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.
摘要：
鼠γ疱疹病毒68（MHV68）主要在B细胞中建立潜伏期，并引起淋巴瘤，让人联想到实验室小鼠的人类γ疱疹病毒疾病。研究病毒感染的分子机制以及病毒决定子如何控制细胞并最终引起肿瘤发生，容易获得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。已知γ疱疹病毒感染发育中的B细胞和巨噬细胞，因此，我们的目标是扩大MHV68潜伏感染的细胞系库。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中分离出潮霉素抗性重组MHV68，HE2.1，并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TaqManqPCR分析这些细胞系的亚克隆的病毒miRNA表达,并评估裂解病毒转录物M3的表达。通过消化-环化PCR分析显示,细胞系将病毒基因组保持为附加体。此处产生的潜伏感染细胞系不表达高于亲本细胞系的病毒miRNA。然而,这些细胞系可能为研究潜伏期机制和miRNA靶标鉴定研究提供替代工具.
