Abstract：
During the late stage of infection, alphabaculoviruses produce many occlusion bodies (OBs) in the nuclei of the insect host\'s cells through the hyperexpression of polyhedrin (POLH), a major OB component encoded by polh. The strong polh promoter has been used to develop a baculovirus expression vector system for recombinant protein expression in cultured insect cells and larvae. However, the relationship between POLH accumulation and the polh coding sequence remains largely unelucidated. This study aimed to assess the importance of polh codon usage and/or nucleotide sequences in POLH accumulation by generating a baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) expressing mutant polh (co-polh) optimized according to the codon preference of its host insect. Although the deduced amino acid sequence of CO-POLH was the same as that of wild-type POLH, POLH accumulation was significantly lower in cells infected with the co-polh mutant. This reduction was due to decreased polh mRNA levels rather than translational repression. Analysis of mutant viruses with chimeric polh revealed that a 30 base-pair (bp) 5\' proximal polh coding region was necessary for maintaining high polh mRNA levels. Sequence comparison of wild-type polh and co-polh identified five nucleotide differences in this region, indicating that these nucleotides were critical for polh hyperexpression. Furthermore, luciferase reporter assays showed that the 30 bp 5\' coding region was sufficient for maintaining the polh promoter-driven high level of polh mRNA. Thus, our whole-gene scanning by codon optimization identified important hidden nucleotides for polh hyperexpression in alphabaculoviruses.
摘要：
在感染后期,通过多角体蛋白（POLH）的过度表达，在昆虫宿主细胞的细胞核中产生许多闭塞体（OB），由polh编码的主要OB分量。强polh启动子已用于开发杆状病毒表达载体系统，用于在培养的昆虫细胞和幼虫中表达重组蛋白。然而,POLH积累与polh编码序列之间的关系仍未阐明。这项研究旨在通过生成杆状病毒Bombyxmori核多角体病毒（BmNPV）来评估POLH积累中polh密码子使用和/或核苷酸序列的重要性，该杆状病毒表达根据其宿主昆虫的密码子偏好进行优化的突变polh（co-polh）。尽管推导出的CO-POLH的氨基酸序列与野生型POLH的氨基酸序列相同,在用co-polh突变体感染的细胞中，POLH的积累显着降低。这种减少是由于polhmRNA水平降低而不是翻译抑制。用嵌合polh对突变病毒的分析表明，30个碱基对（bp）5'近端polh编码区对于维持高polhmRNA水平是必需的。野生型polh和co-polh的序列比较确定了该区域的五个核苷酸差异，表明这些核苷酸对于polh过表达至关重要。此外，荧光素酶报告基因测定表明，30bp的5'编码区足以维持polh启动子驱动的高水平polhmRNA。因此，我们通过密码子优化的全基因扫描确定了字母杆状病毒中polh超表达的重要隐藏核苷酸。
