Walsh Scott R, Gerpe María Carla Rosales, Wootton Sarah K
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
McMaster Immunology Research Centre, McMaster University, Hamilton, Ontario, Canada.
Virol J. 2016 Dec 30;13(1):209. doi: 10.1186/s12985-016-0660-x.
Enzootic nasal tumor virus (ENTV-1) is an ovine betaretrovirus that has been linked to enzootic nasal adenocarcinoma (ENA), a contagious tumor of the ethmoid turbinates of sheep. Transmission experiments performed using virus isolated from cell free nasal tumor homogenates suggest that ENTV-1 is the causative agent of ENA; however, this etiological relationship has not been conclusively proven due to the fact that the virus cannot be propagated in vitro nor is there an infectious molecular clone of the virus.
Here we report construction of a molecular clone of ENTV-1 and demonstrate that transfection of this molecular clone into HEK 293T cells produces mature virus particles.
Analysis of recombinant virus particles derived from the initial molecular clone revealed a defect in the proteolytic processing of Gag; however, this defect could be corrected by co-expression of the Gag-Pro-Pol polyprotein from the highly related Jaagsiekte sheep retrovirus (JSRV) suggesting that the polyprotein cleavage sites in the ENTV-1 molecular clone were functional. Mutagenesis of the molecular clone to correct amino acid variants identified within the pro gene did not restore proteolytic processing; whereas deletion of one proline residue from a polyproline tract located in variable region 1 (VR1) of the matrix resulted in production of CA protein of the mature (cleaved) size strongly suggesting that normal virion morphogenesis and polyprotein cleavage took place. Finally, electron microscopy revealed the presence of spherical virus particles with an eccentric capsid and an average diameter of about 100 nm.
In summary, we have constructed the first molecular clone of ENTV-1 from which mature virus particles can be produced. Future experiments using virus produced from this molecular clone can now be conducted to fulfill Koch's postulates and demonstrate that ENTV-1 is necessary and sufficient to induce ENA in sheep.
地方性鼻肿瘤病毒1型（ENTV-1）是一种绵羊β逆转录病毒，与绵羊筛骨鼻甲的传染性肿瘤——地方性鼻腺癌（ENA）有关。使用从无细胞鼻肿瘤匀浆中分离出的病毒进行的传播实验表明，ENTV-1是ENA的病原体；然而，由于该病毒无法在体外繁殖，也没有其感染性分子克隆，这种病因关系尚未得到确凿证实。
在此，我们报告了ENTV-1分子克隆的构建，并证明将该分子克隆转染到HEK 293T细胞中可产生成熟病毒颗粒。
对源自初始分子克隆的重组病毒颗粒的分析显示，Gag的蛋白水解加工存在缺陷；然而，通过共表达高度相关的绵羊肺腺瘤病毒（JSRV）的Gag-Pro-Pol多蛋白可以纠正这一缺陷，这表明ENTV-1分子克隆中的多蛋白切割位点是有功能的。对分子克隆进行诱变以纠正pro基因内鉴定出的氨基酸变体并不能恢复蛋白水解加工；而从基质可变区1（VR1）中的多聚脯氨酸序列中删除一个脯氨酸残基导致产生成熟（切割）大小的CA蛋白，这强烈表明发生了正常的病毒体形态发生和多蛋白切割。最后，电子显微镜显示存在平均直径约为100 nm、衣壳偏心的球形病毒颗粒。
总之，我们构建了第一个可产生成熟病毒颗粒的ENTV-1分子克隆。现在可以使用从该分子克隆产生的病毒进行进一步实验，以满足科赫法则，并证明ENTV-1是诱导绵羊ENA所必需且充分的。
