Chang K O, Kim Y J, Saif L J
Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster 44691, USA.
Virus Genes. 1999;18(3):229-33. doi: 10.1023/a:1008068218966.
The NSP4 protein of rotavirus is a nonstructural glycoprotein and has a crucial function in virus morphogenesis during infection of host cells. It was recently reported that NSP4 may also function as a viral enterotoxin in the induction of rotavirus diarrhea by causing Ca++ influx in the cytoplasm of the infected cells. We sequenced and analyzed two (Wa and M strains) pairs of NSP4 genes of virulent (v) and attenuated (a) (after 30 to 40 passages in cell culture) human group A rotaviruses and a pair of NSP4 genes of virulent and attenuated porcine group C rotavirus (Cowden strain). These strains were previously identified as virulent (induce diarrhea) or attenuated (no diarrhea) in a gnotobiotic pig model of rotavirus infection [Bohl et al. (4), Saif et al. (13), Ward et al. (17)]. The NSP4 genes of the Wa, M and Cowden strains were amplified with RT-PCR using a proof reading polymerase (Tli) and the RT-PCR product was sequenced directly. Analysis of the NSP4 deduced amino acid sequences showed that only 3 (Wa) and 2 (M and Cowden) amino acids differed between the virulent and attenuated strains. For the Wa strain, the changes from the virulent to attenuated strain were in amino acids 13 (V to A), 16 (L to S) and 34 (P to L); in the M strain, the difference was in amino acids 53 (T to I) and 104 (K to E), and in the Cowden strains, amino acids 50 (L to F) and 97 (D to N) differed between virulent and attenuated strains. To our knowledge, this is the first sequence comparison between NSP4 of a virulent and attenuated pair of group C rotaviruses. The potential impact of these few amino acid changes on the pathogenesis of the NSP4 protein for piglets is unclear, relative to previous findings in mice (1), but requires further study using purified recombinant NSP4 proteins or peptides.
轮状病毒的NSP4蛋白是一种非结构糖蛋白，在宿主细胞感染期间的病毒形态发生中起关键作用。最近有报道称，NSP4通过引起感染细胞胞质中的Ca++内流，在诱导轮状病毒腹泻方面也可能作为一种病毒肠毒素发挥作用。我们对两株（Wa和M株）强毒株（v）和减毒株（a）（在细胞培养中传代30至40次后）的人A组轮状病毒的NSP4基因对以及一对强毒株和减毒株猪C组轮状病毒（考登株）的NSP4基因进行了测序和分析。这些毒株先前在轮状病毒感染的悉生猪模型中被鉴定为强毒株（诱导腹泻）或减毒株（不引起腹泻）[博尔等人（4），赛义夫等人（13），沃德等人（17）]。使用具有校对功能的聚合酶（Tli）通过RT-PCR扩增Wa、M和考登株的NSP4基因，并对RT-PCR产物进行直接测序。对推导的NSP4氨基酸序列的分析表明，强毒株和减毒株之间仅3个（Wa株）和2个（M株和考登株）氨基酸不同。对于Wa株，从强毒株到减毒株的变化发生在第13位氨基酸（V变为A）、第16位氨基酸（L变为S）和第34位氨基酸（P变为L）；在M株中，差异发生在第53位氨基酸（T变为I）和第104位氨基酸（K变为E），而在考登株中，强毒株和减毒株之间第50位氨基酸（L变为F）和第97位氨基酸（D变为N）不同。据我们所知，这是首次对一组强毒株和减毒株C组轮状病毒的NSP4进行序列比较。相对于先前在小鼠中的研究结果（1），这少数氨基酸变化对仔猪NSP4蛋白发病机制的潜在影响尚不清楚，但需要使用纯化的重组NSP4蛋白或肽进行进一步研究。
