Virus reconstituted from infectious bacterial artificial chromosome (BAC)-Cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences
Virus reconstituted from infectious bacterial artificial chromosome (BAC)-Cloned murine gammaherpesvirus 68 acquires wild-type properties in vivo only after excision of BAC vector sequences
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DOI：
10.1128/jvi.75.12.5692-5696.2001
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发表时间：
2001-06-01
影响因子：
5.4
通讯作者：
Koszinowski, UH
中科院分区：
文献类型：
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作者：
Adler, H;Messerle, M;Koszinowski, UH
以传染性细菌人工染色体（BAC）克隆的鼠γ -疱疹病毒68 （MHV-68）基因组为载体，研究了重组病毒的体内生物学特性。含有BAC载体序列的重组病毒R γ HV68A98.01在体内是减毒的，这是通过以下方法确定的：(i)感染急性期肺部病毒滴度，（ii）脾大程度，以及（iii）在体外再激活试验中潜伏感染的脾脏细胞重新激活病毒的数量。由于BAC载体序列两侧有loxP位点，因此将病毒在表达Cre重组酶的成纤维细胞中传代，产生了重组病毒R γ HV68A98.02，其生物学特性与野生型MHV-68相当。基于这些数据，我们得出以下结论：(1)从克隆的MHV-68基因组中切除BAC载体序列对于重建该病毒的野生型表型特性至关重要；(2)bag克隆的MHV-68基因组适合于构建突变体和突变文库，这些突变体和突变文库的表型可以在体内可靠地评估。
We studied the in vivo biological properties of viruses reconstituted from the genome of murine gamma-herpesvirus 68 (MHV-68) cloned as an infectious bacterial artificial chromosome (BAC). Recombinant virus R gamma HV68A98.01, containing BAC vector sequences, is attenuated in vivo as determined by (i) viral titers in the lungs during the acute phase of infection, (ii) the extent of splenomegaly, and (iii) the number of latently infected spleen cells reactivating virus in an ex vivo reactivation assay. Since the BAC vector sequences were flanked by loxP sites, passaging the virus in fibroblasts expressing Cre recombinase resulted in the generation of recombinant virus R gamma HV68A98.02, with biological properties comparable to those of wild-type MHV-68, On the basis of these data we conclude (i) that excision of BAC vector sequences from cloned MHV-68 genomes is critical for reconstitution of the wild-type phenotypic properties of this virus and (ii) that the BAG-cloned MHV-68 genome is suitable for the construction of mutants and mutant libraries whose phenotypes can be reliably assessed in vivo.