A cell-impermeable kinase inhibitor uncovers outside-in signaling pathways that promote HIV-1 infection.

Studies of herpes simplex virus (HSV) entry revealed a previously unrecognized "outside-in" signaling pathway involving phosphatidylserine (PS) scrambling and associated translocation and subsequent extracellular activation of canonical intracellular proteins, including Akt. We hypothesized that HIV-1, which activates a different scramblase, TMEM16F, to induce PS externalization, may similarly trigger an "outside-in" signaling response to promote viral entry. To study this process, we utilized a cell-impermeable staurosporine analog, alkyl-CIMSS, which is a broadly active kinase inhibitor that blocks HSV-induced exofacial Akt phosphorylation, and HSV entry. We show that TMEM16F-mediated PS externalization in response to HIV is not associated with Akt translocation; however, surprisingly, pretreatment of cells with alkyl-CIMSS enhanced HIV-1 infection post-entry. To identify potential biological processes that mediated this enhancement, we performed whole-cell total and phosphoproteomics, and bulk RNA sequencing. Cells treated with alkyl-CIMSS exhibited increased cyclin-dependent kinase (CDK) activity, resulting in higher levels of phosphorylated SAMHD1. Further, alkyl-CIMSS treatment robustly upregulated the cell surface density of the proteoglycan glypican-1 (GPC1). Lentivirus-driven GPC1 overexpression or shRNA knockdown demonstrated that, independent of alkyl-CIMSS treatment, GPC1 expression promotes HIV infection. Collectively, these findings demonstrate that alkyl-CIMSS modulates the exofacial plasma membrane to promote susceptibility to HIV infection by increasing CDK activity and upregulating GPC1. IMPORTANCE: The cell-impermeable pan-kinase inhibitor alkyl-CIMSS inhibits HSV infection by blocking phosphorylation of exofacial Akt. In contrast, alkyl-CIMSS enhances HIV infection. This increase was associated with enhanced cyclin-dependent kinase activity and upregulation of surface-presented glypican-1. These results illustrate that distinct processes associated with the exofacial proteome can either promote viral infection, as observed for HIV, or impede infection, as in the case of HSV, and may facilitate the identification of pathways that can be targeted for future antiviral drug development.