Hepatitis B Virus Evades Immune Recognition Via RNA Adenosine Deaminase ADAR1-mediated Viral RNA Editing In Hepatocytes

Hepatitis B virus（HBV）infection is the most important risk factor of hepatocellular carcinoma（HCC）.Hence,HBV control is critical for prevention of HCC.Despite the success of HBV vaccination and available treatments such as interferon（IFN）and nucleotide（NUC）,the chronically infected population remains high at 257 million worldwide with an annual death toll from HBV at 800,000.Pattern recognition receptors（PRRs）play a crucial role in the initiation of innate immune response by detecting potential harmful pathogens.HBV is a hepatotropic,partially double-stranded DNA virus with a unique life cycle producing both viral DNA and RNA intermediates,which should be detected by PRRs to induce IFN responses.However,HBV has long been considered as a "stealth" virus since little or no IFN production is detected in most HBV-infected patients with high quantities of viral particles and antigens compared with HCV and SeV.Although hepatocytes lack a functional DNA-sensing pathway,there are abundant RNA sensors in hepatocytes.Treatment with poly（I:C）or infection of HCV results in robust expression of IFNs in hepatocytes,suggesting that PRRs could recognize exogenous RNAs.To date,the mechanisms of innate immune recognition and evasion of HBV are still poorly understood.RNA modification such as N6-methyladenosine（m6A）,m5C methylation,pseudouridine（Ψ）and RNA editing,are a common mechanism for shielding dsRNA away from PRRs.Enzymes related to adenosine deaminases acting on RNA 1（ADAR1）,the RNA-editing enzyme converting adenosine（A）to inosine（I）of duplex RNA regions,is one of the most important strategies controlling innate immune responses to endogenous RNAs.We identified ADAR1 from previous reported data showing HBV pgRNA interacted proteins,indicating that ADAR1 may account for extensive editing of HBV RNAs and regulate innate immunity against HBV.However,it remains unclear whether ADAR1 manipulates HBV RNAs editing to facilitate HBV innate immune evasion and viral replication.This study aims to reveal the regulatory role and mechanisms of ADAR1 in HBV immune escape.Our data provide a new therapeutic target for HBV clearance.The main research methods and results are as following:Part 1 Hepatocytes fail to respond to natural HBV infectionHBV has long been considered as a "stealth" virus,however,a recent report described that the immune recognition of HBV pgRNA by RIG-I triggered a low level of IFN-λ in hepatocytes.To verify whether HBV infection trigger IFN response in hepatocytes,different HBV infected models were used to detect IFN expression.1.HBV infection/transfection triggers low levels of IFNs in hepatocytesIFNs were detected in HBV infected hepatocytes including HepG2NTCP,HLCZ-01 cells and primary human hepatocytes（PHH）.The results showed that HBV infection triggered low levels of IFNs expression in all hepatocytes compared with the inactivated HBV.Considering the relatively low efficiency of HBV infection,HepG2 cells were transfected with HBV1.1（genotype D）or HBV1.3（genotype C）plasmids to drive high levels of viral replication.However,HBV plasmids still induced weak production of IFNs even when pgRNA abundance had reached a plateau,and there are no obvious differences of IFNs expression between HBV genotypes.2.RNA sensors are abundant in hepatocytes to recognize exogenous RNAsPRRs expression levels were dectected in human hepatocytes with those in differentiated macrophage cell line THP-1,which expresses high levels of various PRRs.Compared with differentiated THP-1 cells,DNA sensors including cGAS,STING and IFI16 were barely detectable in hepatocytes.However,RNA sensors,such as TLR3,RIG-I and MDA5,were expressed at comparable levels in all hepatocyte cell lines and PHH as compared to THP-1 cells.Consistently,the DNA sensor agonist CTDNA（deoxyribonucleic acid sodium salt from calf thymus）and 2’3’-cGAMP（cyclic GMP-AMP）failed to induce intense IFN responses in HepG2 cells.Stimulation with either RNA mimic poly（I:C）or RNA virus（VSV）triggered mass production of IFNs in HepG2 cells.Above data suggest that the RNA sensing