Su Y H, Meegalla R L, Chowhan R, Cubitt C, Oakes J E, Lausch R N, Fraser N W, Block T M
Department of Biochemistry and Molecular Pharmacology, Jefferson Center for Biomedical Research of Thomas Jefferson University, Doylestown, Pennsylvania, USA.
J Virol. 1999 May;73(5):4171-80. doi: 10.1128/JVI.73.5.4171-4180.1999.
A two-cell system for the stimulation of herpes simplex virus type 1 (HSV-1) from an in vitro model of long-term (quiescent) infection is described. Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor were infected with HSV-1 strain 17. Little, if any, cytotoxicity was observed, and a quiescent infection was established. The long-term infection was characterized by the absence of all detectable virus in the culture medium and little, if any, detectable early or late viral-gene expression as determined by reverse transcriptase PCR analysis. The presence of HSV-1 DNA was determined by PCR analysis. This showed that approximately 180 viral genomes were present in limiting dilutions where as few as 16 cells were examined. The viral DNA was infectious, since cocultivation with human corneal fibroblasts (HCF) or human corneal epithelial cells (HCE) resulted in recovery of virus from most, if not all, clusters of PC12 cells. Following cocultivation, viral antigens appeared first on PC12 cells and then on neighboring inducing cells, as determined by immunofluorescent staining, demonstrating that de novo viral protein synthesis first occurred in the long-term-infected PC12 cells. Interestingly, the ability to induce HSV varied among the cell lines tested. For example, monkey kidney CV-1 cells and human hepatoblastoma HepG2 cells, but not mouse neuroblastoma cells or undifferentiated PC12 cells, mediated stimulation. This work thus shows that (i) quiescent HSV infections can be maintained in PC12 cells in vitro, (ii) HSV can be induced from cells which do not accumulate significant levels of latency-associated transcripts, and (iii) the activation of HSV gene expression can be induced via neighboring cells. The ability of adjacent cells to stimulate HSV gene expression in neuron-like cells represents a novel area of study. The mechanism(s) whereby HCF, HCE, and HepG2 and CV-1 cells communicate with PC12 cells and stimulate viral replication, as well as how this system compares with other in vitro models of long-term infection, is discussed.
本文描述了一种用于从长期（静止）感染的体外模型中刺激单纯疱疹病毒1型（HSV-1）的双细胞系统。用神经生长因子分化的大鼠嗜铬细胞瘤（PC12）细胞感染HSV-1 17株。观察到几乎没有细胞毒性，从而建立了静止感染。长期感染的特征是培养基中没有所有可检测到的病毒，并且通过逆转录酶PCR分析确定几乎没有可检测到的早期或晚期病毒基因表达。通过PCR分析确定HSV-1 DNA的存在。这表明在有限稀释中存在约180个病毒基因组，其中仅检测了16个细胞。病毒DNA具有传染性，因为与人类角膜成纤维细胞（HCF）或人类角膜上皮细胞（HCE）共培养导致从大多数（如果不是全部）PC12细胞簇中回收病毒。共培养后，通过免疫荧光染色确定病毒抗原首先出现在PC12细胞上，然后出现在相邻的诱导细胞上，这表明从头病毒蛋白合成首先发生在长期感染的PC12细胞中。有趣的是，在所测试的细胞系中诱导HSV的能力各不相同。例如，猴肾CV-1细胞和人肝癌HepG2细胞介导刺激，而小鼠神经母细胞瘤细胞或未分化的PC12细胞则不能。因此，这项工作表明：（i）静止的HSV感染可以在体外的PC12细胞中维持；（ii）HSV可以从没有积累大量潜伏相关转录本的细胞中诱导出来；（iii）HSV基因表达的激活可以通过相邻细胞诱导。相邻细胞在神经元样细胞中刺激HSV基因表达的能力代表了一个新的研究领域。本文讨论了HCF、HCE以及HepG2和CV-1细胞与PC12细胞通信并刺激病毒复制的机制，以及该系统与其他长期感染体外模型的比较。
