Hepatitis B virus confers innate immunity evasion through hepatitis B virus-miR-3 down-regulation of cGAS-Sting-IFN signaling

Abstract

BACKGROUND

Hepatitis B virus (HBV) evades the innate immunity and leads to persistent chronic infection, but the molecular mechanism is still not well known.

AIM

To investigate whether HBV-miR-3 is involved in HBV immune evasion.

METHODS

HBV-miR-3 agomir and antagomir were employed to verify the effectiveness of HBV-miR-3 on cGAS-Sting-IFN pathway through the experiments on relative luciferase activity, cGAS protein expression, Sting phosphorylation and interferon (IFN) production.

RESULTS

HBV-miR-3 down-regulates cGAS protein expression post-transcriptionally by inhibition of cGAS 3’-untranslated region (3’-UTR) activity, which results in lower Sting phosphorylation and IFN production. HBV-miR-3 antagomir rescued cGAS protein expression, Sting phosphorylation and IFN-β production.

CONCLUSION

HBV-miR-3 plays an important role in HBV immunity evasion by targeting cGAS 3’-UTR and interfering with cGAS-Sting-IFN pathway.

Key Words: Hepatitis B virus; HBV-miR-3; cGAS; Sting; IFN-β

Core Tip: This study uncovers a novel mechanism of hepatitis B virus (HBV) immunity evasion through HBV-miR-3, a recently identified HBV-encoded microRNA. HBV-miR-3 down-regulates the cGAS-STING-IFN pathway by inhibiting cGAS 3’ untranslated region activity, leading to decreased cGAS protein expression, STING phosphorylation, and interferon production. The findings highlight the crucial role of HBV-miR-3 in modulating host immune responses, providing insights into potential therapeutic targets for HBV infection.

INTRODUCTION

Hepatitis B virus (HBV) infection, leading to liver cirrhosis and hepatocellular carcinoma (HCC), is a major global health problem[1]. The interaction between HBV and the host immune system plays a critical role in establishing persistent HBV infection[2]. HBV has developed many strategies to evade and weaken the host immunological defenses[3], including disrupting antigen processing and presentation, preventing MHC molecules from binding peptide segments, producing various viral proteins that inhibit antibody, cytokine production, and T cell activation[4-6]. The cGAS-Sting pathway is crucial in innate immunity against viruses[7]. When viral DNA is sensed, cGAS generates cGAMP, which binds Sting, triggering cascades leading to type I interferon (IFN) production and antiviral molecules. Compounds like Schisandrin C or other Sting agonists can activate the cGAS-Sting pathway, resulting in the suppression of HBV replication[8,9]. Conversely, disrupting the cGAS-Sting pathway can facilitate the establishment of persistent viral infections[10,11]. HBV is a double-stranded DNA virus that theoretically serve as a substrate for activating the cGAS-Sting pathway, and then inhibiting HBV replication. In actuality, HBV can evade the innate immunity by inhibiting cGAS pathway and establishing persistent infection[3,12]. Such as HBx mediates cGAS ubiquitination, leading to the downregulation of the cGAS-Sting pathway[13]. In the present study, we aim to explore the role of HBV-miR-3 in modulating the cGAS-Sting pathway and its impact on host immune response for HBV replication.

MATERIALS AND METHODS

Plasmids and cell culture

pHBV 1.3 was preserved by the Infectious Disease Laboratory of the Second Xiangya Hospital. To perform target gene assays, a wild-type (WT) fragment of the 3’-untranslated region (3’-UTR) of cGAS containing the predicted HBV-miR-3 binding sequence was inserted into the pmirGLO Dual-luciferase miRNA target expression vector (Promega, Madison, Wisconsin, United States); pmirGLO-cGAS-WT, and a mutant (MUT) fragment of the 3’-UTR of cGAS was also cloned into the vector to generate a pmirGLO-cGAS-MUT construct with a mutated binding site; The dual-fluorescence plasmids were generated, then confirmed by sequencing.

