The development of effective viroimmunotherapies for cancer relies on the ability of replicating viruses to infect the entire tumor tissue and induce comprehensive inflammation within the tumor microenvironment (TME). In this study, we investigate the impact of mutations in the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) on tumor cell infectivity, replication, and the immune response. Specifically, we focus on two key positions in GP1 (181 and 185) that significantly enhance tumor cell infectivity and improve CD8⁺ T cell function in the GP-mutated LCMV strain LCMV P52. Our findings demonstrate that LCMV strain LCMV P52 replicates preferentially in vivo in tumors, exhibits improved tumor penetration, attracts functional CD8⁺ T cells more effectively to the tumor, and reprograms the tumor toward a more immunogenic microenvironment to allow for effective checkpoint inhibitor combination.

The lymphocytic choriomeningitis virus (LCMV) represents a strong T cell activating virus with anti-tumoral properties. In recent work, we developed an attenuated cancer cell-adapted reassortant LCMV strain carrying tumor-tropic mutations. The immunological mechanisms underlying these mutations remained unknown. Here, we passaged wild-type LCMV-WE in human lung cancer H1975 cells for 52 passages. After 29 passages, the virus acquired the GP1 mutations I181M and R185W, involved in viral receptor interactions, which remained stable throughout the subsequent 33 passages. Compared with wild-type LCMV-WE, LCMV-P52 showed increased infectivity, partly concomitant with enhanced propagation in human tumor cells. In vivo, LCMV-P52 induced reduced type I interferon (IFN-I) responses and showed accelerated expansion in CD169⁺ macrophages. Propagation within the splenic marginal zone correlated with accelerated priming of virus-specific CD8⁺ T cells and limited liver tissue damage. Mechanistic in vitro experiments showed that the I181M and R185W mutations were associated with substantially reduced infection of plasmacytoid dendritic cells (pDCs). Collectively, the I181M and R185W mutations limit systemic IFN-I induction and accelerate LCMV propagation in CD169⁺ macrophages, correlating with improved CD8⁺ T cell function and reduced liver damage. These findings provide mechanistic insight into how defined GP mutations can modulate viral tropism and antiviral immune responses.