DENGUE VACCINE DEVELOPMENT--MUTATIONS IN NONSTRUCTURAL PROTEIN REGION OF DEN2

登革热病毒蛋白是非结构蛋白2B的异源二聚体 (NS2B)和NS3。NS3的氨基末端的三分之一包含四个区域 与类胰酶丝氨酸蛋白酶的有限序列同源性，包括 三个活性中心残基。NS3中的这四个区域中的两个 (“同源方框”3和4)含有预测对 基于类比的底物结合(AA 1604、1605、1625、1627和1628) 具有结构特异性的胰酶P1口袋。我们表演了 日本血吸虫同源框3和4中部分残基的诱变研究 为了描述他们对登革2型病毒的需求 并鉴定具有部分切割缺陷的突变体。 最初，我们分析了46个突变对分子内的影响。 NS2B-NS3连接处的切割。框4中的大多数突变，包括 AA1625、1627和1628中的菌株取消了蛋白水解酶活性。这是 与框4构成部分的假设一致 衬底捆绑袋。相比之下，框3中的许多突变， 包括AA 1604和1605中的那些，保留了显著的蛋白酶 活动，甚至在少数情况下是野生活动。令人惊讶的是，asp 1604，预计将在卵裂处接触带正电荷的残基 可改变为具有显著残留活性的赖氨酸或精氨酸。 胰酶中类似的突变可以消除卵裂。框中的其他突变 3、聚集在催化丝氨酸周围，丧失了蛋白酶活性。 这证实了框3中的一些残基对蛋白酶是重要的。 功能，但与AA 1604和1605的拟议角色不一致 在底物结合中。同源框外的对照突变 都被很好地容忍了。最近，这46个突变也是 分析它们对NS4B-NS5分子间切割的影响 交叉口。一般来说，对于野生型和突变型结构，切割 在反式中比在顺式中的切割效率更高。然而， 突变对土壤中蛋白水解酶活性影响的相对顺序 顺式或反式是相同的，例如严重减弱的突变 顺式蛋白水解酶活性在反式蛋白中也存在严重缺陷。 目前正在进行在NS3中引入一些突变的工作，这使得 显著残留的蛋白酶活性回到登革热基因组，在 为了确认观察到的对蛋白酶功能的影响，以及 可能是为了减弱病毒。作为迈向这一目标的第一步，两个 沉默突变成功导入DEN感染性 克隆以创建位于诱变物两侧的唯一限制酶切位点 区域，这将有助于将NS3突变引入 基因组。 出版：Valle，R.P.C.和B.Falgout。NS3的诱变作用 登革2型病毒的蛋白酶。1998。J.维罗尔。72：624-632。

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. Initially, 46 mutations were analyzed for their effect on intramolecular cleavage at the NS2B-NS3 junction. Most mutations in box 4, including those in aa 1625, 1627, and 1628, abolished protease activity. This is consistent with the hypothesis that box 4 forms part of the substrate-binding pocket. In contrast, many of the mutations in box 3, including those in aa 1604 and 1605, retained significant protease activity, even wild-type activity in a few cases. Surprisingly, asp 1604, predicted to contact a positively charged residue at the cleavage site, could be changed to lys or arg with significant residual activity. Analogous mutations in trypsin abolish cleavage. Other mutations in box 3, clustered around the catalytic serine, abolished protease activity. This confirmed that some residues in box 3 are important for protease function, but is inconsistent with the proposed role for aa 1604 and 1605 in substrate binding. Control mutations outside of the homology boxes were well-tolerated. More recently, these 46 mutations were also analyzed for their effects on intermolecular cleavage at the NS4B-NS5 junction. In general, for both wild-type and mutant constructs, cleavage in trans was somewhat more efficient than cleavage in cis. However, the relative order of the effects of the mutations on protease activity in cis or in trans were the same, e.g. mutations which severely diminished protease activity in cis were also severely defective in trans. Work is 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. As a first step toward this goal, two silent mutations were successfully introduced into the DEN2 infectious clone to create unique restriction sites which flank the mutagenized region, which will facilitate the introduction of the NS3 mutations into the genome. Publications: Valle, R. P. C., and B. Falgout. Mutagenesis of the NS3 protease of dengue virus type 2. 1998. J. Virol. 72:624-632.