Ribosome quality control activity potentiates vaccinia virus protein synthesis during infection

Abstract

Ribosomes are highly abundant cellular machines that perform the essential task of translating the genetic code into proteins. Cellular translation activity is finely tuned and proteostasis insults, such as those incurred upon viral infection, activate stress signaling pathways that result in translation reprogramming. Viral infection selectively shuts down host mRNA while redistributing ribosomes for selective translation of viral mRNAs. The intricacies of this selective ribosome shuffle from host to viral mRNAs are poorly understood. Here, we uncover a role for the ribosome associated quality control (RQC) factor ZNF598, a sensor for collided ribosomes, as a critical factor for vaccinia virus mRNA translation. Collided ribosomes are sensed by ZNF598, which ubiquitylates 40S subunit proteins uS10 and eS10 and thereby initiates RQC-dependent nascent chain degradation and ribosome recycling. We show that vaccinia infection in human cells 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 which either lack ZNF598 or contain a ubiquitylation deficient version of uS10. Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that host translation of vaccinia virus mRNAs is compromised in cells that lack RQC activity as compared to control cells whereas there was little evidence of differences in host or viral transcription. Additionally, vaccinia virus infection resulted in a loss of cellular RQC activity, suggesting that ribosomes engaged in viral protein production recruit ZNF598 away from its function in host translation. Thus, co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.

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

“…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.…”

The Respiratory Syncytial Virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly

“…Nevertheless, these functions do not directly correlate to proteomic data obtained, nor were we able to identify the granular structures to which M2-2 is recruited. Because many of the proteins identified by co-IP were mainly associated with translation elongation, we chose to investigate potential paths connecting it with viral infection, raising a possible association of M2-2 with ribosome quality control (RQC), among other mechanisms involved with this step of translation [41, 42, 43]. M2-2 potential partners encompass, beyond ribosomal proteins, numerous chaperones, including VCP, recently described as necessary for proper RSV infection [44].…”

“…On the other hand, M2-2 interaction with DRiPs, as well as its association with the proteasome machinery, implies it could be involved with translation elongation, directing prematurely terminated peptides for degradation (Fig 8b). Such events occur during ribosome quality control activation, where stalled ribosomes are ubiquitinated and dismounted in response to errors in translation or peptides not reaching proper folding, a path utilized by some viruses to favor translation of its own transcripts [41, 42, 43]. Moreover, RQC was also described as an eiF2α antagonist [55], and the premature termination of elongation could contribute for the observed inhibition results obtained in both SUnSET and reporter luciferase assays.…”

The 2’,3’ cyclic phosphatase Angel1 facilitates mRNA degradation during human ribosome-associated quality control

“…While our reporter assays show that Angel1 is rate-limiting for decay of engineered human NSD substrates (Figure 5), our eCLIP experiments suggest that Angel1 associates broadly with protein coding regions of mRNAs (Figure 2). Indeed, depletion of RQC factors such as ZNF598 have shown broad, low-level, effects on the transcriptome (Tuck et al 2020; Kalisiak et al 2017; Weber et al 2020; Sundaramoorthy et al 2021). Identification of endogenous substrates of RQC has been generally unsuccessful with only a few potential examples, including the ER stress-induced XBP1, which in human cells is upregulated at the protein level upon depletion of ZNF598 (Han et al 2020).…”