Department of Microbial Sciences, University of Surrey, Guildford, GU2 7XH, UK.
Present address: Division of Rheumatology, Department of Medicine, University of Pennsylvania, Perelman School of Medicine, Pennsylvania, PA, USA.
J Gen Virol. 2024 Oct;105(10). doi: 10.1099/jgv.0.002036.
Poxviruses are dsDNA viruses infecting a wide range of cell types, where they need to contend with multiple host antiviral pathways, including DNA and RNA sensing. Accordingly, poxviruses encode a variety of immune antagonists, most of which are expressed early during infection from within virus cores before uncoating and genome release take place. Amongst these antagonists, the poxvirus immune nuclease (poxin) counteracts the cyclic 2'3'-GMP-AMP (2'3'-cGAMP) synthase (cGAS)/stimulator of interferon genes DNA sensing pathway by degrading the immunomodulatory cyclic dinucleotide 2'3'-cGAMP, the product of activated cGAS. Here, we use poxviruses engineered to lack poxin to investigate how virus infection triggers the activation of STING and its downstream transcription factor interferon-responsive factor 3 (IRF3). Our results demonstrate that poxin-deficient vaccinia virus (VACV) and ectromelia virus (ECTV) induce IRF3 activation in primary fibroblasts and differentiated macrophages, although to a lower extent in VACV compared to ECTV. In fibroblasts, IRF3 activation was detectable at 10 h post-infection (hpi) and was abolished by the DNA replication inhibitor cytosine arabinoside (AraC), indicating that the sensing was mediated by replicated genomes. In macrophages, IRF3 activation was detectable at 4 hpi, and this was not affected by AraC, suggesting that the sensing in this cell type was induced by genomes released from incoming virions. In agreement with this, macrophages expressing short hairpin RNA (shRNA) against the virus uncoating factor D5 showed reduced IRF3 activation upon infection. Collectively, our data show that the viral genome is sensed by cGAS prior to and during genome replication, but immune activation downstream of it is effectively suppressed by poxin. Our data also support the model where virus uncoating acts as an immune evasion strategy to simultaneously cloak the viral genome and allow the expression of early immune antagonists.
痘病毒是感染多种细胞类型的 dsDNA 病毒，需要应对多种宿主抗病毒途径，包括 DNA 和 RNA 感应。因此，痘病毒编码多种免疫拮抗剂，其中大多数在脱壳和基因组释放发生之前，从病毒核心内早期表达。在这些拮抗剂中，痘病毒免疫核酸酶（poxin）通过降解免疫调节环二核苷酸 2'3'-cGAMP，即激活的 cGAS 的产物，来对抗环鸟苷酸-腺苷酸合酶（cGAS）/干扰素基因刺激物 DNA 感应途径。在这里，我们使用缺乏 poxin 的痘病毒来研究病毒感染如何触发 STING 的激活及其下游转录因子干扰素反应因子 3（IRF3）。我们的结果表明，poxin 缺陷型牛痘病毒（VACV）和细弱病毒（ECTV）在原代成纤维细胞和分化的巨噬细胞中诱导 IRF3 激活，尽管与 ECTV 相比，VACV 的程度较低。在成纤维细胞中，IRF3 激活可在感染后 10 小时（hpi）检测到，并被 DNA 复制抑制剂胞嘧啶阿拉伯糖苷（AraC）消除，表明该感应是由复制基因组介导的。在巨噬细胞中，IRF3 激活可在 4 hpi 检测到，且不受 AraC 影响，表明在这种细胞类型中，感应是由来自传入病毒粒子的基因组释放引起的。与这一观点一致的是，表达针对病毒脱壳因子 D5 的短发夹 RNA（shRNA）的巨噬细胞在感染后显示出 IRF3 激活减少。总的来说，我们的数据表明，病毒基因组在基因组复制之前和期间被 cGAS 感应，但 poxin 有效地抑制了其下游的免疫激活。我们的数据还支持这样的模型，即病毒脱壳作为一种免疫逃避策略，同时掩盖病毒基因组并允许早期免疫拮抗剂的表达。
