Single-Cell Transcriptome Analysis of CD34+ Stem Cell-Derived Myeloid Cells Infected With Human Cytomegalovirus

Myeloid cells are important sites of lytic and latent infection by human cytomegalovirus (CMV). We previously showed that only a small subset of myeloid cells differentiated from CD34+ hematopoietic stem cells is permissive to CMV replication, underscoring the heterogeneous nature of these populations. The exact identity of susceptible and resistant cell types, and the cellular features characterizing permissive cells, however, could not be dissected using averaging transcriptional analysis tools such as microarrays and, hence, remained enigmatic. Here, we profile the transcriptomes of ~ 7000 individual cells at day one post-infection using the 10X genomics platform. We show that viral transcripts are detectable in the majority of the cells, suggesting that virion entry is unlikely to be the main target of cellular restriction mechanisms. We further show that viral replication occurs in a small but specific sub-group of cells transcriptionally related to, and likely derived from, a cluster of cells expressing markers of Colony Forming Unit – Granulocyte, Erythrocyte, Monocyte, Megakaryocyte (CFU-GEMM) oligopotent progenitors. Compared to the remainder of the population, CFU-GEMM cells are enriched in transcripts with functions in mitochondrial energy production, cell proliferation, RNA processing and protein synthesis, and express similar or higher levels of interferon-related genes. While expression levels of the former are maintained in infected cells, the latter are strongly down-regulated. We thus propose that the preferential infection of CFU-GEMM cells may be due to the presence of a pre-established pro-viral environment, requiring minimal optimization efforts from viral effectors, rather than to the absence of specific restriction factors. Together, these findings identify a potentially new population of myeloid cells susceptible to CMV replication, and provide a possible rationale for their preferential infection. Overall design: The 10x Genomics Chromium platform was used for single cell RNA sequencing of a population of myeloid cells differentiated from CD34+ hematopoietic progenitors and infected with CMV strain TB40/E.

髓系细胞(myeloid cells)是人类巨细胞病毒(human cytomegalovirus, CMV)发生裂解性与潜伏性感染的重要宿主位点。我们此前的研究证实，仅少数由CD34阳性造血干细胞(CD34+ hematopoietic stem cells)分化而来的髓系细胞可允许CMV复制，这凸显了该细胞群体的异质性。然而，受限于基因芯片(microarrays)等整体转录组分析工具，我们无法解析易感与抗性细胞类群的确切身份，以及允许CMV复制的细胞的特征性细胞属性，相关问题长期悬而未决。本研究借助10X Genomics平台(10X genomics platform)，对感染后1天的约7000个单个细胞进行转录组分析。结果显示，大多数细胞中均可检测到病毒转录本，这表明病毒粒子进入细胞不太可能是细胞抗病毒限制机制的主要作用靶点。我们进一步证实，病毒复制发生于一小部分具有特异性转录特征的细胞亚群——这些细胞在转录层面与表达粒细胞-红细胞-单核细胞-巨核细胞集落形成单位(colony forming unit – granulocyte, erythrocyte, monocyte, megakaryocyte, CFU-GEMM)多能祖细胞标志物的细胞簇高度相关，且大概率源自该细胞簇。与其余细胞群体相比，CFU-GEMM细胞的转录本富集于线粒体能量产生、细胞增殖、RNA加工及蛋白质合成等功能通路，且干扰素相关基因的表达水平与其余细胞相当或更高。在感染细胞中，前述功能相关转录本的表达水平得以维持，但干扰素相关基因的表达则被显著下调。据此我们提出，CFU-GEMM细胞更易被感染，可能是因为其本身已存在利于病毒复制的前病毒微环境，病毒仅需最少的效应分子调控即可完成复制，而非因为该类细胞缺乏特异性抗病毒限制因子。综上，本研究发现了一类可能新的、可被CMV复制感染的髓系细胞群，并为其优先感染现象提供了合理的解释。总体实验设计：采用10x Genomics Chromium平台，对由CD34阳性造血祖细胞分化而来、经CMV毒株TB40/E(CMV strain TB40/E)感染的髓系细胞群体进行单细胞RNA测序。