pathways are relatively abundant in human hepatocytes,and hepatocytes respond to exogenous RNAs effectively.3.Hepatocytes efficiently respond to in vitro transcribed HBV RNAsTo identify whether PRRs could recognize the in vitro transcribed HBV RNAs in hepatocytes.HBV pgRNA was transcribed in vitro,dephosphorylated and restored its secondary structure（tpgRNA）.Surprisingly,tpgRNA transfection activated IFN signalling pathways dose-dependently in both HepG2 and PHH cells,indicating that HBV RNAs evade from immune system.Part 2 ADAR1 mediated editing of HBV RNAs inhibites IFN response to regulate HBV replicationI ADAR1 editing of HBV RNAs blocks host immune recognitionTo investigate the mechanism of HBV evasion,HBV RNAs-interacting proteins were selected in pgRNA pulled-down database,and the mechanisms were detected.1.ADAR1 interacts with HBV RNAs intermediates depending on its dsRNA binding domainsBased on re-analysis of the published data,proteins related to RNA processing and infectious disease were enriched,and ADAR1 is included in both pathways.RNA pulldown and RIP assays demonstrated the interaction of ADAR1 and HBV RNAs.IFA further verified the co-localization of ADAR1 and biotinylated-tpgRNA.A serious of ADAR1 truncations and mutants were constructed and transfected with HBV 1.3 palsmids into Huh7 cells.RIP analysis showed that dsRNA binding domains are required for ADAR1 binding with HBV RNAs.2.ADAR1 editing of HBV RNAs inhibites IFN response in hepatocytesTo address whether ADAR1 accounts for HBV editing,HBV infected HepG2NTCPTet-shADAR1 cells were treated with Dox to induce ADAR1 knockdown and then RNA-sequencing was performed.RNA-seq results showed that mutations including A>G and U>C transitions of host genes and HBV RNAs in HepG2NTCP-Tet-shADAR1 cells without Dox treatment.GSEA showed a significant enrichment of differentially expressed gene signatures on Type Ⅰ IFN production pathway in Dox treated HepG2NTCP-Tet-shADAR1 cells.Consistently,HBV infection significantly enhanced IFNs expression in Dox induced HepG2NTCP-Tet-shADAR1 cells or 8-aza treated HepG2 cells.To verify the role of ADAR1 mediated HBV RNAs editing in regulation of IFN responses and consequent HBV immune evasion,we constructed HBV1.3-edited plasmid carrying ADAR1 mediated 11 A>G mutant sites identified by RNA-seq.HBV1.3-edited lost the ability to activate IFN response in Dox-treated HepG2NTCP-TetshADAR1 cells.Consistently,the tpgRNA-edited stimulated IFN expression weakly.RIP analysis revealed that ADAR1 inhibited the enrichment of RIG-I and MDA5 on HBV RNAs depending on the editing activity.Furthermore,intervention of MAVS significantly destroyed the augmented production of IFNs in Dox treated HepG2NTCPTet-shADAR1 cells.Above data indicating that ADAR1 mediated RNA editing enables HBV RNAs escape from RNA sensing and IFNs responses.II ADAR1 promotes HBV replication depending on its editing activityTo investigate the role of ADAR1 on HBV replication,different HBV infected models were included.1.ADAR1 promotes HBV replicationADAR1 overexpression or ADAR1 knockdown was performed in different HBV infected hepatocytes,including HepG2.2.15,HBV infected Huh7NTCP cells/HepG2NTCP cells,Dox treated HepG2.2.15-Tet-shADAR1 cells and HBV infected HepG2NTCP-TetshADAR1 cells.ELISA,RT-qPCR,Western blot,IFA and Northern blot were used to detect HBV related antigens and HBV pgRNA levels.Overexpression of ADAR1 strongly promoted expression of viral proteins and viral RNA transcripts,while ADAR1 knockdown greatly decreased the levels of all detected viral antigens and viral RNA transcripts.2.ADAR1 promotes HBV replication through its editing activityIn order to define the key domain of ADAR1 responsible for promoting HBV infection,serial truncates and mutants were involved.The results showed that ADAR1 promoted HBV replication depending on dsRNA binding domains and editing activity.Consistently,8-Aza treatment significantly inhibited HBV replication.3.ADAR1 promotes HBV replication via immune regulationTo verify whether ADAR1 regulates HBV replication depending on IFN responses,HBV infected HepG2NTCP-Tet-shADAR1 cells were treated with mixed IFN neutralizing antibodies or siMAVS.Dox induced ADAR1 knockdown significantly decreased HBV replication,while this inhibition of HBsAg,HBeAg and pgRNA was almost completely rescued by either IFN blocking antibodies or silence of MAVS.Similarly,HBV1.3-edited plasmid lost the ability to activate IFN signaling pathways,but significantly increased pgRNA transcription.