Turner Mallory A, Middha Sumit, Hofherr Sean E, Barry Michael A
Virology and Gene Therapy Program, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota, USA.
Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, Minnesota, USA.
J Virol. 2015 Dec;89(24):12401-17. doi: 10.1128/JVI.01534-15. Epub 2015 Sep 30.
Our understanding of adenovirus (Ad) biology is largely extrapolated from human species C Ad5. Most humans are immune to Ad5, so lower-seroprevalence viruses like human Ad6 and Ad26 are being tested as therapeutic vectors. Ad6 and Ad26 differ at the DNA level by 34%. To better understand how this might impact their biology, we examined the life cycle of the two viruses in human lung cells in vitro. Both viruses infected A549 cells with similar efficiencies, executed DNA replication with identical kinetics within 12 h, and began killing cells within 72 h. While Ad6-infected cells remained adherent until death, Ad26-infected cells detached within 12 h of infection but remained viable. Next-generation sequencing (NGS) of mRNA from infected cells demonstrated that viral transcripts constituted 1% of cellular mRNAs within 6 h and 8 to 16% within 12 h. Quantitative PCR and NGS revealed the activation of key early genes at 6 h and transition to late gene activation by 12 h by both viruses. There were marked differences in the balance of E1A and E1B activation by the two viruses and in the expression of E3 immune evasion mRNAs. Ad6 was markedly more effective at suppressing major histocompatibility complex class I (MHC I) display on the cell surface and in evading TRAIL-mediated apoptosis than was Ad26. These data demonstrate shared as well as divergent life cycles in these genetically distant human adenoviruses. An understanding of these differences expands the knowledge of alternative Ad species and may inform the selection of related Ads for therapeutic development.
A burgeoning number of adenoviruses (Ads) are being harnessed as therapeutics, yet the biology of these viruses is generally extrapolated from Ad2 and Ad5. Here, we are the first to compare the transcriptional programs of two genetically distant Ads by mRNA next-generation sequencing (NGS). Species C Ad6 and Ad26 are being pursued as lower-seroprevalence Ad vectors but differ at the DNA level by 34%. Head-to-head comparison in human lung cells by NGS revealed that the two viruses generally conform to our general understanding of the Ad transcriptional program. However, fine mapping revealed subtle and strong differences in how these two viruses execute these programs, including differences in the balance of E1A and E1B mRNAs and in E3 immune evasion genes. This suggests that not all adenoviruses behave like Ad2 and Ad5 and that they may have unique strategies to infect cells and evade the immune system.
我们对腺病毒（Ad）生物学的理解很大程度上是从人类C型Ad5推断而来的。大多数人对Ad5具有免疫力，因此像人类Ad6和Ad26这样血清阳性率较低的病毒正被作为治疗载体进行测试。Ad6和Ad26在DNA水平上有34%的差异。为了更好地理解这可能如何影响它们的生物学特性，我们在体外研究了这两种病毒在人肺细胞中的生命周期。两种病毒以相似的效率感染A549细胞，在12小时内以相同的动力学进行DNA复制，并在72小时内开始杀死细胞。虽然感染Ad6的细胞在死亡前一直保持贴壁状态，但感染Ad26的细胞在感染后12小时内就会脱离，但仍保持存活。对感染细胞的mRNA进行下一代测序（NGS）表明，病毒转录本在6小时内占细胞mRNA的1%，在12小时内占8%至16%。定量PCR和NGS显示，两种病毒在6小时时激活关键早期基因，并在12小时时转变为晚期基因激活。两种病毒在E1A和E1B激活的平衡以及E3免疫逃避mRNA的表达方面存在显著差异。Ad6在抑制细胞表面主要组织相容性复合体I类（MHC I）展示以及逃避TRAIL介导的细胞凋亡方面比Ad26明显更有效。这些数据表明，这些遗传距离较远的人类腺病毒具有共同的以及不同的生命周期。对这些差异的理解扩展了对其他腺病毒种类的认识，并可能为治疗性开发中相关腺病毒的选择提供参考。
越来越多的腺病毒（Ads）正被用作治疗药物，然而这些病毒的生物学特性通常是从Ad2和Ad5推断而来的。在这里，我们首次通过mRNA下一代测序（NGS）比较了两种遗传距离较远的腺病毒的转录程序。C型Ad6和Ad26正被作为血清阳性率较低的腺病毒载体进行研究，但在DNA水平上有34%的差异。通过NGS在人肺细胞中进行的直接比较表明，这两种病毒总体上符合我们对腺病毒转录程序的一般理解。然而，精细定位揭示了这两种病毒在执行这些程序方面存在细微和显著的差异，包括E1A和E1B mRNA平衡以及E3免疫逃避基因的差异。这表明并非所有腺病毒的行为都像Ad2和Ad5，它们可能有独特的感染细胞和逃避免疫系统的策略。
