UL45 of Human Cytomegalovirus Promotes Macrophage Survival and Modulates RIPK3-Associated Cell Death

Programmed necrotic cell death (necroptosis) is a key antiviral defense pathway, yet its regulation during human cytomegalovirus (HCMV) infection remains incompletely defined in physiologically relevant cell types. The M45 protein of murine cytomegalovirus, a homologue of UL45, inhibits necroptosis and contributes to MCMV dissemination, providing a rationale for examining UL45 in HCMV. Previous studies of UL45 have largely relied only on fibroblasts or immortalized cell lines, which lack the necroptotic machinery necessary to reveal viral anti-necroptotic functions. To investigate the role of the HCMV UL45 protein, we generated a UL45 stop-frameshift mutant (UL45st) in the TB40E strain and compared its phenotype to that of the wild-type virus in primary human fibroblasts and monocyte-derived macrophages. In parallel, transfection-based assays in RIPK3-competent HT29 cells were used to assess the intrinsic cell-death-inhibitory activity of UL45. UL45st-infected primary macrophages exhibited significantly reduced numbers of viable cells and showed morphological features suggesting necroptotic death compared to wild-type-infected cells, whereas infected fibroblasts showed only modest defects. Expression of UL45 alone in HT29 cells partially protected cells from both apoptotic and RIPK3-dependent necroptotic stimuli, indicating a direct anti-death function of the protein. These findings demonstrate that HCMV UL45 promotes cellular survival in primary human macrophages and reveal that this function is likely associated with RIPK3-associated cell death. Collectively, this work identifies a never-before-shown function of HCMV UL45 as a key determinant of viral survival in myeloid compartment cells and underscores the importance of using physiologically relevant models to uncover cell-type-specific viral immune evasion mechanisms.