Methods for evaluating attenuated vaccine candidate viruses.

登革病毒（DEN）蛋白酶是非结构蛋白2B（NS 2B）和NS 3的异源二聚体。NS 3的氨基末端的第三个是胰蛋白酶样丝氨酸蛋白酶。以前，我们进行了诱变研究的一些残基在NS 3的DEN 2型（DEN 2）预测参与底物结合，以表征其对蛋白酶活性的要求，并确定突变体与部分切割缺陷。总共分析了46个突变对体外NS 2B-NS 3连接处的分子内切割和NS 4 B-NS 5连接处的分子间切割的影响。随后，Padmanabhan和Murthy报道了DEN 2 NS 3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一致。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NS 3蛋白，看看结构是否确实如预测的那样受到干扰。还进行了将这些NS 3突变中的一些引入DEN 2基因组中的工作，以努力证实观察到的对蛋白酶功能的影响，并可能减弱病毒。13个突变体，野生型或接近野生型的活性在体外进行了分析，通过逆向工程，他们到一个全长DEN 2 cDNA克隆和测试突变体RNA转录的感染性：5个是非感染性的，7个被回收的病毒，在1例回收的病毒是假回复突变体。这些病毒的表型目前正在研究中的生长曲线和受感染细胞的蛋白酶加工的生化分析。在这组13个突变中，致死的5个在体外具有较小的活性，表明DEN不能耐受蛋白酶活性的大幅降低。 在一组不相关的实验中，我们观察到由在3'端具有短缺失的DEN 2 cDNA克隆制备的转录物是感染性的。缺失多达7个nt的构建体是可行的，而缺失8个或更多个nt是致命的。在除一种情况外的所有情况下，回收的病毒的3'端序列是野生型的，这表明病毒具有修复不正确的3'端结构的某种机制。从7 nt缺失中回收的病毒具有在1.5个位置处不同于野生型的3'端序列，并且该突变体病毒似乎比野生型DEN 2生长更慢。目前正在进一步研究该突变体的表型，并试图了解3'端修复的机制。

The dengue virus (DEN) protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. The amino-terminal third of NS3 is a trypsin-like serine protease. 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 order to characterize their requirement for protease activity and to identify mutants with partial cleavage defects. In all, 46 mutations were analyzed for their effect on intramolecular cleavage at the NS2B-NS3 junction and intermolecular cleavage at the NS4B-NS5 junction in vitro. 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 has also been done to introduce some of these NS3 mutations back into the DEN2 genome, in an effort to confirm the observed effect on protease function and possibly to attenuate the virus. Thirteen mutants that had wild-type or nearly wild-type activity in vitro were analyzed by reverse engineering them into a full-length DEN2 cDNA clone and testing mutant RNA transcripts for infectivity: 5 were non-infectious, 7 were recovered as virus, and in 1 case the recovered virus was a pseudorevertant. The phenotypes of these viruses are currently being investigated in growth curves and by biochemical analyses of protease processing in infected cells. Among this set of 13 mutations, the 5 that were lethal had less activity in vitro, suggesting that DEN is not able to tolerate much reduction in protease activity. In an unrelated set of experiments, we have observed that transcripts made from DEN2 cDNA clones with short deletions at the 3' end are infectious. Constructs missing up to 7 nt are viable, while deletion of 8 or more nt is lethal. In all but one case, the 3' end sequences of the recovered viruses are wild-type, suggesting that the virus has some mechanism to repair incorrect 3' end structures. Virus recovered from the 7 nt deletion has a 3' end sequence that differs from wild-type at 1.5 positions, and this mutant virus appears to grow more slowly than wild-type DEN2. Work is in progress to further investigate the phenotype of this mutant, and to attempt to understand the mechanism involoved in the 3' end repair.