Abstract

Murine gammaherpesvirus 68 (MHV68) establishes latency mainly in B cells and causes lymphomas reminiscent of human gammaherpesvirus diseases in laboratory mice. To study the molecular mechanism of virus infection and how the viral determinants control cell and eventually cause tumorigenesis, readily available latently infected cell lines are essential. For in vitro MHV68 latency studies, only two cell culture systems have been available. Gammaherpesviruses are known to infect developing B cells and macrophages, therefore we aimed to expand the MHV68 latently infected cell line repertoire. Here, several latently infected immature B cell and macrophage-like cell line clones were generated. Hygromycin-resistant recombinant MHV68 was isolated from a laboratory-made latent cell line, HE2.1, and propagated to develop stable cell lines that carry the viral genome under hygromycin selection. Subclones of these cells lines were analyzed for viral miRNA expression by TaqMan qPCR and assessed for expression of a lytic viral transcript M3. The cell lines maintain the viral genome as an episome shown by the digestion-circularization PCR assay. Latently infected cell lines generated here do not express viral miRNAs higher than the parental cell line. However, these cell lines may provide an alternative tool to study latency mechanisms and miRNA target identification studies.

中文翻译：

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抗潮霉素鼠丙型疱疹病毒 68 基因组产生的潜伏巨噬细胞和未成熟 B 细胞系表达适度水平的病毒 miRNA

摘要

鼠伽马疱疹病毒 68 (MHV68) 主要在 B 细胞中建立潜伏期，并在实验室小鼠中引起类似于人类伽马疱疹病毒疾病的淋巴瘤。为了研究病毒感染的分子机制以及病毒决定簇如何控制细胞并最终导致肿瘤发生，现成的潜伏感染细胞系是必不可少的。对于体外 MHV68 潜伏期研究，只有两种细胞培养系统可用。众所周知，伽玛疱疹病毒会感染发育中的 B 细胞和巨噬细胞，因此我们的目标是扩大 MHV68 潜伏感染细胞系库。在这里，产生了几个潜伏感染的未成熟 B 细胞和巨噬细胞样细胞系克隆。从实验室制造的潜伏细胞系 HE2.1 中分离出潮霉素抗性重组 MHV68，并进行增殖以开发在潮霉素选择下携带病毒基因组的稳定细胞系。通过 TaqMan qPCR 分析这些细胞系亚克隆的病毒 miRNA 表达，并评估裂解病毒转录物 M3 的表达。消化环化 PCR 测定显示，细胞系将病毒基因组维持为附加体。这里产生的潜伏感染细胞系不表达比亲代细胞系更高的病毒 miRNA。然而，这些细胞系可能为研究潜伏机制和 miRNA 靶点识别研究提供替代工具。