VIDO-Intervac, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3, Canada.
VIDO-Intervac, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3, Canada; Vaccinology and Immunotherapeutics, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3, Canada.
Virus Res. 2015 Feb 2;197:116-26. doi: 10.1016/j.virusres.2014.12.017. Epub 2014 Dec 18.
The UL47 gene product, VP8, is one of the most abundant tegument proteins of bovine herpesvirus-1 (BoHV-1). Deletion of VP8 leads to impaired growth in tissue culture, and VP8 is indispensable for BHV-1 replication in cattle. To elucidate the biological functions of VP8, we explored its interaction with mRNAs of immediate early (bICP0), early (gB, gD) and late (gC) genes of BoHV-1. FLAG-tagged VP8 was pulled down from COS-7 cells co-transfected with plasmids encoding VP8 and either gB, gC, gD or bICP0. This was followed by RNA extraction, cDNA synthesis and qPCR, which demonstrated binding of VP8 to bICP0, gB, gC and gD mRNAs in the cytoplasm and nucleus. These results were supported by co-localization of VP8 with bICP0, gB, gC and gD mRNAs in the nucleus as determined by confocal microscopy. Amino acids 259-342, located in the conserved portion of UL47 homologues, were found to contain the RNA binding region on VP8. To further characterize these interactions, Northwestern blotting was performed by immobilizing VP8 on a nitrocellulose membrane followed by hybridization with in vitro transcribed bICP0 mRNA. The results demonstrated binding of VP8 to intron-less mRNA but not intron-containing mRNA of bICP0. In addition, the interaction of VP8 with bICP0 mRNA was confirmed in vitro by RNA electrophoretic mobility shift assay, which also showed that the zinc finger and acidic domains both interact with VP8. Based on these results, we concluded that VP8 binds to intron-less mRNAs of bICP0, gB, gC and gD.
UL47 基因产物 VP8 是牛疱疹病毒 1（BoHV-1）中最丰富的衣壳蛋白之一。VP8 缺失会导致组织培养中的生长受损，并且 VP8 对于 BHV-1 在牛中的复制是必不可少的。为了阐明 VP8 的生物学功能，我们探讨了它与 BoHV-1 的即刻早期（bICP0）、早期（gB、gD）和晚期（gC）基因的 mRNA 的相互作用。FLAG 标记的 VP8 从共转染编码 VP8 和 gB、gC、gD 或 bICP0 的质粒的 COS-7 细胞中下拉。接下来进行 RNA 提取、cDNA 合成和 qPCR，证明 VP8 与 bICP0、gB、gC 和 gD mRNA 在细胞质和核内结合。这些结果通过共聚焦显微镜确定 VP8 与 bICP0、gB、gC 和 gD mRNA 在核内的共定位得到支持。位于 UL47 同源物保守部分的氨基酸 259-342 被发现包含 VP8 的 RNA 结合区。为了进一步表征这些相互作用，通过将 VP8 固定在硝酸纤维素膜上，然后用体外转录的 bICP0 mRNA 进行杂交，进行 Northwestern 印迹。结果表明 VP8 与无内含子的 mRNA 结合，但不与 bICP0 的内含子包含的 mRNA 结合。此外，通过 RNA 电泳迁移率变动分析在体外证实了 VP8 与 bICP0 mRNA 的相互作用，该分析还表明锌指和酸性结构域都与 VP8 相互作用。基于这些结果，我们得出结论，VP8 与 bICP0、gB、gC 和 gD 的无内含子 mRNA 结合。
