Guirakhoo F, Bolin R A, Roehrig J T
Division of Vector-Borne Infectious Diseases, Centers for Disease Control, Fort Collins, Colorado 80522.
Virology. 1992 Dec;191(2):921-31. doi: 10.1016/0042-6822(92)90267-s.
To study the role of the precursor to the membrane protein (prM) in flavivirus maturation, we inhibited the proteolytic processing of the Murray Valley encephalitis (MVE) virus prM to membrane protein in infected cells by adding the acidotropic agent ammonium chloride late in the virus replication cycle. Viruses purified from supernatants of ammonium chloride-treated cells contained prM protein and were unable to fuse C6/36 mosquito cells from without. When ammonium chloride was removed from the cells, both the processing of prM and the fusion activity of the purified viruses were partially restored. By using monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of MVE virus, we found that at least three epitopes were less accessible to their corresponding antibodies in the prM-containing MVE virus particles. Amino-terminal sequencing of proteolytic fragments of the E protein which were reactive with sequence-specific peptide antisera or MAb enabled us to estimate the site of the E protein interacting with the prM to be within amino acids 200 to 327. Since prM-containing viruses were up to 400-fold more resistant to a low pH environment, we conclude that the E-prM interaction might be necessary to protect the E protein from irreversible conformational changes caused by maturation into the acidic vesicles of the exocytic pathway.
为了研究膜蛋白前体（prM）在黄病毒成熟过程中的作用，我们在病毒复制周期后期加入亲酸剂氯化铵，抑制感染细胞中墨累河谷脑炎（MVE）病毒prM向膜蛋白的蛋白水解加工。从氯化铵处理细胞的上清液中纯化的病毒含有prM蛋白，且无法从外部融合C6/36蚊细胞。当从细胞中去除氯化铵后，prM的加工和纯化病毒的融合活性均部分恢复。通过使用针对MVE病毒包膜（E）糖蛋白的单克隆抗体（MAb），我们发现至少三个表位在含prM的MVE病毒颗粒中与其相应抗体的可及性较低。对与序列特异性肽抗血清或MAb反应的E蛋白蛋白水解片段进行氨基末端测序，使我们能够估计E蛋白与prM相互作用的位点在氨基酸200至327范围内。由于含prM的病毒对低pH环境的抗性高达400倍，我们得出结论，E-prM相互作用可能是保护E蛋白免受由成熟进入外排途径酸性囊泡引起的不可逆构象变化所必需的。