The human hepatoma cell lines HepG2 and HepG2.2.15 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HepG2.2.15 cells are derived from HepG2 cells and stably transfected with the HBV genome, continuously producing infectious HBV particles, making them suitable for studying HBV replication. The HepG2-NTCP cell line, stably expressing the sodium taurocholate cotransporting polypeptide (NTCP), was purchased from BLUEFBIO™ (Cat. No. BFN 60805958, Shanghai, China). HepG2-NTCP cells are highly permissive to HBV infection, serving as an in vitro HBV infection model.

All cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Gibco, New York, United States) and 100 U/mL penicillin and streptomycin (Cat. No. ST488, Beyotime, Beijing, China) at 37 °C in a humidified atmosphere with 5% CO₂. The cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/well. After 24 hours, a concentration gradient of HBV-miR-3 agomir or negative control (NC) RNA (synthesized by GenePharma, Shanghai, China) was transfected into HepG2 cells.

To establish an HBV infection model, collect the culture supernatant from HepG2.2.15 cells and measure the HBV DNA concentration using quantitative PCR. Count the HepG2-NTCP cells to ensure accurate seeding. Infect the HepG2-NTCP cells with the HBV-containing supernatant at an multiplicity of infection of 300.

Luciferase reporter assay

HepG2 cells, grown to 60% confluence, were transfected with pmirGLO-cGAS-WT or pmirGLO-cGAS-MUT plasmids and either HBV-miR-3 agomir or NC using Lipofectamine™ 2000 Transfection Reagent (Cat. No. 11668-019, Invitrogen, Carlsbad, CA, United States). After 48 hours, luciferase activity was analyzed with the luciferase assay kit (Cat. No. N1610, Promega, Madison, WI, United States).

Western blot

Protein extraction was performed using RIPA buffer (Cat. No. P0013C, Beyotime). After 12% SDS-PAGE and transfer to nitrocellulose membranes (Cat. No. FFN02, Beyotime), membranes were blocked, incubated with cGAS antibody (1:1000, Cat. No. 26416-1-AP, Proteintech), and then with HRP-conjugated secondary antibody (Cat. No. sc-2005, Santa Cruz). Detection used enhanced chemiluminescence (Beyo ECL Star, Cat. No. P1008AS, Beyotime). The membrane was washed three times for five minutes each in wash buffer. After that, it was incubated with p-Sting antibody solution (1:1000, Cat. No. 50907, Cell Signaling, MA, United States), followed by secondary antibody incubation and exposure steps. The membrane was washed again, then incubated with Sting and IFN-β antibodies (1:1000, Cat. No. 19851-1-AP/27506-1-AP, Proteintech, Chicago, United States), and finally, incubated with the HRP-conjugated internal control GAPDH antibody solution (1:3000, Cat. No. HRP-60004, Proteintech, Chicago, United States). Each experiment was conducted in triplicate, with each sample also tested in triplicate.

qRT-PCR

RT-PCR analysis of cGAS mRNA was performed using the following steps. Firstly, total RNA was extracted from cells or tissues using TRIzol reagent (Takara, Japan; Cat. No. 9109). Subsequently, the total RNA was reverse transcribed into cDNA using a reverse transcription kit (Thermo Fisher, United States; Cat. No. K1622). During the reverse transcription process, random primers and reverse transcriptase were used to synthesize cDNA. Next, PCR amplification was carried out using the TB Green PCR Master Mix (Takara, Japan; Cat. No. RR820A) on a 7500 real-time PCR system. The PCR reaction conditions were as follows: Stage 1: Pre-denaturation, with 1 cycle of 95 °C for 30 seconds; Stage 2: PCR reaction, with 40 cycles of 95 °C for 5 seconds and 60 °C for 30-34 seconds. In this study, the mRNA expression level of cGAS was quantitatively detected and analyzed under different conditions using the described RT-PCR method. The primers used for cGAS mRNA were as follows: cGAS: Forward primer (F): 5'-GGGAGCCCTGCTGTAACACTTCTTAT-3'; Reverse primer (R): 5'-CCTTTGCATGCTTGGGTACAAGGT-3'. GAPDH: Forward primer (F): 5'-CCACTCCTCCACCTTTGAC-3'; Reverse primer (R): 5'-ACCCTGTTGCTGTAGCCA-3'.

