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

我们以前报道过Pin 1通过磷酸化的Ser 16-Pro 17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Ser 16磷酸化的特定激酶仍然未知。在这里，我们表明，病毒体相关的胞外信号调节激酶2（ERK 2）磷酸化丝氨酸16。将CEM/LA V-1细胞暴露于10 μM沙奎那韦和HIV-1蛋白酶的D25 N失活突变体中产生的不成熟病毒粒子的特征表明，Ser 16在Pr 55 gag加工开始后被磷酸化。此外，基于质谱的体外激酶分析表明，ERK 2特异性磷酸化Ser 16-Pro 17基序的底物中的Ser 16残基。用ERK 2抑制剂sc-222229处理CEM/LA V-1细胞后，病毒颗粒内Ser 16磷酸化水平降低，Ser 16磷酸化部分缺陷的病毒由于减弱了Pin 1依赖性脱包被而表现出逆转录受损和复制减弱。RNA干扰抑制CEM/LA V-1细胞中ERK 2的表达，可抑制病毒颗粒内ERK 2的包装，降低病毒颗粒内丝氨酸16的磷酸化水平。有趣的是，ERK 2包装缺陷病毒显示逆转录受损和HIV-1复制减弱。总之，这些发现提供了对Pin 1依赖的HIV-1去包被过程的深入了解。

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.