PDF(Pubmed)
Abstract：
Marek\'s disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek\'s disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.
摘要：
马立克氏病（MD），由Gallidα疱疹病毒2（GaAHV2）或Marek病疱疹病毒（MDV）引起，是鸡的一种毁灭性疾病，其特征是全身淋巴瘤的发展。针对MD的疫苗株包括gallidα疱疹病毒3（GaAHV3），与MDV同源的非致癌鸡甲疱疹病毒，和同源melegridα疱疹病毒1（MeAHV1）或火鸡疱疹病毒（HVT）。先前的工作表明，尽管预测的蛋白质包含跨膜结构域，但在体外传代过程中产生的大多数MDVgC被分泌到受感染细胞的培养基中。我们以前鉴定了在体外MDV复制过程中分泌的两种可变剪接的gCmRNA，基于每个UL44(gC)转录物去除的内含子的大小,称为gC104和gC145。由于gC在Alphaherpesvirinae亚科中保守，我们假设GaAHV3（菌株301B/1）和HVT也由于mRNA剪接而分泌gC。为了解决这个问题，我们从301B/1和HVT感染的细胞培养物中收集培养基,并使用蛋白质印迹分析,确定301B/1和HVT均产生分泌的gC.接下来,我们从301B/1和HVT感染的细胞培养物和鸡羽毛毛囊上皮（FFE）皮肤细胞中提取了RNA。RT-PCR分析证实了301B/1gC的一个剪接变体(gC104)和HVTgC的两个变体(gC104和gC145)。有趣的是,所有三种病毒之间的剪接非常保守。预测和验证的mRNA剪接供体的进一步分析，分支点(BP)，和受体位点表明301B/1UL44转录物序列内单核苷酸多态性（SNP）导致不产生gC145。然而,301B/1gC145供体的修饰，BP,和MDVUL44序列的受体位点没有导致gC145mRNA剪接变体，表明mRNA剪接比最初假设的更复杂。总之,我们的结果表明,禽疱疹病毒的mRNA剪接是保守的,这些信息对于开发下一代MD疫苗或阻断传播的疗法可能很重要.
