Emerging Technologies Applied to the Discovery of Human Tumor Viruses

描述（由申请人提供）：感染导致全球约20%的人类癌症。然而，已知的致癌传染因子的清单却出奇地短，而且在过去的十年里没有发现新的人类肿瘤病毒。目前的肿瘤病毒发现方法并不全面，很可能会错过新的药物家族的发现。使用这些技术的阴性研究不能排除肿瘤病毒的存在，可能会遗漏以前未知的因子。我们建议修改基因表达的长序列分析（SAGE）作为一种无偏倚的手段，在二氧化硅数字转录减法（DTS）中发现病毒。我们在这里展示了一种实用的方法来执行转录组范围内的短转录标记的硅减法，允许区分人类和非人类序列。一旦发现候选肿瘤病毒序列，就可以作为病毒基因组行走和表征的起点。为了证明这种方法的可行性，我们在感染潜伏KSHV病毒的肿瘤细胞系上进行了DTS的初步研究。DTS快速且独特地鉴定了5个KSHV转录本，占细胞转录组总数的0.44%。通过将感染kshv的细胞系RNA注入未感染的人肿瘤组织RNA，定量再现了该技术。我们还使用DTS确定了区分大多数人类多态性与病毒SAGE标签的实际截止水平。最后，我们对3例鳞状细胞结膜癌（SCCC）肿瘤（一种免疫缺陷相关的恶性肿瘤）进行了试点DTS。对108,000个SAGE标签进行计算机减法，生成46个候选序列，其中包括12个高概率标签，这些标签正在被评估为可能的SCCC代理序列。我们表明，这种技术对RNA降解具有惊人的免疫力，因此它可以用于发生部分RNA降解的稀有或档案材料。我们寻求II期R33资助，对4个SCCC样本进行DTS，将我们的试点研究扩展到每百万5-10个转录本水平的SCCC全面分析。这将使我们能够确定或排除可能导致这种免疫缺陷相关癌症的肿瘤病毒。这也完成了DTS技术的发展，使我们能够充分优化其性能，应用于其他疑似感染性肿瘤

DESCRIPTION (provided by applicant): Infection contributes to ~20% of human cancers worldwide. The list of known carcinogenic infectious agents, however, is surprisingly short and no new human tumor viruses have been discovered over the past decade. Current tumor virus discovery methods are not comprehensive and are likely to miss discovery of new families of agents. Negative studies using these techniques do not rule out the presence of a tumor virus and may miss a previously unknown agent. We propose modifying long Serial Analysis of Gene Expression (SAGE) as an unbiased means for virus discovery using in silica digital transcript subtraction (DTS). We show here a practical method to perform transcriptome-wide in silico subtraction of short transcript tags, allowing discrimination between human and nonhuman sequences. Once a candidate tumor virus sequence is found, it can be used as a start point for viral genome walking and characterization. To demonstrate the feasibility of this method, we performed pilot studies of DTS on a tumor cell line infected with latent KSHV virus. DTS rapidly and uniquely identified 5 KSHV transcripts de novo comprising 0.44% of the total cell transcriptome. The technique was quantitatively reproduced by spiking KSHV-infected cell line RNA into uninfected human tumor tissue RNA. We also identified practical cut-off levels that distinguish most human polymorphisms from viral SAGE tags using DTS. Finally, we performed pilot DTS on 3 squamous cell conjunctival carcinoma (SCCC) tumors, an immunodeficiency-related malignancy. In silico subtraction of 108,000 SAGE tags generated 46 candidate sequences, including 12 high probability tags that are being evaluated as possible SCCC agent sequences. We show that this technique is surprisingly immune to RNA degradation so that it can be used on rare or archival materials in which partial RNA degradation has occurred. We seek phase II R33 funding to perform DTS on 4 SCCC samples, extending our pilot studies into a full analysis of SCCC at the 5-10 transcripts per million level. This will allow us to either identify or exclude a likely tumor virus causing this immunodeficiency-related cancer. This also completes development of DTS technology, allowing us to fully optimize its performance for application to other suspected infectious tumors ASSESSMENT: