Keelapang Poonsook, Sriburi Roongtawan, Supasa Sanpaechuda, Panyadee Nantaya, Songjaeng Adisak, Jairungsri Aroonroong, Puttikhunt Chunya, Kasinrerk Watchara, Malasit Prida, Sittisombut Nopporn
Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok 10400, USA.
J Virol. 2004 Mar;78(5):2367-81. doi: 10.1128/jvi.78.5.2367-2381.2004.
During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.
在黄病毒颗粒通过分泌途径输出的过程中，一种病毒包膜糖蛋白prM被前体蛋白转化酶弗林蛋白酶切割；这种切割对于随后受体结合E糖蛋白的重排和病毒感染性是必需的。与许多弗林蛋白酶底物相似，虫媒黄病毒中的prM在切割位点近端的P1、P2和P4位置含有碱性残基；此外，在P3位置以及P5和P13位置之间发现了一些带电荷的残基，这些残基在每种黄病毒抗原复合物中都是保守的。通过用蜱传脑炎病毒（TBEV）、黄热病毒（YFV）和日本脑炎病毒（JEV）的13个氨基酸的切割近端区域替换登革病毒（16681株）的该区域，并比较从RNA转染的蚊细胞产生的所得嵌合病毒，研究了额外带电荷残基对pr-M切割和病毒复制的影响。在这三种嵌合病毒中，JEVpr/16681中prM的切割比YFVpr/16681中更大程度地增强，但在TBEVpr/16681中略有降低。出乎意料的是，JEVpr/16681在C6/36、PS和Vero细胞系中表现出焦点大小减小、峰值滴度降低和复制受抑制。JEVpr/16681增殖的减少与感染性病毒粒子从感染细胞中延迟输出相关，但与特异性感染性的变化无关。JEVpr/16681与固定化肝素的结合以及细胞的肝素抑制性感染没有改变。因此，当在登革病毒prM背景中测试时，黄病毒不同的pr-M连接近端序列对pr-M切割有不同的影响。更重要的是，大大增强的prM切割能力对登革病毒输出有不利影响，而对感染性的影响最小。由于在大量登革病毒分离株中未观察到pr-M连接处带电荷残基的广泛变化，这些结果提供了一种可能的机制，通过该机制登革病毒pr-M连接处的序列保守性在自然界中得以维持。
