APPLICATIONS OF INFECTIOUS CDNA TECHNOLOGY TO RNA VIRUS

感染性CDNA技术在RNA病毒中的应用

• 批准号: 6101208
• 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/6101208
• 关键词: complementary DNA; dengue virus; electroporation; genetic manipulation; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein engineering

We made full length "infectious" cDNA clones from a virulent dengue type 1 virus (DEN1 WP) and from a live attenuated DEN1 vaccine candidate, which had been adapted to grow in dog kidney cells (DEN1 PDK20). First, the left and right ends of DEN1 WP were amplified by RT-PCR using virion RNA as a template and synthetic oligonucleotide primers. The primers introduce an SP6 RNA polymerase promoter just upstream of the left-hand end. The cDNA products (approximately 3kb at the left end and 4kb at the right end) were cloned in proper orientation in the polylinker of the plasmid pRS424. Then, a 5kb cDNA was made from the middle of DEN1 WP by RT-PCR; this fragment overlaps both the left and right cloned ends. Homologous recombination between the linearized clone and the 5kb cDNA upon cotransfection of yeast creates a plasmid containing the full-length DEN1 WP cDNA. Plasmid DNA was prepared from E coli, and transcribed in vitro. Electroporation of RNA into LLCMK2 cells produced a dengue infection. Recovered virus was passaged on C6/36 cells. This virus behaved like the parent DEN1 WP in growth curves. Comparison of the nucleotide sequence of the cloned DEN1 WP cDNA to the sequence of the WP virion RNA (determined by sequencing uncloned RT-PCR products) revealed only 5 differences: 3 silent and 2 missense mutations. The DEN1 PDK20 infectious clone was made by replacing the WP sequences in the full-length clone with cDNA made from PDK-20 virion RNA. First, an RT-PCR product representing the right half of PDK-20 was used to replace the corresponding region of WP by homologous recombination in yeast. This chimeric plasmid was grown in E. coli, and transcripts from it were proven infectious. Then, the chimeric plasmid was used as a starting point to introduce the left half of PDK20 as above, and a PDK20 full-length clone which made infectious transcripts was identified. Comparison of the nucleotide sequence of the cloned DEN1 PDK20 cDNA with that of PDK20 virion RNA (determined by sequencing uncloned RT-PCR products) revealed 8 differences, including 4 coding changes. These coding changes were repaired in a further series of homologous recombination steps in yeast, resulting in a final infectious DEN1 PDK20 clone which has only 3 silent changes from its parent. Transcripts were electroporated into Vero cells to make virus, which was passaged on C6/36 cells. This virus has the same small plaque phenotype as its DEN1 PDK20 parent. Growth of these viruses in monkey and dog kidney cells is currently being investigated. In the future, we plan to map the mutations responsible for the adaptation of PDK20 to growth in dog cells, by making chimeras between the WP and PDK20 infectious clones. Also, in human clinical trials the PDK20 virus is slightly too reactogenic, yet a PDK26 virus is overattenuated. We plan to introduce further attenuating mutations into the PDK20 clone in an effort to recover a correctly attenuated vaccine strain, and at first we will use subsets of the mutations found in PDK26 which are not in PDK20.

我们从强毒力登革热 1 型病毒 (DEN1 WP) 和活的减毒 DEN1 候选疫苗中克隆出全长“感染性”cDNA，该疫苗已适应在狗肾细胞 (DEN1 PDK20) 中生长。首先，使用病毒体RNA作为模板和合成寡核苷酸引物通过RT-PCR扩增DEN1 WP的左端和右端。引物在左端上游引入 SP6 RNA 聚合酶启动子。将cDNA产物（左端约3kb，右端约4kb）以正确的方向克隆到质粒pRS424的多接头中。然后，通过RT-PCR从DEN1 WP的中部制备5kb cDNA；该片段与左右克隆末端重叠。共转染酵母后，线性化克隆与 5kb cDNA 之间的同源重组产生含有全长 DEN1 WP cDNA 的质粒。从大肠杆菌中制备质粒DNA，并在体外转录。将 RNA 电穿孔至 LLCMK2 细胞中产生登革热感染。回收的病毒在C6/36细胞上传代。该病毒的生长曲线与亲代 DEN1 WP 相似。将克隆的 DEN1 WP cDNA 的核苷酸序列与 WP 病毒体 RNA 的序列（通过对未克隆的 RT-PCR 产物进行测序确定）进行比较，仅发现 5 个差异：3 个沉默突变和 2 个错义突变。 DEN1 PDK20 感染性克隆是通过用由 PDK-20 病毒粒子 RNA 制成的 cDNA 替换全长克隆中的 WP 序列而制成的。首先，用代表PDK-20右半部分的RT-PCR产物在酵母中通过同源重组替换WP的相应区域。这种嵌合质粒在大肠杆菌中生长，并且其转录物被证明具有感染性。然后，以该嵌合质粒为起点，如上所述导入PDK20的左半部分，鉴定出产生感染性转录物的PDK20全长克隆。克隆的 DEN1 PDK20 cDNA 的核苷酸序列与 PDK20 病毒体 RNA 的核苷酸序列（通过对未克隆的 RT-PCR 产物进行测序确定）的比较发现了 8 个差异，其中包括 4 个编码变化。这些编码变化在酵母中的一系列同源重组步骤中得到修复，产生最终的感染性 DEN1 PDK20 克隆，其与亲本相比仅具有 3 个沉默变化。将转录本电穿孔至 Vero 细胞中以制备病毒，并在 C6/36 细胞上传代。该病毒与其 DEN1 PDK20 亲本病毒具有相同的小噬斑表型。目前正在研究这些病毒在猴子和狗肾细胞中的生长。 未来，我们计划通过在 WP 和 PDK20 感染性克隆之间制作嵌合体，来绘制导致 PDK20 适应狗细胞生长的突变图谱。此外，在人体临床试验中，PDK20 病毒的反应原性稍高，而 PDK26 病毒则过度减毒。 我们计划在 PDK20 克隆中引入进一步的减毒突变，以努力恢复正确的减毒疫苗株，首先我们将使用 PDK26 中发现的、PDK20 中没有的突变子集。