Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates.
Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates.
复制标题
鉴定人副流感病毒 2 型 (HPIV-2) V 蛋白氨基酸残基，可减少 V 与 MDA5 的结合并减弱非人灵长类动物中 HPIV-2 的复制。
DOI：
10.1128/jvi.02542-10
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发表时间：
2011
影响因子：
5.4
通讯作者：
Schmidt,AlexanderC
中科院分区：
文献类型：
--
作者：
Schaap-Nutt,Anne;Higgins,Caraline;Amaro-Carambot,Emerito;Nolan,SheilaM;D'Angelo,Christopher;Murphy,BrianR;Collins,PeterL;Schmidt,AlexanderC
人副流感病毒2型（HPIV-2）是一种重要的儿科呼吸道病原体，编码一种V蛋白，可抑制I型干扰素（IFN）的诱导和信号传导。使用反向遗传学，我们尝试恢复一组V突变病毒，这些病毒分别含有六个半胱氨酸至丝氨酸（残基193，197，209，211，214和218）取代之一，两个成对的电荷至丙氨酸（R175 A/R176 A和R205 A/K206 A）取代之一，或组氨酸至苯丙氨酸（H174 F）取代。使用cDNA衍生的HPIV-2病毒进行该诱变，所述HPIV-2病毒表达来自单独mRNA的V和P编码序列。在半胱氨酸取代中，只有C193 S、C214 S和C218 S产生活病毒，并且只有C214 S突变体复制得足够好以用于进一步分析。H174 F、R175 A/R176 A和R205 A/K206 A突变体是活的并且复制良好。H174 F和R205 A/K206 A突变体与野生型（WT）V在与MDA 5（诱导I型IFN的非自身RNA的细胞质传感器）物理相互作用的能力方面没有差异。与WT HPIV-2一样，这些突变体抑制IFN-β诱导并在非洲绿色猴（AGM）中有效复制。相反，C214 S和R175 A/R176 A突变体不能有效结合MDA 5，不能抑制干扰素调节因子3（IRF 3）二聚化或IFN-β诱导，并且在AGM中减弱。这些发现表明，V与MDA 5的结合对于HPIV-2在非人灵长类动物中的毒力是重要的，并且参与MDA 5结合的一些V蛋白残基对于HPIV-2的体外有效生长不是必需的。使用瞬时表达系统，20个额外的突变体V蛋白筛选MDA 5结合，和跨越残基175至180的区域被发现是必不可少的这种活性。
Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growthin vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.