eIF3d drives cholesterol biosynthesis required for KSHV lytic replication

Herpesvirus genomes are decoded by host RNA polymerase II, generating viral messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5{square}-m7GTP caps and poly(A) tails that enable assembly of host translation initiation factors (TIFs) required for viral protein synthesis. Here we show that maximal production of viral proteins during lytic replication requires both the canonical host TIF eukaryotic initiation factor 4F (eIF4F) and the alternative host TIF eIF3d. Despite this, production of infectious virions is largely dependent on eIF3d, with eIF4F playing a minor role. Investigating the effects of eIF3d silencing on the host proteome revealed that it is required for accumulation of proteins involved in cholesterol metabolism, as well as the accumulation of intracellular cholesterol during KSHV lytic replication. In KSHV infected and uninfected cells alike, eIF3d supports efficient translation of cholesterol biosynthesis enzymes, including squalene epoxidase (SQLE), whose function is required to support productive lytic replication during cholesterol scarcity. These findings position eIF3d as a critical TIF required to support translation of essential host mRNAs required for KSHV replication. AUTHOR SUMMARYAnimal cells tightly regulate uptake, synthesis, storage, and export of cholesterol to ensure optimal membrane fluidity and permeability. Many enveloped viruses require cholesterol to support membrane fusion events during cell entry and cell surface budding events during assembly and egress. Herpesviruses like Kaposis sarcoma-associated virus (KSHV) require cholesterol to support entry, but its role in post-entry events, including budding and fusion at internal membranes, remains incompletely understood. Here, through our studies of KSHV protein synthesis, we uncovered a new mechanism of cellular control of cholesterol metabolism involving the host translation initiation factor (TIF) eIF3d. We demonstrated that eIF3d was required to support efficient translation of host mRNAs encoding key proteins required for cholesterol biosynthesis and uptake. Using a lytic reactivation model that bypasses the entry step, we observed that productive KSHV lytic replication is accompanied by eIF3d-dependent increases in intracellular cholesterol. Preventing cholesterol synthesis and uptake, or silencing eIF3d, strongly inhibited production of infectious virions. These studies shed new light on the importance of intracellular cholesterol to support KSHV replication, and provide motivation for future studies of how cholesterol influences discrete post-entry steps of KSHV infection.