The Effect And Mechanism Of Guanolylate-binding Protein 1(GBP1) Inhibiting Hantaan Virus Infection

Hantaan virus(HTNV),belonging to the Buniavirus family,orthohantavirus genus,is an enveloped negative-stranded RNA virus.The HTNV genome consists of three gene segments: L,M,and S,with L encoding RNA-dependent RNA polymerase(Rd Rp),M encoding glycoprotein(GP),and S encoding nucleocapsid protein(NP).HTNV infection in humans can cause Hemorrhagic Fever with Renal Syndrome(HFRS),a severe disease characterized by acute onset and high mortality rates,posing a serious threat to human health with no current effective treatment methods.Innate immunity is the first line of defense that the host employs to resist viral invasion.Virus infection triggers the host to produce interferon(IFN),which binds to cell surface receptors,inducing the expression of interferon-stimulated genes(ISGs),ultimately exerting direct antiviral and immune-regulatory functions.Guanylate-binding protein 1(GBP1)is an ISG that can defend against various pathogen infections.However,the function of GBP1 in HTNV infection is not entirely clear.In this study,we investigated the role and specific molecular mechanism of GBP1 in inhibiting HTNV infection,as follows:The first part of this study revealed the inhibitory effect of GBP1 on HTNV infection.The results showed that HTNV infection in A549 cells activated the IFN signaling pathway and induced the expression of GBP1.Knocking down GBP1 promoted the expression of HTNV S gene m RNA and HTNV NP,resulting in an increase in the titer of HTNV in cell supernatant.Conversely,overexpression of GBP1 in A549 cells inhibited the expression of HTNV S gene m RNA and HTNV NP,leading to a decrease in the titer of HTNV in cell supernatant.These results demonstrated that GBP1 could inhibit HTNV infection.The second part of our study revealed the specific molecular mechanism by which GBP1 inhibits HTNV infection.We investigated whether GBP1 exerts its antiviral effect by regulating the IFN signaling pathway.Luciferase reporter assay results showed that GBP1 did not affect the IFN signaling pathway.Additionally,we found that GBP1 influenced the HTNV life cycle.RT-q PCR results showed that overexpression of GBP1 in A549 cells reduced the levels of HTNV S gene v RNA and c RNA,indicating that GBP1 might act during the early stages of HTNV replication.Furthermore,we investigated the effect of GBP1 on HTNV attachment and entry.RT-q PCR and luciferase reporter assay results showed that GBP1 could inhibit HTNV entry into cells.FACS experiment results showed that GBP1 inhibited clathrin-mediated endocytosis,and immunoprecipitation assay results indicated that GBP1 could interact with β-actin(ACTB).Immunofluorescence assay results showed that overexpression of GBP1 hindered the co-localization of dynamin-2(DNM2)and clathrin light chain A(CLTA).These results suggest that during HTNV infection,GBP1 inhibits clathrin-mediated endocytosis and HTNV entry into cells by hindering the recruitment of DNM2 by ACTB,ultimately reducing the interaction between DNM2 and CLTA.We also investigated the key amino acid residues involved in GBP1’s antiviral function.We constructed three mutant forms of GBP1,namely GBP1-R240 A,GBP1-K51 A,and GBP1-ΔCAAX,and investigated their interactions with ACTB.Immunoprecipitation assay results showed that GBP1 interacted with ACTB.The R240 A mutation weakened the interaction between GBP1 and ACTB,but ΔCaa X and K51 A mutations did not affect the interaction.We then investigated the antiviral effect of GBP1 mutants on HTNV.Western-blot results showed that overexpression of GBP1-K51 A and GBP1-ΔCAAX significantly reduced the expression of HTNV NP,whereas overexpression of GBP1-R240 A did not affect the expression of HTNV NP.FFA experiment results showed that overexpression of GBP1,GBP1-K51 A,and GBP1-ΔCAAX significantly reduced the titer of HTNV in cell supernatant,while overexpression of GBP1-R240 A did not have any effect on the titer of HTNV in cell supernatant.We also investigated the effect of GBP1 mutants on HTNV entry using a luciferase reporter assay,which showed that overexpression of GBP1-K51 A inhibited HTNV entry into cells,while overexpression of GBP1-R240 A had no effect.These results suggest that arginine at position 240(R240)is crucial for the antiviral function of GBP1 in inhibiting HTNV infection and entry.In summary,GBP1 can inhibit HTNV infection by reducing the interaction between DNM2 and CLTA and inhibiting clathrin-mediated endocytosis,thus preventing HTNV entry into cells.Our study expands upon the antiviral role of GBP1 and provides a theoretical basis for developing antiviral drugs against HTNV based on ISGs.