mTORC1 activity is dispensable for synthesis of KSHV lytic proteins

Herpesvirus genomes are decoded by host RNA polymerase enzymes, generating mRNAs that are post-transcriptionally modified and exported to the cytoplasm through the combined work of host and viral factors. These viral mRNAs bear 5′-m7G caps and poly(A) tails that should permit assembly of canonical eIF4F cap-binding complexes to initiate protein synthesis. However, the precise mechanisms of translation initiation remain to be investigated for most herpesviruses. eIF4F assembly requires mTORC1-dependent phosphorylation of 4E-BP1, which releases eIF4E from repressive protein complexes. Here, we report that mTORC1 is active and eIF4F is readily detectable throughout the Kaposi’s sarcoma-associated herpesvirus (KSHV) replication cycle. Pharmacologic inhibition of mTORC1 activity caused eIF4F disassembly in KSHV-infected cells, indicating that the mTORC1/4E-BP1/eIF4F signalling axis was intact during virus replication. mTORC1 activity contributed to global protein synthesis in infected cells and was required for lytic gene expression immediately upon reactivation from latency. However, once early gene expression had begun, mTORC1 activity was largely dispensable for viral genome replication, late gene expression, and release of infectious viral progeny. Furthermore, polysome fractionation and RNA-sequencing analysis demonstrated that the translational efficiency of viral mRNA was unaltered by changes in the abundance of eIF4F. Accumulating evidence suggests that herpesvirus mRNA translation can be initiated in an eIF4F-independent manner by an alternative mTORC1/4EBP1-resistant initiation complex, thereby facilitating translation of viral mRNA and the production of infectious progeny. Author summary All viruses require complex host cell machinery to convert viral mRNAs into proteins. Efficient protein synthesis is required to meet the high anabolic demands of a growing cell. A protein complex known as mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis. In nutrient-rich conditions, mTORC1 is active and promotes protein synthesis. In nutrient-poor conditions or under stress, mTORC1 is rapidly shut off, and global protein synthesis is arrested. Remarkably, we discovered that synthesis of Kaposi’s sarcoma-associated herpesvirus (KSHV) proteins is largely resistant to drugs that inhibit mTORC1, including rapamycin and a more recently developed drug known as Torin. These surprising findings suggest that herpesviruses may employ previously unknown mechanisms to ensure efficient synthesis of viral proteins in situations when canonical translation initiation machinery would be expected to succumb to stress.


Introduction
150 with rapamycin at the time of dox-induced lytic reactivation (0 hpi) modestly inhibited viral 151 protein accumulation, whereas Torin potently inhibited accumulation of viral proteins across all 152 temporal classes, including IE (RTA), early (E: ORF45) and late (L: K8.1, ORF65) ( Fig 1A). In 153 keeping with this broad inhibition of viral protein accumulation, Torin treatment also inhibited 154 accumulation of the RTA-dependent RFP reporter (Fig 1D), newly-replicated viral genomes (Fig   155 1B), and late viral mRNAs (Fig 1C) when delivered concurrently with dox. If treatment with 156 Torin or rapamycin was delayed to 24 hpi, the phosphorylation status of S6 and 4E-BP1 was 157 similar to that of the treatment at 0 hpi, suggesting that mTORC1 is similarly required for 158 phosphorylation of these canonical target proteins throughout lytic replication. However, 159 delaying treatment with Torin to 24 hpi allowed viral genomes to replicate, and late mRNAs and 160 viral proteins to accumulate to levels comparable to vehicle-treated controls over the subsequent 161 24-48 h (Fig 1A-D). These findings suggest that even though mTORC1 is active during KSHV 162 lytic replication, it may be dispensable for the synthesis of viral proteins in middle and late 163 stages of the lytic replication cycle. 164 To determine whether mTOR inhibition affects virion production, we used a FACS-based 165 titering assay to detect the release of recombinant virions bearing a GFP gene driven by a 166 constitutive EF-1 promoter. Supernatants from iSLK.219 cultures were used to infect a 167 monolayer of recipient 293A. Quantification of GFP-positive cells in this monolayer by FACS 168 revealed that the first virions are produced by dox-treated iSLK.219 cells as early as 48 hpi and 169 virion production was maximal by 96 hpi (Fig 2A). Cells treated with Torin at 0 produced very 170 few infectious virions, as measured by FACS, but delaying Torin treatment to 12 hpi allowed 171 approximately 50% of maximal virion production compared to vehicle control ( Fig 2B).
172 Delaying Torin treatment to 24, 48, and 72 hpi allowed for a steady increased in the release of 173 infectious virions from these cells, such that treatment with Torin at 72 hpi produced nearly as 174 many virions as vehicle control. We corroborated this finding in TRex-BCBL1-RTA cells [  340 We isolated fractions containing the 40S, 60S, and 80S sub-polysomal peaks, as well as light and 341 heavy polysomes. RNA and associated proteins were precipitated using ethanol and a glycogen 342 co-precipitant. We found that the m 7 GTP cap-binding proteins eIF4E1, eIF4E2, and NCBP80 343 were associated with polysomes in all conditions tested (Fig 7).  1C) and can be initiated for 374 translation (Fig 4, 5B) when Torin is applied during early replication. Furthermore, these late 375 transcripts are efficiently translated despite being first transcribed under conditions where eIF4F 376 is disassembled and unavailable (Fig 6B, 7), and generate sufficient protein to produce infectious 377 virions (Fig 1B, 2). This suggests that while mTORC1 is active during lytic replication, it is 378 dispensable for translation of viral mRNA.

379
During lytic replication we observed a change in global protein synthesis consistent with 380 host-shutoff and the need for viruses to prioritize translation of viral mRNA during infection. We 381 found that mTORC1 inhibition in the low-translation lytic environment had minimal effect on 382 the overall translational output of the cell (Figs. 3, 4A).  (Fig 4)

426
Inhibition of mTORC1 during lytic replication leads to disassembly of eIF4G from the 427 cap-binding eIF4E (Figs. 6, 7). We could observe this disassembly both in a cap-analogue 428 pulldown experiments (Fig 6) and

444
During Torin treatment, both the TOP-containing transcript Rps20 and the non-TOP -445 actin transcript were depleted from polysome fractions that retained viral mRNAs (Fig 5B). In 446 these same fractions eIF4G1 and eIFG3 are also lost, but ORF57 is retained. ORF57 and related