The Role Of Interferon-stimulated Genes IFI16 And PFKFB3 In Regulating Host Antiviral Immunity

Viral infection threats human health and lives.Viruses invade and stimulate host antiviral immunity.The recognition and activated immune response are two key steps for host cells to resist virus infection.One side,y-interferon-inducible protein,IFI16 is a unique DNA sensor which recognizes the viral DNA located in nucleus and cytoplasm,and then participates in the antiviral immune response and inflammatory response.There are four isoforms of IFI16,which are IFI16-iso1,2,3,4.Among them,the structure and function of IFI16-iso2 are more clearly studied.IFI16-iso2 is mainly located in nucleus and recognizes viral DNA replicated in the nucleus,for example HSV-1.Meanwhile,IFI16-iso2 also recognizes cytoplasmic viruses,for example VACV,after being acetylated and transported to cytoplasm.Studies have shown that the pyrin domain of IFI16-iso2 N-terminal mainly mediates intermolecular interactions and co-assembly of IFI16 oligomers,and the HIN200 domains at the Cterminal directly bind viral DNA.Our study found that the length of amino acid encoded by IFI 16-iso 1 is exactly the same as that of IFI16-iso2,but the NLS sequence is missing in the N-terminal region and replaced by an extra sequence between the two HIN200 domain.However,the recognition of viral DNA by IFI16-iso 1 and its role in antiviral immunity have not yet been reported.One the other side,the antiviral immune response is triggered in an IFN-I dependent or independent manner after recognition.Previous studies have found that the activation of the innate immune system requires the consumption of a large number of macromolecules and metabolic intermediates.At the same time,cellular metabolism is involved in the proliferation,differentiation and activation of immune cells,such as the metabolism of glucose and serine,and the synthesis of cholesterol and tryptophan.In addition,it has been reported that both virus-infected cells and activated immune cells are prone to glycolysis metabolism for energy supplying.Therefore,targeting of glycolysis will be an effective treatment against virus infection.On the one hand,we applied new designed viral DNA mimics and virus with different subcellular location to analyze the role of IFI16-isol in antiviral immune response and its functional difference with IFI16-iso2.First,the location of exogenous IFI16-iso1 and IFI16-iso2 was analyzed by immunofluorescence.Next,we modified commercial viral DNA mimics which can only enter the cytoplasm by conventional transfection technology.On this basis,the differences between IFI 16iso 1 and IFI16-iso2 in recognition and antiviral immunity were analyzed by using modified virus DNA mimics and DNA viruses with different sub-location.In addition,RT-qPCR and Western blot were used to analyze the differences of IFI16-iso1 and IFI16-iso2 on DNA viruses-induced immune response.Finally,TCID50 was used to evaluate the difference in their ability to resist virus replicated in cytoplasmic and nuclear.On the other hand,we analyzed the effect of cellular glycolysis on host antiviral immunity in vitro and in vivo.First,differentially expressed genes in PBMC of ZIKAor influenza-infected patients were analyzed by transcriptomic sequencing data.And it was found that PFKFB3,a key rate-limiting enzyme of glycolysis,was significantly upregulated.Subsequently,the PFKFB3 expression and its upstream signal molecules were detected by RT-qPCR,Western blot and Luciferase reporting system,revealing that PFKFB3 play a role in the antiviral immune response as an ISG.Next,the antiviral effect of PFKFB3 in vitro was analyzed by transiently overexpressing or stable overexpressing cell lines.Meanwhile,myeloid cell-specific Pfkfb3-/-mice constructed by Cre-Loxp system and the PFKFB3 inhibitor 3PO were applied to analyze the antiviral effect of PFKFB3 in vivo.RT-qPCR and TCID50 were used to measure the viral content in organs,serum and bronchoalveolar lavage fluid of mice;the tissue damage and inflammatory infiltration after virus infection were evaluated by HE staining;finally,the survival state was monitored to evaluated the antiviral effect of PFKFB3 in vivo.Additionally,supernatants of PFKFB3 and its mutant plasmids overexpressed in cells(conditional medium)were collected,and their functions in viral infection were analyzed by Flow cytometry,RT-qPCR and TCID50.The content of amino acids in PFKFB3 conditioned medium was analyzed by mass spectrometry.On this basis,the expression of enzymes related to amino acid metabolism,such as ARG1 and NOS2 were detected by RT-qPCR and Western blot.Finally,the corresponding truncated plasmids were constructed,and their interactions were analyzed by immunoprecipitation,immunofluorescence and PLA.Proteins with Flag or GST tagged were purified from eukaryotic or prokaryotic expression system,and their activation in cell-free system were measured by Western blot to explore the mechanism of PFKFB3 in antiviral immunity.In this study,we firstly found that IFI 16-iso1 has the same number of amino acid as IFI16-iso2 but lacks NLS,and IFI16-iso1 is mainly located in the cytoplasm.Then,we developed new nuclear viral DNA mimics that can be recognized by nucleus DNA receptors,like IFI16-iso2 and hnRNPA2B1,and effectively induced the expression of IFN-β and ISGs.On this basis,we found that the different sub-location of IFI16-iso1 and IFI16-iso2 led to differences in viral DNA recognition and antiviral immune response.IFI16-iso1 preferred to co-locate with cytoplasmic replicating DNA viruses and cytoplasmic viral DNA mimics,such as VACV and HSV60mer,while IFI16-iso2 preferred to co-locate with DNA in the nucleus,such as HSV-1,HSV60-DNLS.Comparing with IFI16-iso2,IFI16-iso1 significantly induced the expression of IFN-βand ISGs after recognition of cytoplasmic DNA.In addition,IFI16-iso2 can also effectively resist against VACV invasion due to its nuclear-cytoplasmic shuttling.However,IFI16-iso2 mediated IFN-I-dependent antiviral immunity was significantly stronger than IFI16-iso1 after HSV60-DNLS or HSV-1 stimulation.At last,we also found that PFKFB3 may be involved in the host antiviral immune response as an ISG.Functional experiments showed that overexpression of PFKFB3 could significantly inhibit the virus replication in vitro.In vivo experiments showed that specific deletion of Pfkfb3 in myeloid cells or drug inhibition of PFKFB3 enzyme activity promoted virus replication in viscera,blood and alveolar lavage fluid,aggravated multiple tissue damage,and ultimately led to a significant decrease in surviva1.Additionally,we found that conditioned medium of PFKFB3 also exhibited significant antiviral activity in an IFN-I independent pathway.The results of mass spectrometry showed that arginine was significantly reduced in the PFKFB3 conditioned medium.However,the content of arginine was significantly increased in alveolar lavage fluid or BMDM of Pfkfb3 knockout.The mechanism analysis showed that the kinase domain of PFKFB3 can directly bind and activate STAT1 and STAT6,thus upregulating the expression of ARG1 and NOS2,and ultimately consuming arginine to exert antiviral effect.In general,we developed a novel nuclear DNA receptor agonist in this study;we found that IFI16-iso1 play roles in viral DNA recognition and antiviral immune response.Combining with the viral DNA mimics and corresponding DNA virus,we demonstrated the difference of IFI16-iso1 and IFI16-iso2 in host antiviral immunity.Finally,our results confirmed that both PFKFB3 gene and its conditional medium have significant antiviral activities,and the PFKFB3 kinase domain directly interacts and activates STAT1 and STAT6 to promote arginine consumption,and ultimately resist viral infection.