GENOME OF THE CELL FUSING AGENT

大约12年前，我们在实验室发现了一种新病毒。 该病毒存在于埃及伊蚊的培养基中 细胞谎言，并被承认，因为它引起融合的伊蚊 白纹伊蚊细胞 我们称之为细胞融合剂CFA。 我们 显示，在那个时候，CFA是一个50纳米的球形，包膜 病毒与（+）链RNA基因组，它复制在 细胞质，它有三个结构蛋白。 虽然在 CFA在这些方面与披膜病毒和黄病毒相似， 在这两种方法中均未显示与病毒的血清学交叉反应 两个家庭 目前，CFA的分类地位仍然是 仍然不稳定。 我们现在建议扩展对终审法院的研究。 有两 现在进行这些研究的令人信服的理由。 内 在过去的6个月里，我们通过终点稀释法分离了CFA的克隆， 复制速度更快，产量提高50-100倍， 我们的原始菌株。 这将极大地促进生物化学 问题研究 其次，由于我们的原始工作已经完成， 重组DNA技术的出现使得新的 研究病毒的方法。 在本提案中，我们描述了实验方法， 帮助我们了解更多关于CFA如何复制以及它的一些 生物学特性 将加大力度 CFA基因组的克隆和测序。 获得的信息 将使我们能够确定开放阅读帧的数量， 推导出病毒蛋白质的氨基酸序列， 如果CFA RNA和其他病毒RNA之间有任何同源性。 在 对CFA病毒RNA的直接研究，我们将确定 RNA的3'末端的核苷酸序列，在该过程中 了解RNA是否是多聚腺苷酸化的，我们将 表征RNA的5'末端的帽结构。 的 CFA蛋白质将在其N-末端测序，使得其 可以在病毒基因组上比对编码序列。 突变体 将选择CFA，就像我们对辛德比斯病毒所做的那样， 编码改变的RNA加帽和甲基化酶。 这些将 最终用于识别编码这些基因的基因， 内切酶 我们将更详细地研究 CFA，测试其在其他蚊子细胞系中复制的能力， 在果蝇和鳞翅目品系中，以及在选定脊椎动物中 包括一条黑头呆鱼线。 我们将测试CFA是否 可以干扰阿尔法病毒和黄病毒。 最后，我们将 在新泽西对蚊子进行类似CFA的病毒筛查。 从这些研究中，我们将1）更好地了解如何 CFA复制，2）帮助确定CFA的分类地位，以及3） 深入了解小包膜RNA病毒的进化， 包括引起人类疾病的节肢动物传播的病毒。

About 12 years ago we discovered a new virus in our laboratory. The virus was present in the culture medium of an Aedes aegypti cell lie and was recognized because it caused fusion of Aedes albopictus cells. We called it CFA for cell fusing agent. We showed, at that time, that CFA was a 50 nm spherical, enveloped virus with a (+) strand RNA genome, that it replicated in the cytoplasm, and that it had three structural proteins. Although in these respects CFA resembled the togaviruses and flaviviruses, it shown no serological cross reaction with viruses in either of these two families. At present, the taxonomic status of CFA still remains unsettled. We propose now to extend our studies of CFA. There are two compelling reasons for undertaking these studies now. Within the past 6 months we have isolated by endpoint dilution, clones of CFA which replicate more quickly and produce yields 50-100 fold higher than our original strain. This will greatly facilitate biochemical studies. Secondly, since our original work was done, the field of recombinant DNA technology has emerged making possible new approaches to the study of viruses. In this proposal we describe experimental approaches which will help us learn more about how CFA replicates and about some of its biological properties. A major effort will be directed to the cloning and sequencing of the CFA genome. The information obtained will enable us to determine the number of open reading frames, to deduce the amino acid sequences of the viral proteins, and to see if there is any homology between CFA RNA and other viral RNAs. In direct studies on the CFA viral RNA we shall determine the nucleotide sequence of the 3' terminus of the RNA, in the process learning whether the RNA is polyadenylated, and we shall characterize the cap structure at the 5' terminus of the RNA. The CFA proteins will be sequenced at their N-termini so that their coding sequences can be aligned on the viral genome. Mutants of CFA will be selected, as we have done with Sindbis virus, which code for altered RNA capping and methylating enzymes. These will eventually be used for identifying the genes which code for these enzymes. We shall investigate in more detail the host range of CFA, testing its ability to replicate in other mosquito cell lines, in Drosophila and lepidopteran lines, and in selected vertebrate cells including a fathead minnow line. We shall test whether CFA can interfere with alpha and flaviviruses. Finally, we shall screen mosquitoes in New Jersey for viruses which resemble CFA. From these studies we shall 1) obtain a better understanding of how CFA replicates, 2) help settle the taxonomic status of CFA, and 3) gain insights into the evolution of small enveloped RNA viruses, including the arthropod-borne viruses which cause disease in man.