Methods for evaluating attenuated vaccine candidate viru

摘要：登革病毒（DEN）蛋白酶是非结构蛋白2B （NS2B）和NS3的异源二聚体。此前，我们对DEN2型（DEN2）的NS3中一些预计与底物结合有关的残基进行了诱变研究。总共分析了46个突变对体外切割的影响。其中13个具有野生型或接近野生型活性的突变体通过反向工程构建全长DEN2 cDNA克隆并检测突变体RNA转录本的传染性进行了分析：6个无传染性，7个恢复为病毒。恢复的病毒具有野生型生长动力学。现在，先前在DEN4 NS2B区域中对蛋白酶活性至关重要的一组突变的8个成员已经被设计成由Robin Levis制造的传染性DEN4克隆。其中5例发现了病毒，3例是致命的。其中一个恢复的病毒是部分逆转录病毒。一些病毒在NS2B的另一个位置发生第二位点突变，将特定的met残基改变为ile或val，使引入突变对表型影响的分析复杂化。其中一个恢复的病毒有明显的生长缺陷。

Summary: The dengue virus (DEN) protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. Previously, we performed a mutagenesis study of some of the residues in NS3 of DEN type 2 (DEN2) predicted to be involved in substrate binding. In all, 46 mutations were analyzed for their effect on cleavage in vitro. Thirteen of these mutants with wild-type or nearly wild-type activity were analyzed by reverse engineering into a full-length DEN2 cDNA clone and testing mutant RNA transcripts for infectivity: 6 were non-infectious, while 7 were recovered as virus. The recovered viruses had wild-type growth kinetics. Now, 8 members of a previously-made set of mutations in the region of DEN4 NS2B essential for protease activity have been engineered into an infectious DEN4 clone made by Robin Levis. Virus was recovered from 5 of these, while 3 were lethal. One of the recovered viruses was a partial revertant. Several of the viruses had a second-site mutation at another location in NS2B, changing a specific met residue to ile or val, complicating the analysis of the the effect of the introduced mutations on the phenotype. One of the recovered viruses had a significant growth defect. We had previously observed that transcripts made from DEN2 cDNA clones with short deletions at the 3' end are infectious. Constructs missing up to 6 nt are viable, while deletion of 8 or more nt is lethal. Deletions of 7 nt are viable or lethal, depending on the presence and composition of a restriction site overhang at the 3' end of the run-off transcript. In all but one case, the 3' end sequences of the recovered viruses are wild-type; virus recovered from the 7 nt deletion has a 3' end sequence that differs from wild-type at 1.5 positions, but this mutant virus appears to grow with wild-type kinetics. To investigate this further, we have continued our collaboration with the Padmanabhan lab, now located at Georgetown. Several small 3' end deletions are being analyzed in their in vitro RNA replication system. So far, it is clear that the deleted RNAs are replicating, and analysis of the 3' end sequences of replicating RNAs is ongoing. We have two other ongoing collaborations with the Padmanabhan group. (1) Several double mutants in DEN2 NS3 were constructed, and are being expressed in E coli and purified, for subsequent crystallographic analysis. We are also trying to express and purify a dimer between NS3 and the protease domain of NS2B. (2) We have attempted to make a DEN2 replicon expressing green fluorescent protein (GFP). The initial results do not look promising, in that the level of GFP expression is very poor. Work is underway to change transgenes. Finally, in a side project, a collaboration was done with Natasha Caplen and Richard Morgan at the NIH, demonstrating the inhibitory effect of small dsRNA on gene expression and on the growth of dengue virus in C6/36 mosquito cells. This has resulted in a publication.