Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Center for Microbial Pathogenesis, UPMC Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA.
J Virol. 2020 Dec 22;95(2). doi: 10.1128/JVI.01571-20.
Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP -acetylneuraminic acid synthetase () and solute carrier family 35 member A1 (), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA and T3SA, were used to evaluate and as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA, which does not engage sialic acid. Furthermore, infectivity of T3SA was diminished to levels similar to those of T3SA in cells lacking and by CRISPR ablation. However, exogenous expression of and into the respective null cells restored sialic acid expression and T3SA binding and infectivity. These results demonstrate that and , which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, and , which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
病毒与细胞表面受体的相互作用是决定病毒嗜性和疾病发生的关键因素。呼肠孤病毒的附着蛋白 σ1 结合唾液酸化糖和蛋白质受体来介导感染，但不同细胞类型的具体要求尚不完全清楚。为了确定重配病毒诱导细胞死亡所需的宿主因子，我们针对小鼠小胶质细胞 BV2 中的超过 20000 个基因进行了 CRISPR 敲除筛选。候选基因需要重配病毒引起细胞死亡，它们高度富集了唾液酸的合成和转运。鉴定出的两个顶级候选基因，CMP -N-乙酰神经氨酸合成酶 () 和溶质载体家族 35 成员 A1 ()，促进了细胞表面唾液酸的表达。两种在结合唾液酸能力上存在差异的呼肠孤病毒株，T3SA 和 T3SA，被用来评估 和 作为重配病毒感染所需的潜在宿主基因。CRISPR-Cas9 敲除这两个基因中的任意一个后，细胞表面的唾液酸表达减少。这些结果与 T3SA 结合能力的降低相关，T3SA 能够与唾液酸结合。T3SA 的低水平结合不受基因敲除的影响，因为 T3SA 不与唾液酸结合。此外，T3SA 的感染性在缺乏 和 的细胞中通过 CRISPR 消融而降低到类似于缺乏 和 的细胞中 T3SA 的水平。然而，外源表达 和 到各自的缺失细胞中，恢复了唾液酸表达和 T3SA 结合及感染性。这些结果表明，介导细胞表面唾液酸表达的 和 ，在小鼠小胶质细胞中是重配病毒有效结合和感染所必需的。附着因子和受体是重配病毒诱导疾病过程中传播和嗜性的重要决定因素。在 CRISPR 细胞存活筛选中，我们发现了两个基因， 和 ，它们编码在细胞表面表达唾液酸所需的蛋白质，并介导重配病毒对小胶质细胞的感染。这项工作阐明了使小胶质细胞易感染重配病毒的宿主基因，并扩展了当前对重配病毒结合的小胶质细胞受体的认识。这些知识可能会为针对小胶质细胞的溶瘤应用提供新的靶向策略。
