METHODS FOR EVALUATING ATTENUATED VACCINE CANDIDATE VIRUSES.

登革热病毒蛋白水解酶是非结构蛋白2B(NS2B)和NS3的异源二聚体。NS3的氨基末端含有4个与胰酶样丝氨酸蛋白酶同源性有限的区域，包括3个活性中心残基。NS3的这四个区域中的两个(“同源框”3和4)包含根据与胰酶P1特异性口袋的结构类似的预测对底物结合重要的残基(AA 1604、1605、1625、1627和1628)。我们对登革2型病毒同源框3和4中的一些残基进行了突变研究，以确定它们对蛋白酶活性的需求，并鉴定具有部分切割缺陷的突变体。此前，46个突变在体外分析了它们对NS2B-NS3连接的分子内切割和NS4B-NS5连接的分子间切割的影响。随后，Padmanabhan和Murthy报道了DEN2NS3蛋白的晶体结构，我们观察到的大多数突变的影响与他们预期的蛋白酶结构扰动是一致的。我们现在正在与这些研究人员合作，表达、纯化和结晶各种突变的NS3蛋白，以确定其结构是否真的如预测的那样受到干扰。 还在努力将NS3中的一些突变引入登革热基因组，以确认观察到的对蛋白酶功能的影响，并可能使病毒减毒。此前已将两个沉默突变引入DEN2感染性克隆，以创建位于诱变区两侧的独特的Nar I限制性内切酶切点。将Nar I酶切的DEN 2感染性cDNA与从突变体NS3克隆中纯化的Nsi I/BamH I片段混合，用于将感受态酵母转化为色氨酸非依赖性酵母。在酵母中，两个DNA片段之间的双重同源重组导致了一个全长的DEN2cDNA克隆，其中包含了感兴趣的NS3突变。这些突变克隆在大肠杆菌中生长，在DEN2基因组的3‘端线性化，然后转录成RNA，然后电穿孔到猴子肾脏细胞上。通过这种方式，我们确定该结构是否仍然具有传染性，并从具有传染性的克隆中分离出突变病毒。到目前为止，我们已经获得了4个突变的全长克隆：3个没有感染性(突变的表型是致死的)，1个突变病毒已经产生。这种病毒的表型将通过生长曲线和蛋白酶加工的生化分析来表征。此外，我们将继续将其他NS3突变重新引入感染性克隆中进行鉴定。

The dengue viral protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. The amino-terminal third of NS3 contains four regions of limited sequence homology to trypsin-like serine proteases, including the three active center residues. Two of these four regions in NS3 ("homology boxes" 3 and 4) contain residues predicted to be important for substrate binding (aa 1604, 1605, 1625, 1627 and 1628) based on analogy with the structure of the trypsin P1 specificity pocket. We performed a mutagenesis study of some of the residues in homology boxes 3 and 4 of dengue virus type 2 in order to characterize their requirement for protease activity and to identify mutants with partial cleavage defects. Previously, 46 mutations were analyzed for their effect in vitro on intramolecular cleavage at the NS2B-NS3 junction and intermolecular cleavage at the NS4B-NS5 junction. Subsequently, the crystal structure of the DEN2 NS3 protein was reported by Padmanabhan and Murthy, and the observed effects of most of our mutations were consistent with their expected perturbations of the protease structure. We are now collaborating with these investigators to express, purify, and crystallize various mutant NS3 proteins, to see if the structures are indeed perturbed as predicted. Work is also underway to introduce some of the mutations in NS3 which permit significant residual protease activity back into the dengue genome, in an effort to confirm the observed effect on protease function and possibly to attenuate the virus. Two silent mutations had previously been introduced into the DEN2 infectious clone to create unique Nar I restriction sites which flank the mutagenized region. DEN2 infectious cDNA digested with Nar I is mixed with an Nsi I/BamH I fragment purified from a mutant NS3 clone, and the mixture is used to transform competent yeast to tryptophan independence. In yeast, double homologous recombination between the two DNA fragments results in a full-length DEN2 cDNA clone into which the NS3 mutation of interest has been incorporated. These mutant clones are grown up in E. coli, linearized at the 3' end of the DEN2 genome, and then transcribed into RNA which is electroporated onto monkey kidney cells. In this way, we determine if the construct is still infectious, and isolate mutant viruses from the clones that are. So far, we have made 4 mutant full-length clones: 3 were not infectious (the phenotype of the mutation was lethal), and 1 mutant virus has been produced. The phenotype of this virus will be characterized in growth curves and by a biochemical analysis of protease processing. Also, we will continue to introduce other NS3 mutations back into the infectious clone for characterization.