Preliminary Studies On The Molecular Mechanism That FMDV 3C Proteinase Inhibits The Formation Of G3BP1-mediated Stress Granules

Foot-and-mouth disease(FMD) is a highly infectious disease of domestic and wild cloven-hoofed animals including cattle, swine, sheep, and goats. The disease is characterized by debilitating oral and pedal vesicles that result in high morbidity but, except for infection in young animals, FMD generally causes low mortality. FMD has caught devastating economic losses to animal industry of the world.As we all know, foot-and-mouth disease virus(FMDV) can evade host immune response during infection, and 3C proteinase played a role in this process. However, the mechanism is not very clear now.Stress granules(SGs) are reversible dynamic structures that rapidly form when cells encounter environmental stress(including viral infection) that reduces global translation rates. SGs form from concentration of stalled assembled 43 S and 48 S ribosomal preinitiation complexes and serve as temporary repositories for these complexes. Thus, these translation complexes can be rapidly released to resume protein synthesis when stress conditions end. Most viruses including picornavirus have been reported to modulate the SGs formation in a variable way, and the impact of SGs on virus replication can be wide-ranging. In this research, we focus on the ability of SGs formation induced by FMDV infection, and shed light on the molecular mechanism of 3Cpro cleavaging G3BP1. Details are as follows:1. FMDV doesn’t induce the formation of SGs and 3Cpro cleavages G3BP1IBRS-2 cells were challenged with FMDV or treated with Arsenite(ARS), and G3BP1 was used as a biomarker to detect the formation of SGs by indirect immunofluorescence assays(IFA). Through confocal microscopy, we observed that SGs aggregate in ARS-stimulating cells, which was not found in cells infected by FMDV, and that the expression level of G3BP1 significantly reduced which suggested viral proteinase may involve in the process. FMDV Lproand 3Cpro(and its precursor protein 3ABC and 3CD) were transfected into HEK293 T cells with G3BP1, respectively, and the Western Blot assay revealed that 3Cpro, as well as 3ABC and 3CD, could cleavage G3BP1 in a dose-dependent manner.2. The proteolytical activity of FMDV 3Cpro involves in cleavaging G3BP1 and inhibiting SGs formationTo demonstrate our suspection that the proteolytical activity of 3Cpro involves in cleavaging G3BP1 and inhibiting SGs formation, we constructed three site-mutants of 3Cpro(3CH41Y, 3CC163 G and 3CH181Y) which abolish its proteolytical ability. Western Blot results showed that none of the three site-mutants could cleavage G3BP1, and IFA revealed that cells transfected with 3CC163 G can produce nomal SGs as HA-control by treating with ARS. Further more, 3Cpro exhibited a G3BP1-cleavaging propertyin a MG132-independent and Z-VAD-independent manners. Taken together, 3Cpro could cleavages G3BP1 and inhibits SGs formation in which processes the proteolytical activity of 3Cpro involves.3. Identification of cleavaging-site of G3BP1 by FDMV 3CproAccording to the molecular weight of G3BP1 and its cleavaged-product, we constructed several site-mutants of G3BP1. WB assay showed that only G3BP1E284 A could resist cleavaging by 3Cpro. And the two cleavaged-fragments of G3BP1 can not aggregate to form SGs with ARS treatment through IFA. Collectively, this study demonstrates that the 284 Glutamic acid is the cleavaging site of G3BP1 by 3Cpro, and 3Cpro inhibits SGs formation by cleavaging G3BP1.