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。在此之前，我们对DEN 2型(DEN2)NS3中的一些残基进行了突变研究，预测这些残基参与底物结合。总共分析了46个突变对体外卵裂的影响。对其中13个具有野生型或接近野生型活性的突变体进行了反向工程，获得了一个全长的DEN2cDNA克隆，并检测了突变的RNA转录本的感染性：6个是非感染性的，7个是病毒的。回收的病毒具有野生型生长动力学。现在，先前在DEN4 NS2B区域对蛋白酶活性至关重要的一系列突变中的8个成员已经被改造成由Robin Levis制造的具有感染性的DEN4克隆。其中5个病毒被发现，而3个是致命的。其中一种被发现的病毒是一种部分变异病毒。其中几种病毒在NS2B的另一个位置有第二位点突变，将特定的MET残基改变为ILE或Val，使引入的突变对表型的影响的分析变得复杂。其中一种恢复的病毒存在严重的生长缺陷。 我们以前观察到，由3‘端有短片段缺失的DEN2cDNA克隆制成的转录本具有感染性。缺失多达6个核苷酸的构建体是可行的，而缺失8个或更多的核苷酸则是致命的。7个核苷酸的缺失是可行的还是致命的，这取决于径流转录本3‘端的限制性位点突出部分的存在和组成。除一例外，所有恢复的病毒的3‘端序列都是野生型；从7个核苷酸缺失恢复的病毒在1.5位处有一个与野生型不同的3’端序列，但该突变病毒似乎以野生型动力学方式生长。为了进一步研究这一点，我们继续与帕德马纳班实验室合作，该实验室现在位于乔治敦。在他们的体外RNA复制系统中，正在分析几个小的3‘端缺失。到目前为止，很明显，被删除的RNA正在复制，对复制RNA的3‘端序列的分析正在进行中。 我们与Padmanabhan小组还有另外两个正在进行的合作。(1)在DEN2NS3中构建了几个双突变体，并在大肠杆菌中进行了表达和纯化，为后续的结晶学分析奠定了基础。我们还试图表达和纯化NS3和NS2B的蛋白酶域之间的一个二聚体。(2)成功构建了表达绿色荧光蛋白(GFP)的DEN2复制子。最初的结果看起来并不乐观，因为GFP的表达水平非常低。改变转基因的工作正在进行中。 最后，在一个附带项目中，与美国国立卫生研究院的娜塔莎·卡普伦和理查德·摩根合作，展示了小dsRNA对基因表达和登革热病毒在C6/36蚊子细胞中生长的抑制作用。这导致了一本书的出版。

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.