The Antiviral Mechanism Of CCCH-Zinc Finger Antiviral Protein Against Avian Leukosis Virus Subgroup J

Avian leukosis virus subgroup J(ALV-J),a tumorigenic virus,usually causes persistent viremia,chronic inflammation,immunosuppression and diversification of tumor in clinical infected chickens and the main reason is ALV-J severely damages and suppresses the host immune response.The widespread prevalence and intermittent outbreak of ALV-J have caused huge economic losses to the chicken industry in China and even the world.Investigations indicated that the infection of immunosuppressive virus without vaccine is widespread in animals and people.Unfortunately,there is still no effective methods for its prevention and control.Therefore,it is very important to explore and develop effective antiviral methods or drugs for the prevention and treatment of ALV-J and even the immunosuppressive virus similar to ALV-J.The host innate defense factors are the important line for restricting foreign pathogens.CCCH-zinc finger antiviral protein(CCCH-ZAP),a host innate defense factor,significantly exerts antiviral activity.However,there are still great limitations in the study of CCCH-ZAP antiviral activity.First,the antiviral spectrum of CCCH-ZAP is still confined to mammals with the study in vitro.It is still not clear whether CCCH-ZAP can exert antiviral activity for virus replication of other species such as avian leukosis virus especially in vivo with complex environment.Second,the antiviral pathway of CCCH-ZAP is not comprehensive enough and only the mechanism of the interaction between CCCH-ZAP and virus mRNA was revealed.Nevertheless,the relationship between CCCH-ZAP and target virus proteins and whether CCCH-ZAP participates in other important antiviral pathway in vivo remains probed.Based on these research,this study proposed to explore the antiviral activity and mechanism of CCCH-ZAP against ALV-J from the perspective of pathology,molecular biology and immunology,in order to provide the new scientific basis and technical support for disease prevention and control and the research of new antiviral drugs.To probe the antiviral activity of CCCH-ZAP against ALV-J,quantitative real-time PCR(qPCR)and Western blot were performed to detect the expression of CCCH-ZAP in DF-1cells post-infection by ALV-J.The results showed that CCCH-ZAP expression was up-regulated by AlV-J infection.DF-1cells with CCCH-ZAP overexpression or knockdown were infected by ALV-J and the results from qPCR and Western blot indicated that overexpression of CCCH-ZAP significantly inhibited ALV-J replication and knockdown of CCCH-ZAP promoted ALV-J replication.In addition,the results from confocal laser scanning microscopy(CLSM)and co-immunoprecipitation(Co-IP)showed that CCCH-ZAP and SU(envelope protein of ALV-J)were co-localized around nucleus and interacted with each other.This results demonstrated that CCCH-ZAP can be induced by ALV-J infection and exhibit antiviral activity via binding SU.To explore the effect of ALV-J infection on the expression of CCCH-ZAP in vivo,intraperitoneal injection of ALV-J was performed to establish the model of virus infection in vivo.The results detected by qPCR,immunohistochemistry(IHC)and CLSM indicated that the expression of endogenous CCCH-ZAP was significantly increased in spleen and bursa of Fabricius and CCCH-ZAP migrated around the nucleus.These above results showed that CCCH-ZAP monitored the invasion of ALV-J and has organizational tendency of expression induced by virus infection.To further explore the antiviral activity of CCCH-ZAP in vivo,the model of CCCH-ZAP overexpression in vivo was established by transfection with lentiviral vectors.GFP fluorescence signal was obtained and qPCR was performed to monitor the expression level of CCCH-ZAP in vivo.The results showed that CCCH-ZAP overexpression was focused on spleen and bursa of Fabricius which was consistent with CCCH-ZAP expression level in tissues after ALV-J infection,and GFP fluorescence signal was weak in other tissues,indicating that CCCH-ZAP overexpression tended to occur in immune organs or immune cells.The results from qPCR and Western blot suggested that the overexpression of CCCH-ZAP remarkably suppressed ALV-J replication in vivo.Histopathology showed that the tissue damage such as liver caused by ALV-J infection was observably reduced by CCCH-ZAP overexpression.To probe the distribution of CCCH-ZAP overexpression in immune organs,fluorescence microscope was used and the results showed that CCCH-ZAP mainly distributed in red pulp or marginal zone of spleen and cortex region of the lymphoid nodule of bursa of Fabricius.The observation of hemogram showed that CCCH-ZAP mainly distributed in high nucleoplasm ratio cells and the proportion of GFP positive cells was significantly increased