| Cellular protein NOP53,also known as PICT-1(protein interacting with carboxyl terminus 1),is encoded by glioma tumor suppressor candidate region gene 2(GLTSCR2).Expression of NOP53 is down-regulated or eliminated in various tumors.Localization of NOP53 is mediated by multiple unique nucleolar localization sequences.Nucleolar NOP53 can translocate to nucleoplasm and stabilize p53 in response to the ribosomal stress.NOP53 interacts with various cellular proteins or viral proteins.Our previous study showed that NOP53 blocks type I IFN induction and deactivates retinoic acid-inducible gene RIG I(not TBK1)by negatively regulating it via K63-linked ubiquitination.Research purposes and significance:This study starts with the expression and localization of cellular protein NOP53,investigates the mechanism of viral hijacking NOP53 to facilitate its own replication,provides important scientific data revealing the mechanism of virus-cell interaction,and new thoughts on designing of broad-spectrum antiviral agents.Methods:(1)simplex herpes virus](HSV-1)was used as a model virus to study the effect of overexpression or gene silencing of NOP53 on viral replication.The effects of overexpression or knockdown of NOP53 on virus replication were investigated by RT-PCR,Western blotting,and plaque assays;(2)confocal microscopy and nucleo-cytoplasmic separation were used to’ studythe cellular localization of NOP53 during HSV infection;(3)puromycin labelling,Western blotting,and Co-immunoprecipitation were used to study the interaction of NOP53 with HSV-1 γ34.5 and the mechanism of NOP53 promoting HSV-1 replication;(4)the NOP53-derived protein N3-T,which contains human immunodeficiency virus(HIV)-derived cell-penetrating Tat peptide at the C-terminal region of N3(residues 330-432),was constructed and expressed.The effect of recombinant protein N3-T on type I interferon antiviral response and on multiple virus replication were detected by RT-PCR,Western blotting,immunofluorescence,and virus titer assay;(5)in vivo experiments were carried out to investigate the function of short hairpin RNA(shRNA)targeting NOP53.Results:(1)ectopic expression of NOP53 or its truncation N4 significantly increases the virus titer of HSV-1 wild-type strain F(HSV-I/F),and more subtly promotes viral replication of γ34.5 deletion mutant virus HSV-1/△γ34.5.Conversely,NOP53 knockdown inhibits viral replication of HSV-1/F and HSV 1/△γ34.5;(2)NOP53 is migrated from nuclei in HSV-1/F infected cells,while redistributed incompletely after infection by either HSV-1/△γ34.5 or ICP4 deletion mutant virus HSV-1/d120(replication inadequate).Blocking cytoplasmic translocation of NOP53,by deletion of its nuclear export sequence(NES),abrogated its ability to support viral replication.Ectopic expression of y34.5,consequently,induces cytoplasmic translocation of NOP53 in response to HSV-1/△γ34.5 infection;(3)increase of NOP53,in two forms of transient transfection and in vitro expression,attenuates the phosphorylation level of eIF2a in HSV-1/F infected cells,but fails to affect eIF2a phosphorylation induced by HSV-1/△γ34.5 infection.Knockdown of NOP53,which impairs the specific interaction between γ34.5 and protein phosphatase PP1α,disrupts the ability of γ34.5 to maintain HSV-1 virulence;(4)the recombinant N3-T protein formed trimers,attenuated expression of IFN-p and IFN-stimulated genes,as well as decreased the phosphorylation level of interferon regulatory factor 3(IRF3).Furthermore,N3-T promotes efficient replication of enveloped and non-enveloped DNA and RNA viruses belonging to 5 families;(5)NOP53 knockdown significantly reduces tissue damage and decreases viral yield in livers of HSV-1 infected mice.Conclusion:(1)various viruses induce cytoplasmic translocation of nucleolar protein NOP53;HSV-1 y34.5 induces NOP53 redistribution,cytoplasmic NOP53 facilitates y34.5 recruitment of PP1αto dephosphorylate eIF2a,for optimal viral replication;(2)NOP53-derived protein N3-T penetrates into cell,suppresses type I IFN antiviral responses and increases replication of DNA and RNA viruses from distinct genera;(3)NOP53 knockdown significantly reduces tissue damage and decreases viral yield in livers of HSV-1 infected mice.These findings expand the understanding of the underlying mechanism by which viruses utilize nucleolar protein NOP53 for optimal viral replication.Understanding this mechanism not only provides important information on the process of viral replication,but also will be useful for developing potential target inhibitors against infection. |
