Norwalk virus open reading frame 3 encodes a minor structural protein
Norwalk virus open reading frame 3 encodes a minor structural protein
复制标题
DOI：
10.1128/jvi.74.14.6581-6591.2000
复制
发表时间：
2000-07-01
影响因子：
5.4
通讯作者：
Estes, MK
中科院分区：
文献类型：
--
作者：
Glass, PJ;White, LJ;Estes, MK
诺瓦克病毒（Norwalk virus，NV）是引起人类急性流行性非细菌性胃肠炎的病原体。不能培养NV需要使用分子技术来检查病毒蛋白的基因组组织和功能。由开放阅读框3（ORF 3）编码的NV蛋白的功能一直是未知的。在本文中，我们报告的NV ORF 3蛋白在无细胞翻译系统和昆虫细胞中表达的表征，并显示其与重组病毒样颗粒（VLP）和NV病毒体的关联。ORF 3编码区在兔网织红细胞裂解物中的表达导致产生表观分子量为23，000的单一蛋白质（23 K蛋白），其未被N-连接的糖基化修饰。通过使用两种不同的杆状病毒重组体在昆虫细胞中表达ORF 3蛋白;一种重组体含有以ORF 2编码序列（ORF 2+3）开始的基因组的整个3'末端，而第二种重组体仅含有ORF 3。从含有ORF 2和ORF 3的构建体的表达导致通过用ORF 3特异性肽抗血清的Western印迹分析检测到的单一蛋白质（23 K蛋白）的表达。然而，从仅含有ORF 3编码序列的构建体表达导致产生多种形式的ORF 3蛋白，大小范围为23，000至35，000。使用ORF 3肽抗血清的间接免疫荧光研究表明ORF 3蛋白定位于受感染的昆虫细胞的细胞质。23 K ORF 3蛋白始终与从用含有NV基因组的整个3'末端的杆状病毒重组体感染的昆虫细胞的培养基中纯化的重组VLP相关。从NV感染的志愿者粪便中纯化的NV的Western印迹分析显示存在35 K蛋白以及由ORF 3肽抗血清特异性识别的多个高分子量条带。这些结果表明ORF 3蛋白是病毒粒子的次要结构蛋白。
Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3' end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3' end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.