个氨基酸对于轮状病毒在体外复制不是必需的。