Ⅲ ADAR1 inhibitor inflames liver and promotes HBV clearanceAbove findings demonstrated ADAR1 as a helper for HBV immune evasion via deaminase activity.We then explored the potential therapeutic effect of ADAR1 inhibitor against HBV in vivo.1.ADAR1 inhibitor promotes HBV clearanceAAV-HBV1.2 plasmid（6 μg/mouse）was injected into the tail veins of six to eightweek-old male C57BL/6 mice.4 days later,mouse received 8-aza（2 mg/kg B.W.）by intraperitoneal injection,or ETV（0.1 mg/kg B.W.）by oral gavage once daily.Serum levels of HBsAg、HBV DNA were detected every three days.As expected,8-aza administration significantly restrained serum levels of HBsAg,HBV particle DNA,as well as hepatic pgRNA level and HBcAg expression.2.ADAR1 inhibitor inflames liver8-aza treatment rescued IFN-y and TNF-α secretion in splenic CD8+T cells and NK cells impaired by HBV infection.8-aza inflames HBV infected livers by increasing the proportions of T cells and decreasing the proportion of MDSC and Treg cells.RTqPCR results revealed that 8-aza treatment increased hepatic levels of IFNs,especially Ifnb1,Ifnl3,ISGs such as Oas1,Mx1 and Tnf-α,revealed that 8-aza stimulated immune responses in livers.Part 3 HBx promotes ADAR1 expression1.HBV infection promotes ADAR1 expressionGiven that HBV can regulate multiple host genes expression to promote selfreplication,we asked whether HBV reversely affected ADAR1 expression.Immunohistochemical（IHC）staining and RT-qPCR assay showed higher ADAR1 expression in the adjacent non-tumour tissues from HBV active patients.Further analysis demonstrated a significant positive correlation between ADAR1 and HBcAg/pgRNA expression.ADAR1 expression was significantly enhanced in either HBV infected HepG2NTCP cells or HBV 1.3 transfected Huh7 cells as well as in HBV transgenic mice.Above data illustrate that ADAR1 was highly expressed in HBV infected hepatocytes.2.HBx promotes ADAR1 expressionTo identify the critical viral proteins of HBV initiating the augmented expression of ADAR1,Huh7 cells was transfected with vectors expressing the entire HBV genome（HBV1.3）or viral proteins.Western blot and dual luciferase assay showed that,several HBV-encoded proteins led to elevated expression of ADAR1,among which,HBx displayed as the most potent enhancer of ADAR1 comparably to HBV 1.3 in a dose dependent manner.In addition,deletion of HBx in MC-HBV greatly dampened the ability to enhance ADAR1 expression.3.HBx transactivates ADAR1 expression by YY1To better define the regulatory mechanism of HBx-enhanced transcriptional activation of ADAR1,various truncate constructs at 5’-flanking region of the ADAR1 promoter were cloned.Luciferase reporter assay showed that HBx regulated ADAR1 promoter activity depending on-779～-442 region.Further TRANSFAC analysis predicted a binding site of transcriptional factor YY1 in-779～-442 region.Both mutant ADAR1 promoter reporter plasmid with depletion of YY1 binding motif and siRNA against YY1 were included.Deletion of YY1 binding motif in ADAR1 promoter or siYYl suppressed the HBx-mediated enhancement of ADAR1 promoter activity,suggesting that YY1 is crucial for HBx-mediated transcriptionally enhancement of ADAR1 expression.Conclusion and significance:In this study,we demonstrated for the first time that ADAR1 blocks PRRs recognition of HBV RNAs in hepatocytes depending on its editing activity,thereby limits MDA5 and RIG-I recognition of HBV RNAs and subsequently IFN production.HBV X protein transcriptionally promoted ADAR1 expression to upregulate the threshold for intrinsic immune activation,which in turn enhanced HBV immune escape from immune recognition,leading to persistent infection.ADAR1 inhibitor increased hepatic immune response to promote HBV clearance.Our findings provide a new insight into HBV escape from host immune recognition and a therapeutic target for HBV clearance.