Phosphorylation of human immunodeficiency virus type 1 capsid protein at serine 16, required for peptidyl-prolyl isomerase-dependent uncoating, is mediated by virion-incorporated extracellular signal-regulated kinase 2

Abstract

We reported previously that Pin1 facilitates human immunodeficiency virus type 1 (HIV-1) uncoating by interacting with the capsid core through the phosphorylated Ser16-Pro17 motif. However, the specific kinase responsible for Ser16 phosphorylation has remained unknown. Here, we showed that virion-associated extracellular signal-regulated kinase 2 (ERK2) phosphorylates Ser16. The characterization of immature virions produced by exposing chronically HIV-1LAV-1-infected CEM/LAV-1 cells to 10 µM saquinavir indicated that Ser16 is phosphorylated after the initiation of Pr55Gag processing. Furthermore, a mass spectrometry-based in vitro kinase assay demonstrated that ERK2 specifically phosphorylated the Ser16 residue in the Ser16-Pro17 motif-containing substrate. The treatment of CEM/LAV-1 cells with the ERK2 inhibitor sc-222229 decreased the Ser16 phosphorylation level inside virions, and virus partially defective in Ser16 phosphorylation showed impaired reverse transcription and attenuated replication owing to attenuated Pin1-dependent uncoating. Furthermore, the suppression of ERK2 expression by RNA interference in CEM/LAV-1 cells resulted in suppressed ERK2 packaging inside virions and decreased the Ser16 phosphorylation level inside virions. Interestingly, the ERK2-packaging-defective virus showed impaired reverse transcription and attenuated HIV-1 replication. Taken together, these findings provide insights into the as-yet-obscure processes in Pin1-dependent HIV-1 uncoating.