Brennan Greg, Kitzman Jacob O, Rothenburg Stefan, Shendure Jay, Geballe Adam P
Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.
Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.
PLoS Pathog. 2014 Mar 13;10(3):e1004002. doi: 10.1371/journal.ppat.1004002. eCollection 2014 Mar.
The majority of recently emerging infectious diseases in humans is due to cross-species pathogen transmissions from animals. To establish a productive infection in new host species, viruses must overcome barriers to replication mediated by diverse and rapidly evolving host restriction factors such as protein kinase R (PKR). Many viral antagonists of these restriction factors are species specific. For example, the rhesus cytomegalovirus PKR antagonist, RhTRS1, inhibits PKR in some African green monkey (AGM) cells, but does not inhibit human or rhesus macaque PKR. To model the evolutionary changes necessary for cross-species transmission, we generated a recombinant vaccinia virus that expresses RhTRS1 in a strain that lacks PKR inhibitors E3L and K3L (VVΔEΔK+RhTRS1). Serially passaging VVΔEΔK+RhTRS1 in minimally-permissive AGM cells increased viral replication 10- to 100-fold. Notably, adaptation in these AGM cells also improved virus replication 1000- to 10,000-fold in human and rhesus cells. Genetic analyses including deep sequencing revealed amplification of the rhtrs1 locus in the adapted viruses. Supplying additional rhtrs1 in trans confirmed that amplification alone was sufficient to improve VVΔEΔK+RhTRS1 replication. Viruses with amplified rhtrs1 completely blocked AGM PKR, but only partially blocked human PKR, consistent with the replication properties of these viruses in AGM and human cells. Finally, in contrast to AGM-adapted viruses, which could be serially propagated in human cells, VVΔEΔK+RhTRS1 yielded no progeny virus after only three passages in human cells. Thus, rhtrs1 amplification in a minimally permissive intermediate host was a necessary step, enabling expansion of the virus range to previously nonpermissive hosts. These data support the hypothesis that amplification of a weak viral antagonist may be a general evolutionary mechanism to permit replication in otherwise resistant host species, providing a molecular foothold that could enable further adaptations necessary for efficient replication in the new host.
人类中大多数新出现的传染病是由动物的跨物种病原体传播引起的。为了在新的宿主物种中建立有效的感染，病毒必须克服由多种快速进化的宿主限制因子介导的复制障碍，如蛋白激酶R（PKR）。这些限制因子的许多病毒拮抗剂具有物种特异性。例如，恒河猴巨细胞病毒PKR拮抗剂RhTRS1在一些非洲绿猴（AGM）细胞中抑制PKR，但不抑制人类或恒河猴的PKR。为了模拟跨物种传播所需的进化变化，我们构建了一种重组痘苗病毒，该病毒在缺乏PKR抑制剂E3L和K3L的菌株中表达RhTRS1（VVΔEΔK+RhTRS1）。在最低限度允许的AGM细胞中连续传代VVΔEΔK+RhTRS1可使病毒复制增加10至100倍。值得注意的是，在这些AGM细胞中的适应性也使病毒在人类和恒河猴细胞中的复制提高了1000至10000倍。包括深度测序在内的基因分析揭示了适应性病毒中rhtrs1基因座的扩增。通过反式提供额外的rhtrs1证实，仅扩增就足以改善VVΔEΔK+RhTRS1的复制。rhtrs1扩增的病毒完全阻断了AGM的PKR，但仅部分阻断了人类的PKR，这与这些病毒在AGM和人类细胞中的复制特性一致。最后，与可以在人类细胞中连续传代的AGM适应性病毒不同，VVΔEΔK+RhTRS1在人类细胞中仅传代三次后就没有产生子代病毒。因此，在最低限度允许的中间宿主中rhtrs1扩增是一个必要步骤，使病毒范围能够扩展到以前不允许的宿主。这些数据支持这样的假设，即弱病毒拮抗剂的扩增可能是一种普遍的进化机制，以允许在原本有抗性的宿主物种中复制，提供一个分子立足点，从而能够在新宿主中进行有效复制所需的进一步适应。
