DENGUE VIRUS RNA REPLICATION

登革热病毒 RNA 复制

• 批准号: 3792564
• 负责人: B FALGOUT
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792564
• 关键词: RNA biosynthesis; chloramphenicol acetyltransferase; dengue virus; genetic manipulation; genetic regulatory element; helper virus; tissue /cell culture; transfection; virus RNA; virus genetics; virus replication

Replication of dengue virus requires first making a minus-strand copy of the plus-strand genome, followed by making plus-strand genomes from the minus strand. Approximately the first 100 and the last 300 nucleotides of the dengue genome are not part of the open reading frame, yet are conserved. We presume that the 3' untranslated region (UTR) and the complement of the 5' UTR contain cis-acting sequences essential for the initiation of replication of the minus-strand and the plus-strand, respectively. We previously constructed a clone containing the CAT gene surrounded by the 5' and 3' UTRs of dengue virus type 4. An SP6 polymerase promoter was used to make a capped 1.5 kb transcript which resembles a miniature version of the dengue genome, except it encodes CAT instead of the dengue polyprotein. This RNA was transfected into cells alone or along with dengue virus RNA to provide trans-acting functions, and CAT activity was monitored 5 days later. Unfortunately, the CAT activity was higher in the cells transfected without helper. We were surprised at the longevity of the CAT activity in the absence of helper. The reason for the diminution upon co-transfection with dengue RNA is not certain, but it could be due to an effect on transfection efficiency, or perhaps to an effect of dengue replication on the health of the cells. We are in the process of deriving a clone which will express the lacZ gene from the SP6 promoter, with no dengue sequences, for use as an internal standard in transfection experiments. Also, we are deriving another version of the CAT clone, where the SP6 promoter will transcribe the minus-strand. Transfection of such a minus-strand RNA into cells should not result in CAT activity unless plus-strand copies are made. Ultimately, we hope to engineer mutations into the dengue UTRs to better define critical sequences.

登革热病毒的复制需要首先制作负链副本 正链基因组，然后从 负链。 大约前 100 个和最后 300 个核苷酸 登革热基因组的一部分不属于开放阅读框，但 保守的。 我们假设 3' 非翻译区 (UTR) 和 5' UTR 的互补序列含有顺式作用序列，对于 负链和正链复制的起始， 分别。 我们之前构建了一个含有 CAT 基因的克隆 被 4 型登革热病毒的 5' 和 3' UTR 包围。SP6 使用聚合酶启动子来制作加帽的 1.5 kb 转录物， 类似于登革热基因组的微型版本，不同之处在于它编码 CAT 代替登革热多蛋白。 该RNA被转染至 细胞单独或与登革热病毒RNA一起提供反式作用 功能，5天后监测CAT活性。 很遗憾， 没有辅助转染的细胞中 CAT 活性较高。 我们 令我们惊讶的是，在没有 帮手。 与登革热共转染后减少的原因 RNA 不确定，但可能是由于对转染的影响 效率，或者可能是登革热复制对健康的影响 的细胞。 我们正在衍生一个克隆，它将 从 SP6 启动子表达 lacZ 基因，没有登革热序列， 用作转染实验中的内标。 另外，我们 正在衍生 CAT 克隆的另一个版本，其中 SP6 启动子 将转录负链。 这种负链的转染 RNA 进入细胞不应导致 CAT 活性，除非正链 已制作副本。 最终，我们希望通过基因工程改造 登革热 UTR 可以更好地定义关键序列。