Skraban R, Matthíasdóttir S, Torsteinsdóttir S, Agnarsdóttir G, Gudmundsson B, Georgsson G, Meloen R H, Andrésson O S, Staskus K A, Thormar H, Andrésdóttir V
Institute for Experimental Pathology, University of Iceland, Keldur, Iceland.
J Virol. 1999 Oct;73(10):8064-72. doi: 10.1128/JVI.73.10.8064-8072.1999.
Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.
已从两种梅迪 - 维斯纳病毒(MVV)毒株中分离出感染性分子克隆,其中一种(KV1772kv72/67)是另一种(LV1-1KS1)的抗原逃逸突变体。为了定位型特异性中和表位,我们构建了含有嵌合包膜基因的病毒,以KV1772kv72/67为骨架,并用LV1-1KS1的等效序列替换包膜基因的各个部分。发现中和表型定位于包膜基因中的一个区域,该区域在39个氨基酸(Env的第559至597位)内包含两个缺失和四个氨基酸变化。从感染了嵌合病毒VR1的羔羊获得的血清,VR1在KV1772kv72/67骨架中仅含有来自LV1-1KS1的39个氨基酸,能中和LV1-1KS1但不能中和KV1772kv72/67。我们已证明参与逃逸中和的包膜基因区域被克隆到pGEX-3X表达载体中,并在免疫印迹中测试了来自两个分子克隆的所得融合肽与KV1772kv72/67和VR1型特异性抗血清的反应性。型特异性的KV1772kv72/67抗血清仅与来自KV1772kv72/67的融合肽反应,而不与来自LV1-1KS1的融合肽反应,型特异性的VR1抗血清仅与来自LV1-1KS1的融合肽反应,而不与来自KV1772kv72/67的融合肽反应。Pepscan分析表明该区域包含两个线性表位,其中一个对每个分子克隆病毒都是特异性的。然而,并非所有型特异性中和抗血清都能结合这个线性表位,这表明它本身不是中和表位,但可能是其中一部分。这些发现表明,MVV病毒包膜基因中第559至597位氨基酸内的突变导致逃逸中和。此外,该区域包含一个不连续中和表位的一个或多个部分。