bioRxiv2025
Studies of herpes simplex virus (HSV) entry uncovered a previously unappreciated "outside-in" signaling pathway whereby activation of the calcium (Ca2+) responsive enzyme phospholipid scramblase 1 (PLSCR1), which is known to trigger the bidirectional movement of phosphatidylserines (PS) between the inner and outer leaflet of the plasma membrane, also induces the translocation and subsequent extracellular activation of intracellular proteins, including Akt. We hypothesized that HIV-1, which has been shown to elicit scramblase TMEM16F-mediated PS externalization, may trigger a similar "outside-in" signaling cascade involving exofacial kinase activity to promote its entry into CD4+ T cells. To study this process, we utilized a cell impermeable staurosporine analogue, alkyl-CIMSS, which is a broadly active kinase inhibitor that blocks HSV-induced exofacial Akt phosphorylation and HSV infection. Using multiple cell types including TZM-bl, Jurkat T cells, and human peripheral blood mononuclear cells (PBMCs), we show that, in contrast to the effects on HSV, treatment of cells with alkyl-CIMSS enhances HIV-1 infection post-entry that is not dependent on TMEM16F. To identify potential biological processes that are responsive to alkyl-CIMSS, we performed bulk RNA-sequencing and whole cell proteomics and found that alkyl-CIMSS treatment of cells robustly upregulates the cell surface density of the proteoglycan glypican-1 (GPC1). Lentiviral delivery of GPC1 overexpression and shRNA knockdown constructs reveal that the presence and absence of GPC1 independently of alkyl-CIMSS treatment significantly impact HIV-1 infection, with the effect on infection corresponding to GPC1 expression. Further, we demonstrate that the influence of GPC1 on HIV-1 infection is in part mediated by TGF-{beta} signaling. Collectively, these findings implicate a cell surface protein susceptible to alkyl-CIMSS in restricting HIV-1 infection and identify GPC1 as a novel modulator of HIV-1 infection. Author SummaryWe utilized a cell-impermeable pan-kinase tool compound, alkyl-CIMSS, to identify cell surface molecules that might be involved in viral infection. Treatment of CD4+ T cells with alkyl-CIMSS increased the expression of the cell-surface protein glypican-1, which led to an increase in early HIV reverse transcriptase products and promoted viral infection. Conversely, alkyl-CIMSS inhibited herpes simplex virus entry and infection. These findings illustrate that viruses interact with exofacial cell membrane molecules differently to promote or impede infection.