Kettle S, Blake N W, Law K M, Smith G L
Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
Virology. 1995 Jan 10;206(1):136-47. doi: 10.1016/s0042-6822(95)80028-x.
A characterization of genes B13R (SPI-2) and B22R (SPI-1) from vaccinia virus strain Western Reserve (WR) is presented. These genes are transcribed early during infection and the predicted encoded proteins show similarity to the superfamily of serine protease inhibitors (serpins). The 5' transcriptional initiation site of each gene was mapped by primer extension experiments to 71-72 and 31 nucleotides upstream of the B13R and B22R open reading frames (ORFs), respectively. Each ORF was expressed in Escherichia coli and specific antisera were raised against the protein produced. These antisera were used to identify the B13R- and B22R-encoded proteins in vaccinia virus-infected cells as stable, intracellular, nonglycosylated proteins of M(r) 38.5K and M(r) 40K, respectively. The B22R gene product was detected in all orthopoxviruses tested including cowpox, rabbitpox, and vaccinia strains WR, Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W. In contrast, the B13R gene product had a more limited distribution and was not detected in Copenhagen, Tashkent, Lister, and Tian Tan. Viable virus deletion mutants that lacked only B13R or B22R coding sequences (delta B13R and delta B22R) and revertant viruses in which the deleted gene was restored were constructed by transient dominant selection. The growth of the deletion mutants in cell culture was indistinguishable from that of wild-type virus. Additionally the virulence of each deletion mutant was indistinguishable from wild-type and revertant viruses in a murine intranasal model.
本文介绍了来自痘苗病毒西储株（WR）的基因B13R（SPI-2）和B22R（SPI-1）的特征。这些基因在感染早期转录，预测的编码蛋白与丝氨酸蛋白酶抑制剂超家族（丝氨酸蛋白酶抑制剂）相似。通过引物延伸实验将每个基因的5'转录起始位点分别定位到B13R和B22R开放阅读框（ORF）上游71-72和31个核苷酸处。每个ORF在大肠杆菌中表达，并针对产生的蛋白质制备了特异性抗血清。这些抗血清用于鉴定痘苗病毒感染细胞中B13R和B22R编码的蛋白，分别为稳定的、细胞内的、非糖基化的蛋白，分子量分别为38.5K和40K。在所有测试的正痘病毒中都检测到了B22R基因产物，包括牛痘、兔痘以及痘苗病毒株WR、哥本哈根株、塔什干株、天坛株、李斯特株、惠氏株、IHD-J和IHD-W。相比之下，B13R基因产物的分布更为有限，在哥本哈根株、塔什干株、李斯特株和天坛株中未检测到。通过瞬时显性选择构建了仅缺失B13R或B22R编码序列的活病毒缺失突变体（ΔB13R和ΔB22R）以及缺失基因得以恢复的回复病毒。缺失突变体在细胞培养中的生长与野生型病毒无异。此外，在小鼠鼻内模型中，每个缺失突变体的毒力与野生型和回复病毒也没有区别。
