Host Cell Central Carbon Metabolism and Cellular NAD⁺ Pool Regulate Efficient Replication of Vesicular Stomatitis...

Vesicular stomatitis virus (VSV) is a promising oncolytic virus whose replication efficiency and tumor selectivity are strongly influenced by host cell metabolism. Cancer cells, including glioblastoma, exhibit profound rewiring of central carbon metabolism to sustain proliferation, redox balance, and biosynthetic demand, yet how these metabolic states regulate VSV replication remains incompletely defined. Here, we investigated the dependency of VSV replication on glycolysis, the pentose phosphate pathway (PPP), and glutamine metabolism in A172 human glioblastoma cells. Pharmacologic inhibition of glycolysis using 2-DG strongly suppressed VSV replication in a dose-dependent manner, highlighting a robust requirement for glycolytic flux and downstream intermediates. While inhibiting the PPP with 6-AN, a nicotinamide adenine dinucleotide (NAD) analog, markedly impaired viral replication, D-ribose was unable to rescue the inhibition, indicating that nucleotide precursor limitation alone was insufficient to explain this effect. Interestingly, depletion of glucose 6-phosphate dehydrogenase (G6PD), a key enzyme in the PPP, resulted in significant enhancement of VSV replication. Restoration of viral replication by NAD⁺ precursors in the presence of 6-AN or suppression of replication by the NAMPT inhibitor FK866 suggested NAD⁺ availability as a critical determinant of VSV replication. Additionally, blockade of glutaminase activity with BPTES reduced viral replication, underscoring the importance of anaplerotic pathways in glioblastoma cells. Collectively, these findings demonstrate that VSV replication is tightly coupled to metabolic programs, particularly those governing energy production and NAD(P)H balance. This work provides a metabolic framework for optimizing oncolytic VSV therapies and suggests that metabolic interventions in cancer treatment may influence oncolytic virus efficacy.