Sun Aixu, Devi-Rao G V, Rice M K, Gary L W, Bloom D C, Sandri-Goldin R M, Ghazal P, Wagner E K
Department of Molecular Biology and Biochemistry and Center for Virus Research, University of California, Irvine, CA 92717-3900, USA.
J Virol. 2004 Oct;78(19):10470-8. doi: 10.1128/JVI.78.19.10470-10478.2004.
We constructed a promoter mutation altering the immediate-early expression of the herpes simplex virus type 1 (HSV-1) ICP27 transcript and its cognate wild-type rescue viruses in order to assess the role of the ICP27 protein in the earliest stages of viral infection by global transcriptional analysis with a DNA microarray. This mutant, ICP27/VP16, replaces the whole ICP27 promoter/enhancer with the VP16 promoter. It demonstrates loss of immediate-early expression of ICP27 according to the criteria expression in the absence of de novo protein synthesis and earliest expression in the kinetic cascade. Significant differences in relative transcript abundances between the mutant and wild-type rescue viruses were limited at the earliest times measured and not evident at all by 4 h after infection. Consistent with this observation, levels of some critical proteins were reduced in the mutant as compared to rescue virus infections at the earliest times tested, but were equivalent by 8 h postinfection. Further, both single and multistep levels of virus replication were equivalent with both mutant and rescue viruses. Thus, altering the immediate-early kinetics of ICP27 leads to a suboptimal quantitative lag phase in gene expression but without consequence for replication fitness in vitro. Infections in vivo also revealed equivalent ability of mutant and rescue viruses to invade the central nervous system of mice following footpad injections. Limitations to an immediate-early role of ICP27 in the biology of HSV are discussed in light of these observations.
我们构建了一种启动子突变体,改变了单纯疱疹病毒1型(HSV-1)ICP27转录本的立即早期表达及其同源野生型拯救病毒,以便通过DNA微阵列进行全局转录分析来评估ICP27蛋白在病毒感染最早阶段的作用。这个突变体,即ICP27/VP16,用VP16启动子取代了整个ICP27启动子/增强子。根据在无从头蛋白质合成时的表达标准以及动力学级联中的最早表达情况,它显示出ICP27立即早期表达的丧失。在最早测量的时间点,突变体和野生型拯救病毒之间相对转录本丰度的显著差异有限,而在感染后4小时根本不明显。与这一观察结果一致,在最早测试的时间点,与拯救病毒感染相比,突变体中一些关键蛋白的水平降低,但在感染后8小时两者相当。此外,突变体和拯救病毒的单步和多步病毒复制水平相当。因此,改变ICP27的立即早期动力学导致基因表达出现次优的定量滞后阶段,但对体外复制适应性没有影响。体内感染还显示,脚垫注射后,突变体和拯救病毒侵入小鼠中枢神经系统的能力相当。根据这些观察结果,讨论了ICP27在HSV生物学中立即早期作用的局限性。