Abstract
ABSTRACTIn response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6Pro. Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6Pro-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway.IMPORTANCE Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6Pro. This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.
Highlights
In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs)
Flag-G3BP2 was efficiently cleaved by EGFP-feline calicivirus (FCV) NS6Pro while EGFP-PV 3CPro expression resulted in only weak cleavage at several distinct sites (Fig. 6B). These results suggest that the impairment of SG assembly during FCV infection may be due to the NS6Pro-mediated cleavage of both GTPase activating SH3 domain binding protein 1 (G3BP1) and G3BP2
Translational arrest is problematic for all viruses as they depend on the host cell protein synthesis machinery for the production of viral proteins
Summary
In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs) This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In order to promote cell survival and limit the use of energy and nutrients, the stressed host cell induces a global reduction in host protein synthesis [1] This translational arrest can be triggered by the phosphorylation of the eukaryotic initiation factor 2␣ (eIF2␣) subunit, which prevents the recycling of the ternary complex tRNAiMet-GTP-eIF2 and the delivery of the initiator tRNA to the ribosome, thereby stalling the initiation of translation [1,2,3,4]. Given that some viruses trigger the formation of SGs and translational shutoff through the phosphorylation of eIF2␣ [14, 15] and that RIG-I-like receptors and PKR have been shown to localize to SGs [16,17,18], it has been proposed that SGs could exert specific antiviral effects [19]
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