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Developing an anti-HIV drugs that inhibit uncoating for overcomingdrug resistance

开发一种抗艾滋病毒药物，通过抑制脱衣来克服耐药性

基本信息

批准号：
23659066
负责人：
MISUMI Shogo
金额：
$ 2.41万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Challenging Exploratory Research
财政年份：
2011
资助国家：
日本
起止时间：
2011 至 2012
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23659066/
关键词：
ウイルス感染症微生物 HIV 薬剤耐性 Pin1 脱殻キナーゼ

项目摘要

We previously reported that Pin1 facilitates the humanimmunodeficiency virus type 1 (HIV -1) uncoating by interacting with the capsid (CA) corethrough the phosphorylated Ser16-Pro17motif. However , the specific kinase responsible forSer16phosphorylation has remained unknown. Here, we show that virion-associatedextracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser16. The characterization ofimmature virions produced by exposing of CEM/LA V -1 cells to 10 μM saquinavir and a D25Ninactive mutant of HIV -1 protease indicated that Ser16is phosphorylated after the initiationof Pr55gagprocessing. Furthermore, a mass spectrometry-based in vitro kinase assaydemonstrated that ERK2 specifically phosphorylated the Ser16residue in theSer16-Pro17-motif-containing substrate. The treatment of CEM/LA V -1 cells with the ERK2inhibitor sc-222229 decreased the Ser16phosphorylation level inside virions, and the viruspartially defective in Ser16phosphorylation showed an impaired reverse transcription and anattenuated replication owing to an attenuated Pin1-dependent uncoating. Furthermore, thesuppression of ERK2 expression by RNA interference in CEM/LA V -1 cells resulted insuppressed ERK2 packaging inside virions and decreased the Ser16phosphorylation levelinside virions. Interestingly , the ERK2-packaging-defective virus showed an impaired reversetranscription and an attenuated HIV -1 replication. Taken together , these findings provideinsights into the as yet obscure processes in Pin1-dependent HIV -1 uncoating.

我们以前报道过Pin1通过磷酸化的Ser16-Pro17基序与衣壳(CA)核心相互作用，促进人类免疫缺陷病毒1型(HIV-1)的脱壳。然而，负责Ser16磷酸化的特定激酶仍不清楚。在这里，我们展示了病毒粒子相关的细胞外信号调节激酶2(ERK2)磷酸化Ser16。将未成熟病毒粒子暴露于10μM沙奎那韦和HIV-1蛋白酶活性突变株D250中，结果表明Ser16在Pr55蛋白加工开始后被磷酸化。此外，基于质谱学的体外激酶分析表明，ERK2特异性地磷酸化了含Ser16-Pro17基序底物中的Ser16残基。ERK2抑制剂sc-222229处理CEM/LA V-1细胞后，病毒颗粒内Ser16的磷酸化水平降低，部分Ser16磷酸化缺陷的病毒由于依赖PIN1的解包被减弱，导致逆转录和复制减弱。此外，通过RNA干扰抑制ERK2在CEM/LA V-1细胞中的表达，抑制了病毒粒子内ERK2的包装，降低了病毒粒子内Ser16的磷酸化水平。有趣的是，ERK2包装缺陷病毒表现出逆转转录受损和HIV-1复制减弱。综上所述，这些发现为了解依赖Pin1的HIV-1去涂层的过程提供了洞察力。