Kim K H, Strauss E G, Strauss J H
Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
J Virol. 2000 Mar;74(6):2663-70. doi: 10.1128/jvi.74.6.2663-2670.2000.
Alphavirus glycoproteins E2 and E1 form a heterodimer that is required for virus assembly. We have studied adaptive mutations in E2 of Sindbis virus (SIN) and E1 of Ross River virus (RR) that allow these two glycoproteins to interact more efficiently in a chimeric virus that has SIN E2 but RR E1. These mutations include K129E, K131E, and V237F in SIN E2 and S310F and C433R in RR E1. Although RR E1 and SIN E2 will form a chimeric heterodimer, the chimeric virus is almost nonviable, producing about 10(-7) as much virus as SIN at 24 h and 10(-5) as much after 48 h. Chimeras containing one adaptive change produced 3 to 20 times more virus than did the parental chimera, whereas chimeras with two changes produced 10 to 100 times more virus and chimeras containing three mutations produced yields that were 180 to 250 times better. None of the mutations had significant effects upon the parental wild-type viruses, however. Passage of the triple variants eight or nine times resulted in variants that produced virus rapidly and were capable of producing >10(8) PFU/ml of culture fluid within 24 h. These further-adapted variants possessed one or two additional mutations, including E2-V116K, E2-S110N, or E1-T65S. The RR E1-C433R mutation was studied in more detail. This Cys is located in the putative transmembrane domain of E1 and was shown to be palmitoylated. Mutation to Arg-433 resulted in loss of palmitoylation of E1. The positively charged arginine residue within the putative transmembrane domain of E1 would be expected to alter the conformation of this domain. These results suggest that interactions within the transmembrane region are important for the assembly of the E1/E2 heterodimer, as are regions of the ectodomains possibly identified by the locations of adaptive mutations in these regions. Further, the finding that four or five changes in the chimera allow virus production that approaches the levels seen with the parental SIN and exceeds that of the parental RR illustrates that the structure and function of SIN and RR E1s have been conserved during the 50% divergence in sequence that has occurred.
甲病毒糖蛋白E2和E1形成一种异二聚体，这是病毒组装所必需的。我们研究了辛德毕斯病毒（SIN）E2和罗斯河病毒（RR）E1中的适应性突变，这些突变使这两种糖蛋白在具有SIN E2但RR E1的嵌合病毒中能更有效地相互作用。这些突变包括SIN E2中的K129E、K131E和V237F以及RR E1中的S310F和C433R。虽然RR E1和SIN E2会形成嵌合异二聚体，但这种嵌合病毒几乎无法存活，在24小时时产生的病毒量约为SIN的10^(-7)，48小时后为10^(-5)。含有一个适应性变化的嵌合体产生的病毒比亲本嵌合体多3至20倍，而含有两个变化的嵌合体产生的病毒多10至100倍，含有三个突变的嵌合体产生的产量则高180至250倍。然而，这些突变对亲本野生型病毒均无显著影响。三重变体传代八次或九次后产生的变体能够快速产生病毒，并且在24小时内能够产生>10^(8) PFU/ml的培养液。这些进一步适应的变体具有一两个额外的突变，包括E2-V116K、E2-S110N或E1-T65S。对RR E1-C433R突变进行了更详细的研究。这个半胱氨酸位于E1的假定跨膜结构域中，并且已被证明是棕榈酰化的。突变为精氨酸-433导致E1的棕榈酰化丧失。E1假定跨膜结构域内带正电荷的精氨酸残基预计会改变该结构域的构象。这些结果表明，跨膜区域内的相互作用对于E1/E2异二聚体的组装很重要，这些区域的胞外域中的区域可能由这些区域中的适应性突变位置确定。此外，嵌合体中四个或五个变化能使病毒产生量接近亲本SIN的水平并超过亲本RR的水平，这一发现表明，在发生了50%的序列差异的情况下，SIN和RR E1的结构和功能仍得以保留。
