KSHV lytic mRNA is efficiently translated in the absence of eIF4F

Abstract

Herpesvirus genomes are decoded by host RNA polymerase II, generating messenger ribonucleic acids (mRNAs) that are post-transcriptionally modified and exported to the cytoplasm. These viral mRNAs have 5'-m7GTP caps and poly(A) tails that should permit assembly of canonical eIF4F cap-binding complexes to initiate protein synthesis. However, we have shown that chemical disruption of eIF4F does not impede KSHV lytic replication, suggesting that alternative translation initiation mechanisms support viral protein synthesis. Here, using polysome profiling analysis, we confirmed that eIF4F disassembly did not affect the efficient translation of viral mRNAs during lytic replication, whereas a large fraction of host mRNAs remained eIF4F-dependent. Lytic replication altered multiple host translation initiation factors (TIFs), causing caspase-dependent cleavage of eIF2alpha; and eIF4G1 and decreasing levels of eIF4G2 and eIF4G3. Non-eIF4F TIFs NCBP1, eIF4E2 and eIF4G2 associated with actively translating messenger ribonucleoprotein (mRNP) complexes during KSHV lytic replication, but their depletion by RNA silencing did not affect virion production, suggesting that the virus does not exclusively rely on one of these alternative TIFs for efficient viral protein synthesis. METTL3, an N6-methyladenosine (m6A) methyltransferase that modifies mRNAs and influences translational efficiency, was dispensable for early viral gene expression and genome replication but required for late gene expression and virion production. METTL3 was also subject to caspase-dependent degradation during lytic replication, suggesting that its positive effect on KSHV late gene expression may be indirect. Taken together, our findings reveal extensive remodelling of TIFs during lytic replication, which may help sustain efficient viral protein synthesis in the context of host shutoff.