【作者】 吕志刚；

【导师】 卢春；

【作者基本信息】 南京医科大学 ， 微生物学， 2008， 硕士

【摘要】 卡波济肉瘤(Kaposi’s sarcoma,KS)是获得性免疫缺陷综合征(acquired immunodeficiency syndrome,AIDS)患者晚期发生率最高的恶性肿瘤。既往研究证实,其病原卡波济肉瘤相关疱疹病毒(Kaposi’s sarcoma-associated herpesvirus,KSHV)不能独立引发KS,还需要协同因子的参与。然而促使KSHV引发KS的因素目前还未完全清楚。已经证明,人类单纯疱疹病毒1型(herpes simplex virus 1,HSV-1)、人巨细胞病毒(human cytomegalovirus,HCMV)、人类疱疹病毒6型(human herpesvirus 6,HHV-6)和人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)反式激活蛋白(transactivative transcription protein,Tat)是激活KSHV裂解性周期复制的重要协同因子。鉴于HIV-1血清阳性人群中人类单纯疱疹病毒2型(human herpes simplex virus-2,HSV-2)阳性率远高于HIV-1阴性人群,HSV-2与HIV-1之间密切相关,本研究我们进一步探讨了HSV-2影响KSHV复制的潜能以及HIV-1 Tat对该过程的影响。实验首先将HSV-2标准株333感染原发性渗出性淋巴瘤【primary effusion lymphoma,PEL,又称体腔渗出性淋巴瘤(bodycavity-based lymphoma,BCBL)】来源的BCBL-1细胞,Western blot结果显示,HSV-2感染BCBL-1细胞72 h后HSV-2型特异性糖蛋白G-2(glycoprotein G-2,gG-2)开始稳定表达。实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-timePCR)检测结果表明,HSV-2感染可以诱导KSHV ORF50(KSHV周期复制的开关基因)、ORF26(编码KSHV次要衣壳蛋白)和ORF29(编码KSHV包装相关蛋白)mRNA转录水平升高。与对照组比较,HSV-2感染组3、6、12、24、48、72和96 h ORF50 mRNA水平分别升高了9.92、2.55、227、31.6、6.6、75和3.02倍(P＜0.05)。类似地,ORF26 mRNA表达水平在3～96 h也呈现不同程度的升高,而ORF29 mRNA表达水平的升高集中在48～96 h。免疫荧光测定(immunofluorescence assay,IFA)结果显示,HSV-2感染BCBL-1同时还可诱导KSHV裂解周期蛋白的表达。虫荧光素酶报告实验验证了HSV-2对ORF50启动子的直接激活作用。进一步将HIV-1 Tat质粒转染HSV-2感染的BCBL-1细胞,采用Real-time PCR和Western blot分别检测KSHV ORF26 mRNA转录和病毒白细胞介素6(Viral Interleukin-6,vIL-6)的表达。与单纯HSV-2感染比较,KSHV ORF26 mRNA转录水平在6、24、48和72 h分别升高了3.92、1.67、2.74和1.82倍(P＜0.05),vIL-6表达水平在6～72 h同样有不同程度的升高。虫荧光素酶报告实验显示,Tat可以增强HSV-2激活KSHV ORF 50启动子活性。以上结果提示,HSV-2可能通过诱导KSHV复制来提高病毒载量从而参与KS的致病过程;HIV-1 Tat能够增强HSV-2对KSHV的激活作用。更多还原

【Abstract】 Patients with acquired immunodeficiency syndrome (AIDS) commonly suffer from opportunistic infections associated with members of the herpes virus family. Kaposi’s sarcoma-associated herpesvirus (KSHV) infection appears to be necessary but not enough for Kaposi’s sarcoma (KS) development without other cofactors. Previously, we identified that both human herpesvirus 6 and human immunodeficiency virus type 1 Tat were important cofactors that activated lytic cycle replication of KSHV. To investigate whether herpes simplex virus type 2 (HSV-2) influence replication of KSHV and what the role of HIV-1 Tat is in this procedure, HSV-2 strain 333 was used to infect BCBL-1 cell. Production of lytic phase mRNA transcripts, viral proteins and infectious viral particles in BCBL-1 cells were determined with reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative PCR (Real-time PCR) and Western blot. As expected, we showed that HSV-2 was a potentially important factor in the pathogenesis of KS. Compared with control, the mRNA transcription level of ORF50 (switch gene of KSHV cycle replication) was increased 9.92, 2.55, 227, 31.6, 6.6, 75 and 3.02-fold, respectively (P < 0.05) in HSV-2 infected BCBL-1 cells at 3, 6, 12, 24, 48 and 96 h. Similarly, ORF26 (encoding KSHV minor capsid protein) mRNA transcription level was increased from 3 to 96 h, and ORF29 (encoding KSHV package associated protein) mRNA transcription level was increased from 48 to 96 h. The results were further confirmed by a luciferase reporter assay testing ORF50 promoter-driven luciferase activity.Furthermore, we showed that HIV-1 Tat significantly increased the lytic replication of KSHV induced by HSV-2. Compared with corresponding control, ORF26 mRNA level in Tat-transfected BCBL-1 cells infected by HSV-2 was increased 3.92, 1.67, 2.74 and 1.82-fold (P < 0.05) at 6, 24, 48 and 72 h, respectively. The expression level of KSHV vIL-6 was also increased from 6 to 72 h. These findings suggest that HSV-2 may participate in KS pathogenesis by inducing KSHV replication and increasing KSHV viral load. These data also suggest that HSV-2 collaborates with HIV-1 Tat in the pathogenesis of AIDS-related KS patients.更多还原