Archaic translation initiation factor eIF5B supports KSHV late lytic replication and viral oncogenesis by mimicking a hypoxic cellular landscape

Kaposis Sarcoma (KS) Herpesvirus (KSHV) is the etiological agent of KS, an AIDS-defining illness. Recent studies have demonstrated that even during normoxic conditions, KSHV facilitates replication by modulating hypoxia-inducible factors (HIFs), creating a hypoxia-like environment that promotes cellular transformation. KSHV lytic viral genes are favored for protein synthesis during infection by upregulating HIF2 and utilizing the hypoxic eIF4E2 translation initiation complex in oxygen-replete conditions. This translation initiation plasticity (TRIP) links viral replication strategies to angiogenic signaling and oncogenicity. However, the molecular basis of this plasticity remains poorly understood. This study reveals that viral inhibition of eukaryotic initiation factor 2 (eIF2) induces the use of another alternative initiation factor, eIF5B, which canonically mediates delivery of initiator methionine-tRNAiMet during hypoxia. We demonstrate through ribosome density fractionation that eIF2 accumulates in translationally inactive monosome fractions while eIF5B redistributes its translation activity toward polysomes during lytic replication. Progressive dependence on eIF5B during KSHV infection was illustrated by impaired late lytic gene expression, diminished virion production, and altered polysome profiles following eIF5B knockdown. Transcriptomic analyses further reveal that the mRNA landscape in KSHV-infected cells depleted of eIF5B mirrors that of uninfected hypoxic cells. Moreover, silencing of eIF5B in a natural infection model reduces both VEGF secretion and anchorage-independent growth--two hallmarks of KSHV viral oncogenicity. These results demonstrate that eIF5B functions as an essential component of alternative translation initiation machinery activated in response to eIF2 inactivation during KSHV lytic replication, emphasizing its key role in viral pathogenesis and its potential as a novel therapeutic target. IMPORTANCEKaposis Sarcoma Herpesvirus (KSHV) is a human cancer virus that causes severe malignancies in immunocompromised individuals, including the blood vessel cancer Kaposis Sarcoma and a highly aggressive body-cavity lymphoma. Although cells mount a response to infection by shutting down global protein synthesis, how viruses like KSHV continue making viral proteins when this cellular machinery is shutdown remains unclear. This study uncovers that KSHV exploits an ancient cellular factor typically used for protein production under low-oxygen conditions. We identify a previously unrecognized role for the translation factor, eIF5B, in supporting KSHV replication and select production of viral proteins during infection. Our work further demonstrates that KSHVs utilization of eIF5B contributes to a hypoxia-like environment during infection, ultimately contributing to cancer-promoting changes within the cell. These findings highlight a new strategy for tumor-virus reprogramming of host cells, opening an avenue for novel therapeutic targets to eliminate virus-associated cancers.