Fig. 7.

The antiviral activity of CysC95-146 is conserved in monkeys and affects HIV-2 and SIV infection in human T cells. (A) Alignment of cystatin C amino acid sequences from the indicated species. Dots indicate identity to the human sequence and dashes identify gaps introduced to optimize the alignment. The region corresponding to the CysC95-146 is shaded. (B) Antiviral activity of human and monkey-derived CysC95-146 peptides. GHOST-GPR15 cells were incubated with increasing amounts of human and monkey CysC95-146 for 2 h at 37 °C prior to infection with SIVmac239 Firefly luciferase (F-Luc). At 3 dpi, infection was analyzed via F-Luc reporter assay. The experiment was performed in triplicates. (C) CysC95-146 shows antiviral activity against GPR15-mediated SIVmac239 replication in human primary cells. To examine possible effects on spreading infection, we isolated and stimulated human PBMCs and treated them with the various compounds (CysC95-146, GPR15L, AMD3100, MVC, and CXCL16) prior to virus exposure. Infectious virus production was determined by infection of TZM-bl indicator cells with PBMC culture supernatants obtained at different days post-infection. (D) Calculated area under the curve (AUC) for the virus replication data obtained in C. ***P < 0.001, **P < 0.01 (Welch’s t test, unpaired). (E) Verification of homozygous deletions in the CCR5 gene of a Δ32/Δ32 PBMC donor. (F) Replication kinetics of SIVmac239 in Δ32/Δ32 PBMCs in the presence of various antiviral agents. Experimental details and symbols are provided in C. (G) Calculated AUC for the virus replication of SIVmac239 (see F) and X4 HIV-1 in Δ32/Δ32 PBMCs.