Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.
PLoS Pathog. 2021 Jan 26;17(1):e1009088. doi: 10.1371/journal.ppat.1009088. eCollection 2021 Jan.
Cytomegaloviruses (CMVs) are generally unable to cross species barriers, in part because prolonged coevolution with one host species limits their ability to evade restriction factors in other species. However, the limitation in host range is incomplete. For example, rhesus CMV (RhCMV) can replicate in human cells, albeit much less efficiently than in rhesus cells. Previously we reported that the protein kinase R (PKR) antagonist encoded by RhCMV, rTRS1, has limited activity against human PKR but is nonetheless necessary and sufficient to enable RhCMV replication in human fibroblasts (HF). We now show that knockout of PKR in human cells or treatment with the eIF2B agonist ISRIB, which overcomes the translational inhibition resulting from PKR activation, augments RhCMV replication in HF, indicating that human PKR contributes to the inefficiency of RhCMV replication in HF. Serial passage of RhCMV in HF reproducibly selected for viruses with improved ability to replicate in human cells. The evolved viruses contain an inverted duplication of the terminal 6.8 kb of the genome, including rTRS1. The duplication replaces ~11.8 kb just downstream of an internal sequence element, pac1-like, which is very similar to the pac1 cleavage and packaging signal found near the terminus of the genome. Plaque-purified evolved viruses produced at least twice as much rTRS1 as the parental RhCMV and blocked the PKR pathway more effectively in HF. Southern blots revealed that unlike the parental RhCMV, viruses with the inverted duplication isomerize in a manner similar to HCMV and other herpesviruses that have internal repeat sequences. The apparent ease with which this duplication event occurs raises the possibility that the pac1-like site, which is conserved in Old World monkey CMV genomes, may serve a function in facilitating rapid adaptation to evolutionary obstacles.
巨细胞病毒（CMV）通常无法跨越物种屏障，部分原因是与一个宿主物种的长期共同进化限制了它们逃避其他物种限制因子的能力。然而，宿主范围的限制并不完全。例如，恒河猴 CMV（RhCMV）可以在人类细胞中复制，尽管效率远低于恒河猴细胞。我们之前曾报道，RhCMV 编码的蛋白激酶 R（PKR）拮抗剂 rTRS1 对人 PKR 的活性有限，但对于 RhCMV 在人成纤维细胞（HF）中的复制仍然是必要和充分的。我们现在表明，敲除人细胞中的 PKR 或用 eIF2B 激动剂 ISRIB 处理，该激动剂克服了 PKR 激活导致的翻译抑制，可增强 HF 中 RhCMV 的复制，表明人 PKR 有助于 RhCMV 在 HF 中复制效率低下。RhCMV 在 HF 中的连续传代可重复性地选择出在人细胞中复制能力提高的病毒。进化后的病毒包含基因组末端 6.8kb 的反向重复，包括 rTRS1。该重复取代了 pac1 样内部序列元件下游约 11.8kb 的位置，pac1 样与基因组末端附近发现的 pac1 切割和包装信号非常相似。从进化而来的病毒中纯化的斑块产生的 rTRS1 至少是亲本 RhCMV 的两倍，并且在 HF 中更有效地阻断 PKR 途径。Southern blot 显示，与亲本 RhCMV 不同，具有反向重复的病毒以类似于 HCMV 和其他具有内部重复序列的疱疹病毒的方式发生异构化。这种重复事件发生的明显容易性提出了这样一种可能性，即 pac1 样位点在旧世界猴 CMV 基因组中保守，可能在促进快速适应进化障碍方面发挥作用。
