EVALUATION OF ONCOGENIC FACTORS RELEVANT TO DEVELOPING SAFE HERPESVIRUS VACCINES

疱疹病毒、人巨细胞病毒的基因组和亚基因组克隆 研究了人巨细胞病毒（HCMV）和人疱疹病毒-6（HHV-6）的致癌性。 为了评估这些病毒在某些人类中的作用， 癌症，特别是HHV-6在淋巴瘤中的作用， 淋巴组织增生性疾病 CMV： 我们之前确认了两个 CMV内的mtrII（980 bp）和mtrIII（7.5kb） 通过将病毒DNA克隆引入啮齿动物细胞（NIH 3T3或Rat-3T3）， 2），并通过在培养基中生长来测定所选细胞的致瘤性。 琼脂糖或在裸鼠中。 绘制了mtrII的转化活性图 79个氨基酸的开放阅读框（ORF）。 最近，我们发现 通过氯霉素乙酰转移酶（CAT）测定的启动子活性 3T3细胞中mtrII编码的特异性mRNA。 此外，我们还绘制了 转化结构域mtrIII转化为270个氨基酸的ORF。 89-氨基- 需要酸性ORF以最大化转化效率。 这些 目前正在将ORF克隆到表达载体中，以促进 研究这些蛋白质的转化机制。 的cmv 主要立即早期基因启动子和基因片段没有诱导 转型 HHV-6：用HHV-6进行了类似的研究， 在人类淋巴瘤中的作用。 两个DNA片段（21 kb和8.7 kb） GS株（分离自淋巴细胞白血病患者） 发现转化NIH 3T3细胞和人角质形成细胞。 其他HHV-6 从其他患者分离的菌株也能转化3T3细胞。 转化 3T3细胞在免疫活性细胞中诱导具有转移潜力的肿瘤 小鼠 诱导肿瘤产生的细胞毒性T细胞可溶解 自体肿瘤 在另一项研究中，HHV-6 DNA与8.7 kb片段在来自免疫母细胞性白血病患者的组织中被鉴定， 淋巴瘤中检测到的EBV DNA的情况下。 最近，6名 PCR检测非霍奇金淋巴瘤HHV-6 DNA阳性， 其中2例免疫组化检测HHV-6抗原阳性。 HHV-6可能含有一个以上的转化基因。 对CMV和其他病毒的开发和安全性评价至关重要 疱疹病毒疫苗和疱疹病毒载体的开发 基因治疗 肿瘤抗原的鉴定可能导致 CMV-或HHV-6诱导的特异性诊断试剂或疫苗 恶性疾病。

Genomes and subgenomic clones of herpesviruses, human cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6), were studied for oncogenic potential in order to evaluate the role of these viruses in certain human cancers, especially the role of HHV-6 in lymphomas and lymphoproliferative disorders. CMV: We previously identified two transforming domains, mtrII (980 bp) and mtrIII (7.5kb) within the CMV genome by introducing viral DNA clones into rodent cells (NIH 3T3 or Rat- 2), and assaying the selected cells for tumorigenicity by growth in agarose or in nude mice. The transforming activity of mtrII was mapped to a 79-amino-acid open reading frame (ORF). Recently, we identified promoter activity by a choramphenicol acetyl transferase (CAT) assay and specific mRNA encoded by mtrII in 3T3 cells. In addition, we mapped the transforming domain, mtrIII, to a 270-amino-acid ORF. A 89-amino- acid ORF was required to maximize the transforming efficiency. These ORFs are currently being cloned into expression vectors to facilitate studies on the mechanism of transformation by these proteins. The CMV major immediate early gene promoter and the gene segment did not induce transformation. HHV-6: Similar studies were performed with HHV-6 to define its role in human lymphomas. Two DNA segments (21 kb and 8.7 kb) of GS strain ( isolated from a patient with lymphocytic leukemia) were found to transform NIH 3T3 cells and human keratinocytes. Other HHV-6 isolates from other patients could also transform 3T3 cells. Transformed 3T3 cells induced tumors with metastatic potential in immunocompetent mice. Cytotoxic T cells derived from induced tumors could lyse autologous tumors. In a separate study, HHV-6 DNA homologous to the 8.7 kb segment was identified in tissues from a patient with immunoblastic lymphoma in the absence of detectable EBV DNA. Recently, six additional Non-Hodkin's lymphomas examined were positive for HHV-6 DNA by PCR and two of them were positive for HHV-6 antigens by immunohistochemistry. HHV-6 may contain more than one transforming gene.These studies are important for the development and safety evaluation of CMV and other herpesvirus vaccines, and the development of herpesvirus-based vectors for gene therapy. Identification of a tumor antigen may lead to a specific diagnostic reagent or a vaccine for CMV- or HHV-6 induced malignant diseases.