Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Abstract

Viral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome-associated quality control (RQC) factor ZNF598 during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 (RPS20) and eS10 (RPS10), initiating RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection enhances uS10 ubiquitylation, indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells that either lack ZNF598 or express a ubiquitylation-deficient version of uS10. Using SILAC-based proteomics and concurrent RNA-seq analysis, we determine that translation, but not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

Translational regulation by ribosome-associated quality control in neurodegenerative disease, cancer, and viral infection

“…However, it remains uncertain whether the RQC pathways are deployed the same way in viral infected cells as they are used to handle stalled ribosomes in non-infected cells. Perhaps the best evidence supporting RQC pathway involvement in viral infection came from studies of vaccinia virus protein synthesis during infection, where it was shown that viral infection enhanced ubiquitination of Rps20, an event catalyzed by ZNF598 during RQC of collided ribosomes, suggesting that RQC was activated during vaccinia virus infection ( Sundaramoorthy et al, 2021 ). Supporting a functional role of RQC in vaccinia viral infection, viral replication was impaired in cells deficient in ZNF598 activity or expressing a ubiquitination-deficient version of Rps20.…”

“…Supporting a functional role of RQC in vaccinia viral infection, viral replication was impaired in cells deficient in ZNF598 activity or expressing a ubiquitination-deficient version of Rps20. Moreover, cellular RQC activity was found declined with vaccinia virus infection, suggesting that co-option of ZNF598 by vaccinia virus confers translational reprogramming that is needed for optimal viral replication ( Sundaramoorthy et al, 2021 ). This result is consistent with an earlier study implicating ZNF598 E3 ligase activity and the ubiquitination of Rps20, but not Rps10, in vaccinia virus replication and the synthesis of vaccinia viral proteins whose encoding mRNAs contain unusual 5’ poly(A) leaders ( DiGiuseppe et al, 2018 ).…”

Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome

“…Early studies reported that such leaders have reduced dependence on eIFs or scanning but lack the structural complexity of true IRES elements ( Chandrasekaran et al, 2019 ; Dhungel et al, 2017 ; Dorokhov et al, 2002 ; Mulder et al, 1998 ; Shirokikh and Spirin, 2008 ; Tang and Passmore, 2019 ). Poly(A) leaders are foreign to their mammalian hosts, and their maximal activity requires either poxvirus infection or expression of phosphomimetic S 278 E RACK1 ( Dhungel et al, 2017 ; Dorokhov et al, 2002 ; Jha et al, 2017 ; Mulder et al, 1998 ; Rollins et al, 2019 ; Shirokikh and Spirin, 2008 ; Sundaramoorthy et al, 2021 ; Tang and Passmore, 2019 ). Our findings suggest that S 278 E RACK1 likely primes 40S subunits in a similar way to IRESs to initiate on mRNAs with little to no scanning.…”

Minding the message: tactics controlling RNA decay, modification, and translation in virus-infected cells

“…In response to detecting stall-inducing A-rich sequences on translating mRNA, ribosome-bound ZNF598 E3 ubiquitin ligase triggers RQC and restricts ribosome read-through by ubiquitination of select 40S RPs ( Garzia et al 2017 ; Juszkiewicz and Hegde 2017 ; Sundaramoorthy et al 2017 ). Unexpectedly, ZNF598 E3 ligase and uS10 (RPS20) site-specific ubiquitination were required for poxvirus replication and translation of virus mRNAs, many of which contain an unusual A-rich 5′ UTR ( DiGiuseppe et al 2018 ; Sundaramoorthy et al 2021 ). Stimulation of translation by ZNF598 is in contrast to its role in 80S stalling and RQC, although both might involve ZNF598-sensing poly(A) or A-rich sequences.…”

“…Instead, ZNF598 might potentially impact scanning or possibly be repurposed to stimulate translation by poxviruses ( DiGiuseppe et al 2018 ). Virus infection might also induce ribosome collisions, and ZNF598-mediated RQC may rescue stalled ribosomes and recycle the subunits to stimulate virus mRNA translation ( Sundaramoorthy et al 2021 ). Further studies are needed to elucidate the underlying mechanism.…”