The Mechanism Of GLTSCR2 Negatively Regulates Type?Interferon And Antiviral Response

Glioma tumour-suppressor candidate region gene 2(,GLTSCR2)is located on chromosome 19,which is encoded the cytosolic protein of 60 kDa.GLTSCR2 was shown to directly interact with viral proteins,such as ICP22 and ICPO of HSV-1 and KS-Bcl-2 of KSHV in infected cells.However,it was not clear whether GLTSCR2 might be involved in viral replication.In this work,we pursued the role of GLTSCR2 in viral replication.Translocation from nucleus to cytoplasm enabled GLTSCR2 to attenuate the ability of RIG-I to induce IFN-P in cells responding to viral infection.To investigate whether GLTSCR2 might play any role in viral replication,we used small interfering RNA(siRNA)to knock down GLTSCR2 in cells,followed by infection with Rhabdoviridae vesicular stomatitis virus(VSV),Paramyxoviridae Newcastle disease virus(NDV),and Coronaviridae infectious bronchitis virus(IBV).The results indicating an important role of GLTSCR2 for production of various viruses.Further,amino acids at positions 330-432 of the GLTSCR2 carboxyl terminal,especially the amino acid at the nucleus of the nucleus,were essential for the replication of the virus by truncation,the mutant test.In the case of viral infection or poly(I:C)stimulation,the localization of GLTSCR2 cells is altered:from the nucleus to the cytoplasm,whereas the GLTSCR2 lacking nucleus sites can not migrate out of the nucleus,whether the virus is infected or not.In order to explore the mechanism of GLTSCR2 in promoting viral replication,we can demonstrate that GLTSCR2 significantly inhibits the activation of IFN-? and NF-?B by a dual-luciferase assay.Further immunoprecipitation and luciferase assay results indicate that GLTSCR2,Terminal GLTSCR2 targets RIG-I,rather than MAVS,TBK-1,negatively regulates the activation of IFN-?,whereas GLTSCR2 lacking nucleus sites does not significantly promote IFN-? activation.To further reveal the molecular mechanism of GLTSCR2 negatively regulating IFN-?,the GLTSCR2 effects on RIG-I phosphorylation or ubiquitination mutants assay have shown that GLTSCR2 is not regulated the phosphorylated mutant of RIG-I.To further study whether GLTSCR2 modulated ubiquitination of RIG-I,HEK-293T cells were co-transfected with plasmids to express Flag-tagged RIG-I-N and GFP-tagged GLTSCR2,along with HA-tagged ubiquitin,K63-linked ubiquitin,or K48-linked ubiquitin.We detected that ectopic expression of GLTSCR2 was able to induce de-conjugation of K63-linked,but not K48-linked polyubiquitin chains from the constitutively-active variant RIG-I-N.Knockdown of endogenous GLTSCR2 resulted in increased K63-linked ubiquitination of RIG-I-N.These results taken together indicated that GLTSCR2 was able to downregulate RIG-I,via K63-linked ubiquitination.We ectopically co-expressed Flag-tagged GLTSCR2 and Myc-tagged TRIM25,USP21,or USP3 in cells with and without being infected with VSV to detect if they were in the immune complexed with GLTSCR2.It was showed no evidence of the interaction regardless of viral infection.To further address whether TRIM25 or USP21 were responsible for GLTSCR2 regulation of K63-linked ubiquitination of RIG-I,we co-expressed Flag-tagged RIG-I-N and HA-tagged K63 ubiquitin in the presence or absence of TRIM25 or USP21 in HEK-293T or GLTSCR2 knockdown cells.No evidence was presented to support TRIM25 or USP21 mediated GLTSCR2 removal of K63-linked polyubiquitin chains from RIG-I.Finally,the recombinant plasmid G4-TAT,which mimics GLTSCR2 protein,could penetrate the cell membrane and inhibit the type I interferon signaling pathway,promotes efficient proliferation of viruses.In summary,we presented evidence that viral infection induced translocation of GLTSCR2 from nucleus to cytoplasm,and cytoplasmic translocation enabled GLTSCR2 to effectively attenuate IFN-?and support viral replication.Our studies revealed a novel role the nucleolar protein GLTSCR2 played in attenuation of IFN-?,via cytoplasmic translocation to induce the ability of USP15 to deactivate RIG-I.Besides,carboxy terminal of GLTSCR2 can also promote the replication of virus by prokaryotic expression.