Hornung Veit, Schlender Jörg, Guenthner-Biller Margit, Rothenfusser Simon, Endres Stefan, Conzelmann Karl-Klaus, Hartmann Gunther
Department of Internal Medicine, Division of Clinical Pharmacology, Ludwig-Maximillians-University, Ziemssenstrasse 1, 80336 Munich, Germany.
J Immunol. 2004 Nov 15;173(10):5935-43. doi: 10.4049/jimmunol.173.10.5935.
Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-alpha induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-alpha induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-alpha induction in PDC. HRSV-induced IFN-alpha production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-alpha by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-alpha induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.
浆细胞样树突状细胞通过Toll样受体7/8（TLR7/8）感知病毒单链RNA（ssRNA），并通过TLR9感知病毒DNA中的CpG基序及其降解产物。尽管这两条内体途径独立于病毒复制发挥作用，但对于浆细胞样树突状细胞（PDC）中活跃复制病毒的检测知之甚少。单链RNA病毒以及许多DNA病毒的病毒基因组的复制和转录会导致非感染细胞中不存在的胞质双链RNA（dsRNA）的形成。在本研究中，我们使用编码融合（F）蛋白的人呼吸道合胞病毒（HRSV）进行直接胞质内进入。HRSV感染以及65个核苷酸的dsRNA的胞质递送均导致PDC中强效的α干扰素（IFN-α）诱导，但在髓样树突状细胞中则不然。紫外线照射使HRSV失活可消除PDC中的IFN-α诱导。对携带HRSV或牛呼吸道合胞病毒F蛋白的两种呼吸道合胞病毒（RSV）构建体的比较表明，F介导的RSV胞质内进入是PDC中IFN-α诱导绝对必需的。HRSV诱导的IFN-α产生分别不受氯喹和PKR抑制剂2-氨基嘌呤所证明的内体酸化和蛋白激酶R（PKR）激酶活性的影响。相反，TLR7/8配体R848、TLR9配体CpG-A寡脱氧核苷酸2216以及灭活流感病毒（依赖TLR7/8）诱导的IFN-α被2-氨基嘌呤完全阻断。小鼠致病性仙台病毒诱导的IFN-α在PKR和髓样分化因子88（MyD88）缺陷小鼠中不受影响，证实一种能够通过质膜融合直接进入宿主细胞的单链RNA病毒可被PDC独立于PKR、TLR7/8和TLR9检测到。
