Faculty of Veterinary Medicine, Institute of Virology, University of Zurich, Winterthurerstrasse 266A, CH-8057 Zurich, Switzerland.
Vet Microbiol. 2010 Jun 16;143(1):37-44. doi: 10.1016/j.vetmic.2010.02.012. Epub 2010 Feb 20.
Bovine herpesvirus 1 (BoHV-1) and BoHV-5 are closely related (82% amino acid identity) but differ strongly in neuropathogenesis. The immediate-early gene for BICP0 is less conserved (70% amino acid identity) and may contribute to a dissimilar phenotype. A peculiar difference is a guanosine hexamer in the BICP0-1 gene which aligns with only five guanosines in the BICP0-5 gene and therefore results in a frameshift in the latter open reading frame. Thus, the C-terminal amino acid sequence (residues 643-676 of BICP0-1 vs. 655-720 of BICP0-5) is completely different. We introduced the BICP0-5 frameshift into the BoHV-1 genome cloned as a bacterial artificial chromosome (BoHV-1 BAC) using the Red recombination system with galK selection and counterselection. Transfection of MDBK cells with the resulting BAC produced recombinant virus that replicated like wild type BoHV-1 in vitro. Attempts to exchange the entire BICP0-1 gene by the BoHV-5 homolog using the same approach failed repeatedly. Therefore, we cotransfected purified BICP0(-)/galK(+)-BoHV-1 BAC DNA with a recombination plasmid coding for BICP0-5 with or without a HA tag into MDBK cells. BoHV-1 recombinants expressing the respective proteins were characterized. In vitro, all recombinants grew to similar titers as the parental viruses, which demonstrates that BICP0-5 compensates for the growth defect of BICP0(-)/galK(+)-BoHV-1 and functionally complements BICP0-1 of BoHV-1. We conclude that BICP0 may be suitable to positively select BoHV-1 recombinants with deletions or insertions of additional genes of interest.
牛疱疹病毒 1（BoHV-1）和 BoHV-5 密切相关（82%的氨基酸同一性），但在神经发病机制上有很大的不同。BCP0 的早期基因（BCP0-1）的保守性较低（70%的氨基酸同一性），可能导致表型不同。一个特殊的差异是 BICP0-1 基因中的一个鸟嘌呤六聚体，只与 BICP0-5 基因中的五个鸟嘌呤对齐，因此导致后者开放阅读框发生移码。因此，BCP0-1 的 C 末端氨基酸序列（BCP0-1 的 643-676 位与 BICP0-5 的 655-720 位）完全不同。我们使用 Red 重组系统和 galK 选择和反选择，将 BICP0-5 移码引入作为细菌人工染色体（BoHV-1 BAC）克隆的 BoHV-1 基因组中。用所得 BAC 转染 MDBK 细胞产生了类似于野生型 BoHV-1 的重组病毒，在体外复制。使用相同的方法，试图用 BoHV-5 同源物交换整个 BICP0-1 基因，但多次失败。因此，我们将纯化的 BICP0(-)/galK(+)-BoHV-1 BAC DNA 与编码 BICP0-5 的重组质粒共转染到 MDBK 细胞中，该质粒带有或不带有 HA 标签。表征了表达相应蛋白的 BoHV-1 重组体。在体外，所有重组体的生长滴度与亲本病毒相似，这表明 BICP0-5 补偿了 BICP0(-)/galK(+)-BoHV-1 的生长缺陷，并在功能上补充了 BoHV-1 的 BICP0-1。我们得出结论，BCP0 可能适合于阳性选择具有感兴趣的额外基因缺失或插入的 BoHV-1 重组体。
