Schumacher D, Tischer B K, Fuchs W, Osterrieder N
Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany.
J Virol. 2000 Dec;74(23):11088-98. doi: 10.1128/jvi.74.23.11088-11098.2000.
The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.
马立克氏病病毒1型（MDV-1）584Ap80C株的全基因组作为细菌人工染色体（BAC）克隆于大肠杆菌中。通过同源重组将BAC载体序列引入MDV-1基因组的U(S)2基因座。含有BAC载体的病毒DNA用于转化大肠杆菌DH10B菌株，鉴定出几个含有完整MDV-1基因组作为F质粒（MDV-1 BACs）的菌落。将来自各种MDV-1 BACs的DNA转染到鸡胚成纤维细胞中，转染后3天获得感染性MDV-1。通过噬斑形成和生长曲线测定评估，从BACs中回收的MDV-1的生长与亲本病毒无异。在一个MDV-1 BAC克隆中，使用PCR扩增的线性DNA片段通过一步诱变删除了编码糖蛋白B（gB）的序列。转染含有2.6kbp gB基因2.0kbp缺失的BAC DNA后回收的突变型MDV-1仅能在反式提供MDV-1 gB的细胞上生长并诱导MDV-1特异性噬斑。本文报道的gB阴性病毒代表首个缺失必需基因的MDV-1突变体，并证明了BACs在分析生长缓慢且严格细胞相关的疱疹病毒中的基因和基因产物方面的强大功能和实用性。
