Lopez S, Yao J S, Kuhn R J, Strauss E G, Strauss J H
Division of Biology, California Institute of Technology, Pasadena 91125.
J Virol. 1994 Mar;68(3):1316-23. doi: 10.1128/JVI.68.3.1316-1323.1994.
We have studied interactions between nucleocapsids and glycoproteins required for budding of alphaviruses, using Ross River virus-Sindbis virus chimeras in which the nucleocapsid protein is derived from one virus and the envelope glycoproteins are derived from the second virus. A virus containing the Ross River virus genome in which the capsid protein had been replaced with that from Sindbis virus was almost nonviable. Nucleocapsids formed in normal numbers in the infected cell, but very little virus was released from the cell. There are 11 amino acid differences between Ross River virus and Sindbis virus in their 33-residue E2 cytoplasmic domains. Site-specific mutagenesis was used to change 9 of these 11 amino acids in the chimera from the Ross River virus to the Sindbis virus sequence in an attempt to adapt the E2 of the chimera to the nucleocapsid. The resulting mutant chimera grew 4 orders of magnitude better than the parental chimeric virus. This finding provides direct evidence for a sequence-specific interaction between the nucleocapsid and the E2 cytoplasmic domain during virus budding. The mutated chimeric virus readily gave rise to large-plaque variants that grew almost as well as Ross River virus, suggesting that additional single amino acid substitutions in the structural proteins can further enhance the interactions between the disparate capsid and the glycoproteins. Unexpectedly, change of E2 residue 394 from lysine (Ross River virus) to glutamic acid (Sindbis virus) was deleterious for the chimera, suggesting that in addition to its role in nucleocapsid-E2 interactions, the N-terminal part of the E2 cytoplasmic domain may be involved in glycoprotein-glycoprotein interactions required to assemble the glycoprotein spikes. The reciprocal chimera, Sindbis virus containing the Ross River virus capsid, also grew poorly. Suppressor mutations arose readily in this chimera, producing a virus that grew moderately well and that formed larger plaques.
我们利用罗斯河病毒 - 辛德毕斯病毒嵌合体研究了甲病毒出芽所需的核衣壳与糖蛋白之间的相互作用，其中核衣壳蛋白来自一种病毒，包膜糖蛋白来自第二种病毒。一种含有罗斯河病毒基因组且衣壳蛋白被辛德毕斯病毒的衣壳蛋白取代的病毒几乎无法存活。在受感染细胞中形成的核衣壳数量正常，但从细胞中释放的病毒很少。罗斯河病毒和辛德毕斯病毒在其33个残基的E2细胞质结构域中有11个氨基酸差异。使用位点特异性诱变将嵌合体中这11个氨基酸中的9个从罗斯河病毒序列改变为辛德毕斯病毒序列，试图使嵌合体的E2适应核衣壳。所得的突变嵌合体比亲本嵌合病毒生长得好4个数量级。这一发现为病毒出芽过程中核衣壳与E2细胞质结构域之间的序列特异性相互作用提供了直接证据。突变的嵌合病毒很容易产生大斑块变体，其生长情况几乎与罗斯河病毒一样好，这表明结构蛋白中额外的单个氨基酸取代可以进一步增强不同衣壳与糖蛋白之间的相互作用。出乎意料的是，E2残基394从赖氨酸（罗斯河病毒）变为谷氨酸（辛德毕斯病毒）对嵌合体有害，这表明除了其在核衣壳 - E2相互作用中的作用外，E2细胞质结构域的N端部分可能参与组装糖蛋白刺突所需的糖蛋白 - 糖蛋白相互作用。反向嵌合体，即含有罗斯河病毒衣壳的辛德毕斯病毒，生长也很差。这种嵌合体很容易出现抑制突变，产生一种生长适度良好且形成较大斑块的病毒。
