CONSTRUCTION OF AN INFECTIOUS DNA CLONE OF THE FULL-LENGTH HUMAN SPUMARETROVIRUS GENOME AND MUTAGENESIS OF THE BEL-1 GENE
CONSTRUCTION OF AN INFECTIOUS DNA CLONE OF THE FULL-LENGTH HUMAN SPUMARETROVIRUS GENOME AND MUTAGENESIS OF THE BEL-1 GENE
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DOI：
10.1016/0042-6822(91)90820-2
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发表时间：
1991-09-01
期刊：
影响因子：
3.7
通讯作者：
FLUGEL, RM
中科院分区：
文献类型：
--
作者：
LOCHELT, M;ZENTGRAF, H;FLUGEL, RM
构建了人类SpumaretroVirus(HSRV)基因组DNA感染性全长分子克隆。通过将HSRV特异的细胞病变作用作为无细胞培养上清，通过对PHSRV13 DNA转染细胞中HSRV颗粒的电子显微镜观察，通过检测HSRV转录本，以及通过间接免疫荧光试验和蛋白质印迹鉴定HSRV编码的蛋白，证实了PHSRV 13克隆在感染敏感细胞后的感染性。用Bel-1和Bel-2特异性抗血清在免疫印迹中检测到主要的HSRV蛋白，其表观分子量为56 kDa，对应于Bet。Bet的氨基端由Bel 1特异的部分RNA编码，较大的Bet结构域由bel 2基因的RNA物种编码(Muranyi，W.和Flu？Gel，R.M.J.Virology65,727-735,1991)。HSRV的36和43 kDa蛋白分别与Bel-1和Bel-2抗血清反应，与Bel-1和Bel-2基因产物的计算值一致。对转录HSRV特异性反式激活子b1和bet2基因的缺失突变，完全消除了PHSRV13克隆的感染性。RNA、蛋白质和病毒粒子合成的缺陷-通过共转染含有完整编码区的表达克隆来补充。这一结果表明，bel-1基因是病毒复制所必需的。是否其他HSRV基因产物，如与bel 1有共同区域的bet1，是否导致了观察到的缺陷，仍有待确定。
An infectious and full-length molecular clone of genomic human spumaretrovirus (HSRV) DNA was constructed. The infectivity of the pHSRV 13 clone was demonstrated after transfection into susceptible cells by passage of HSRV-specific cytopathic effects as a cell-free culture supernatant, by electron microscopy of HSRV particles in pHSRV13 DNA-transfected cells, by detection of HSRV transcripts, and by identification of HSRV-encoded proteins with Env- and Bel-specific antisera in indirect immunofluorescence assays and in protein blotting. The predominant HSRV protein detected in immunoblots by both Bel 1- and Bel 2-specific antisera had an apparent molecular weight of 56 kDa and corresponds to Bet. The amino-terminus of Bet is encoded by part of a Bel 1-specific RNA and the larger Bet domain by an RNA species from the bel 2 gene (Muranyi, W., and Flu¨gel, R. M.J. Virology65, 727–735, 1991). HSRV-specific proteins of 36 and 43 kDa reacted with Bel 1 and Bel 2 antisera, consistent with the values calculated for thebel 1andbel 2gene products, respectively. Deletion mutagenesis of the transcriptional HSRV-specifictrans-activatorbel 1and thebetgenes completely abolished the infectivity of the pHSRV13 clone. The defect in RNA, protein, and virion synthesis wastrans-complemented by cotransfection of an expression clone harboring the completebelcoding region. This result demonstrates that thebel 1gene is required for viral replication. It remains to be determined whether other HSRV gene products, likebetthat share a common region withbel 1, contributed to the defect observed.