after ALV-J infection.The results from flow cytometry(FCM)showed that GFP positive cell belongs to T lymphocyte and CCCH-ZAP overexpression significantly up-regulated the number of T cells but not B cells.In addition,the proliferation of GFP~+CD3~+cells were induced by ALV-J infection with CCCH-ZAP overexpression condition.To clarify the effect of CCCH-ZAP overexpression on T cell differentiation,the detection of CCK-8 was performed on T lymphocyte line(MSB-1)after ALV-J infection under CCCH-ZAP overexpression condition.The results indicated that CCCH-ZAP overexpression recovered the proliferation of T cell with ALV-J infection.ELISA detection showed that CCCH-ZAP overexpression up-regulated the secretion of IL-2,IL-4 and IL-21,but down-regulated IL-10secretion and had no significantly effect on IFN-?secretion.Furthermore,anti-ALV-J antibodies were up-regulated by CCCH-ZAP overexpression.To further confirm the effect of CCCH-ZAP on T cell activation,a series of biological techniques such as FCM,qPCR and so on were performed and the results showed that CCCH-ZAP overexpression up-regulated the mRNA level of IL-2 and promoted the proportion of IL-2 positive cell.CCCH-ZAP overexpression removed the inhibition of IL-2 secretion by ALV-J.Western blot detection suggested that CCCH-ZAP overexpression facilitated the nuclear translocation of nuclear factor of activated T cells(NFAT),indicating that CCCH-ZAP overexpression activated T cell and indirectly promoted antiviral antibodies by activating NFAT.Taken together,CCCH-ZAP inhibited ALV-J replication by activating T cells and indirectly promoted the secretion of antiviral antibodies independent of IFN-?.To explore the molecular mechanism of CCCH-ZAP removed the inhibition of T cell activation by ALV-J,this study detected the effect of ALV-J infection on the immune response and activation of T cells in the absence of CCCH-ZAP and the results showed that knockdown of CCCH-ZAP promoted the virus titer and replication of ALV-J in T cells.Furthermore,the susceptibility of T cell to ConA was reduced in the absence of CCCH-ZAP.Western blot showed that knockdown of CCCH-ZAP promoted the phosphorylation of NFAT.These above results suggested that knockdown of CCCH-ZAP significantly reduced the antiviral immune response of T cells and CCCH-ZAP is crucial for NFAT activation.To explore the molecular mechanism of CCCH-ZAP activates T cell with ALV-J infection,proteomics analysis was performed to find the differential proteins influenced by CCCH-ZAP overexpression.Norbin-like-protein(NLP)and protein kinase C?(PKC-?)were selected.To confirm the activity of NLP and PKC-?involved in T cell activation,this study first overexpressed NLP and CCCH-ZAP.Western blot showed that NLP interacted with SU and this interaction was reduced by CCCH-ZAP overexpression.The detection of phosphatase activity indicated that CCCH-ZAP overexpression removed the inhibition of NLP phosphatase activity by ALV-J.Taken together,CCCH-ZAP extricated NLP via competitively binding with SU.To confirm the relationship between NLP and PKC-?,the total protein and phosphorylation of PKC-?were detected under NLP overexpression.The results indicated that NLP overexpression significantly reduced the total protein level and phosphorylation of PKC-?and promoted the translocation from membrane to cytoplasm of it.Furthermore,knockdown of NLP removed the down-regulation of the total protein level and phosphorylation of PKC-?by CCCH-ZAP overexpression,indicating that NLP is essential for ZAP regulating PKC-?.Subsequently,to confirm the relationship between PKC-?and NFAT,Western blot was performed in MSB-1 cells with knockdown of PKC-?and the results showed that knockdown of PKC-?reduced the phosphorylation of NFAT and promoted the nuclear translocation of it.Moreover,PKC-?overexpression have no significantly effect on NFAT phosphorylaton under rottlerin(inhibitor of PKC-?phosphorylation)stimulation,indicating that reduction of PKC-?phosphorylation was necessary for reduction of NFAT phosphorylation and nuclear traslocation which was essential for T cell activation.In conclusion,this study identified the antiviral activity that CCCH-ZAP not only can exerts its antiviral activity by binding viral protein,but also selectively be expressed in T cells to mediate the host antiviral immune response after virus induction.More importantly,CCCH-ZAP extricates NLP phosphatase activity via competitively binding with SU,thus relieving the immunosuppresson caused by ALV-J and recovering the immune response of T cell against foreign pathogens.The discovery of the diversified antiviral pathways and mechanisms of CCCH-ZAP provide a novel idea for the study of host innate defenses factors and provide a novel theoretical basis for the prevention and control of immunosuppressive virus.