Kaposi's sarcoma-associated herpesvirus can productively infect primary human keratinocytes and alter their growth properties
Kaposi's sarcoma-associated herpesvirus can productively infect primary human keratinocytes and alter their growth properties
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DOI：
10.1128/jvi.75.5.2435-2443.2001
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发表时间：
2001-03-01
影响因子：
5.4
通讯作者：
Flore, O
中科院分区：
文献类型：
--
作者：
Cerimele, F;Curreli, F;Flore, O
以往的研究表明，Kaposi肉瘤相关疱疹病毒(KSHV/HHV8)DNA存在于皮肤Kaposi肉瘤斑块期结节病变(KS)的内皮细胞、表皮基底层的角质形成细胞和KS病变内的外分泌腺上皮细胞中。我们用KSHV/HHV8感染原代培养的人角质形成细胞，感染后6d，用逆转录聚合酶链式反应(RT-PCR)检测病毒基因的转录，用抗LANA单抗的免疫荧光法检测蛋白的表达。用12-O-十四酰佛波醇-13-乙酸酯(TPA)处理KSHV/HHV8感染的角质形成细胞，RT-PCR检测病毒裂解基因的转录情况，如编码衣壳蛋白的开放阅读框26。最后，用KSHV感染的TPA诱导的角质形成细胞的浓缩上清液感染其他原代人细胞(人脐静脉内皮细胞)，RT-PCR证实存在病毒转录物。未感染的角质形成细胞在模拟感染后3-5周衰老，而感染KSHV/HHV8的角质形成细胞继续增殖，至今仍在培养。然而，感染8周后，套式PCR检测不到病毒基因组，尽管先前感染KSHV/HHV8的角质形成细胞仍表达上皮标记物，但它们获得了新的特征，如接触抑制丧失、端粒酶活性、锚定非依赖性生长和细胞因子产生的变化。这些结果表明，KSHV/HHV8和其他疱疹病毒一样，可以在体外感染和复制上皮细胞，提示在体内这些细胞可能在KSHV/HHV8感染和病毒传播中发挥重要作用。
Previous studies have shown the presence of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) DNA in endothelial cells, in keratinocytes in the basal layer of the epidermis overlying plaque-stage nodular lesions of cutaneous Kaposi's sarcoma (KS), and in the epithelial cells of eccrine glands within KS lesions. We infected primary cell cultures of human keratinocytes with KSHV/HHV8, At 6 days post infection, transcription of viral genes was detected by reverse transcriptase PCR (RT-PCR), and protein expression was documented by an immunofluorescence assay with an anti-LANA monoclonal antibody. To determine whether the viral lytic cycle was inducible by chemical treatment, KSHV/HHV8-infected keratinocytes were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and RT-PCR was performed to confirm the transcription of lytic genes such as open reading frame 26, (which encodes a capsid protein). Finally, to assess infectious viral production, other primary human cells (human umbilical vein endothelial cells), were infected with concentrated supernatant of KSHV-infected, TPA-induced keratinocytes and the presence of viral transcripts was confirmed by RT-PCR The uninfected keratinocytes senesced 3 to 5 weeks after mock infection, while the KSHV/HHV8-infected keratinocytes continued to proliferate and to date are still in culture. However, 8 weeks after infection, viral genomes were no longer detectable by nested PCR Although the previously KSHV/HHV8-infected keratinocytes still expressed epithelial markers, they acquired new characteristics such as contact inhibition loss, telomerase activity, anchorage-independent growth, and changes in cytokine production. These results show that KSHV/HHV8, like other herpesviruses, can infect and replicate in epithelial cells in vitro and suggest that in vivo these cells may play a significant role in the establishment of KSHV/HHV8 infection and viral transmission.