Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events
Human cytomegalovirus replicates abortively in polymorphonuclear leukocytes after transfer from infected endothelial cells via transient microfusion events
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DOI：
10.1128/jvi.74.12.5629-5638.2000
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发表时间：
2000-06-01
影响因子：
5.4
通讯作者：
Revello, MG
中科院分区：
文献类型：
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作者：
Gerna, G;Percivalle, E;Revello, MG
利用最近开发的体外产生pp 65阳性多形核白细胞（PMNLs）的模型，我们证明了来自免疫活性受试者的PMNLs可能携带传染性人巨细胞病毒（HCMV）和病毒产物（第65页，第72页，DNA和立即早期[IE]和pp 67晚期mRNA），最早在与人脐静脉内皮细胞（HUVEC）共培养后60分钟或用临床HCMV分离株（VR 6110）或其它野生型毒株感染的人胚肺成纤维细胞（HELF）。使用实验室适应的HCMV株感染的HELF，在PMNL中仅检测到非常少量的病毒DNA和IE和晚期mRNA。在与VR 6110感染的细胞共培养的PMNL中检测到大量存在于感染和未感染的HUVEC中的细胞mRNA，即血管细胞粘附分子-1 mRNA，其量比对照组大得多。病毒DNA合成抑制剂显示，在共培养过程中，只有IE基因在PMNLs中转录，PMNLs中IE转录本的合成也得到了以下发现的支持，即只有IE mRNA的拷贝数在PMNLs中才能表达。（而不是DNA或pp 67 mRNA）随时间显著增加，pp 67与IE mRNA的拷贝数比从感染的HUVEC中的大于10变化到共培养的PMNL中的小于1。荧光探针转移实验和电子显微镜研究表明，感染性病毒和病毒产物从感染细胞转移到PMNLs可能仅由野生型株诱导的微融合事件介导。此外，HCMV pp 65和p72均显示定位于核的相同的PMNLs的双重免疫染色。两种不同的机制可以解释病毒在PMNL中的存在：（i）一种主要机制包括短暂的微融合事件（仅由野生型菌株诱导）HUVEC或HELF和PMNL，并将活病毒和生物活性病毒物质转移至PMNL;和（ii）一种次要机制，即，内吞作用，在野生型和实验室菌株中都发生，并导致获得非常少量的病毒核酸。总之，HCMV复制流产PMNLs，和野生型菌株及其产品（以及细胞代谢产物和荧光染料）被转移到PMNLs，从而提供证据的一个潜在的机制，HCMV在体内传播。
Using a recently developed model for in vitro generation of pp65-positive polymorphonuclear leukocytes (PMNLs), we demonstrated that PMNLs from immunocompetent subjects may harbor both infectious human cytomegalovirus (HCMV) and viral products (pp65, p72, DNA and immediate-early [IE] and pp67 late mRNAs) as early as 60 min after coculture with human umbilical vein endothelial cells (HUVEC) or human embryonic lung fibroblasts (HELF) infected with a clinical HCMV isolate (VR6110) or other wild-type strains, The number of PMNLs positive for each viral parameter increased with coculture time. Using HELF infected with laboratory-adapted HCMV strains, only very small amounts of viral DNA and IE and late mRNAs were detected in PMNLs. A cellular mRNA, the vascular cell adhesion molecule-1 mRNA, which is abundantly present in both infected and uninfected HUVEC, was detected in much larger amounts in PMNLs cocultured with VR6110-infected cells than in controls, Coculture of PMNLs with VR6110-infected permissive cells in the presence or absence of RNA, protein, and viral DNA synthesis inhibitors showed that only IE genes were transcribed in PMNLs during coculture, Synthesis of IE transcripts in PMNLs was also supported by the finding that only the copy number of IE mRNA (and not the DNA or the pp67 mRNA) per infected PMNL increased markedly with time, and the pp67 to IE mRNA copy number ratio changed from greater than 10 in infected HUVEC to less than 1 in cocultured PMNLs. Fluorescent probe transfer experiments and electron microscopy studies indicated that transfer of infectious virus and viral products from infected cells to PMNLs Is likely to he mediated by microfusion events induced by wild-type strains only. In addition, HCMV pp65 and p72 were both shown to localize in the nucleus of the same PMNLs by double immunostaining. Two different mechanisms may explain the virus presence in PMNLs: (i) one major mechanism consists of transitory microfusion events (induced by wild type strains only) of HUVEC or HELF and PMNLs with transfer of viable virus and biologically active viral material to PMNLs; and (ii) one minor mechanism, i,e,, endocytosis, occurs with both wild-type and laboratory strains and leads to the acquisition of very small amounts of viral nucleic acids. In conclusion, HCMV replicates abortively in PMNLs, and wild-type strains and their products (as well as cellular metabolites and fluorescent dyes) are transferred to PMNLs, thus providing evidence for a potential mechanism of HCMV dissemination in vivo.