HBV DNA levels were assessed using fluorescent quantitative PCR with kits from (Sansure Biotech Inc. China). HBV pgRNA was extracted from 200 μL of sample using a magnetic bead-based kit (Sansure Biotech Inc. China) and reverse-transcribed into cDNA at 50 °C for 30 minutes. cDNA amplification included an initial step at 95 °C for 2 minutes, 50 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds, and a final cooling at 25 °C for 10 seconds. Fluorescence signals were quantified using the 7500 real-time PCR System (Applied Biosystems^®). PCR data were normalized to the expression of GAPDH as an internal control. The relative expression levels were calculated using the ^ΔΔCt method, where Ct represents the threshold cycle. All results are expressed as fold changes relative to the control group. Each experiment was evaluated using three PCR reactions and was repeated three times. Data are presented as mean ± SD.

Statistical analysis

Data were analyzed with IBM SPSS 20.0. Group comparisons used Student's t-test, paired t-tests for cGAS and HBV-miR-3, and one-way ANOVA for other data. Correlation analysis assessed dose-response relationships, with significance at P < 0.05.

RESULTS

cGAS protein expression is down-regulated in the presence of HBV

Our previous studies have confirmed that HBV-miR-3 is specifically expressed during HBV infection[14,15]. cGAS protein and mRNA levels between HepG2 and HepG2.2.15 cells were studied, and we found cGAS protein were evidently lower in HepG2.2.15 cells, but the cGAS mRNA level was observed similar in these two cell line cells (Figure 1A and B). Similar results were obtained from the HepG2-NTCP cells infected or not infected with HBV from the HepG2.2.15 supernatant (Figure 1C and D). To further evidence this finding, we did the experiment with different doses of pHBV1.3 to transiently transfect the HepG2 cells. The results showed with increasing doses of pHBV1.3, HBV-miR-3, HBV DNA, and HBV pgRNA increased gradually, and the cGAS protein expression decreased accordingly, but the cGAS mRNA between these different doses of pHBV1.3 transfected HepG2 cell groups was kept almost similar (Figure 1E and F). These findings indicated that HBV down-regulated cGAS protein expression post-transcriptionally.

Figure 1 CGAS protein expression in presence of hepatitis B virus. A: CGAS protein expression in HepG2.2.15 cells vs HepG2 cells; B: CGAS mRNA levels in HepG2.2.15 cells vs HepG2 cells; C: CGAS protein expression in HepG2-NTCP cells treated with hepatitis B virus (HBV) from HepG2.2.15 cells supernatant after 72 hours; D: CGAS mRNA levels in HepG2-NTCP cells treated with HBV from HepG2.2.15 cells supernatant after 72 hours; E: CGAS protein expression in HepG2 cells transfected with different doses of pHBV1.3; F: CGAS mRNA levels in HepG2 cells transfected with different doses of pHBV1.3. Each experiment was repeated three times. Data are presented as mean ± SD.

HBV-miR-3 interacts with cGAS 3’-UTR to inhibits cGAS protein expression

Using bioinformatics analysis, we predicted a binding site between HBV-miR-3 and cGAS 3’-UTR (Figure. 2A). A fluorescent reporter construct was designed based on this finding. Artificially synthesized HBV-miR-3 agomir was employed in this experiment. HBV-miR-3 agomir suppressed relative luciferase activity from wild-type cGAS 3’-UTR, but no effect was observed on relative luciferase activity from the mutant-type cGAS 3’-UTR (Figure 2B). Subsequently, we performed a dose dependent experiment to further confirm the inhibitory effect of HBV-miR-3 agomir on cGAS 3’-UTR activity. The result showed with increasing doses of HBV-miR-3 agomir, the relative luciferase activity from wild-type cGAS 3’-UTR decreased gradually, accompanying with cGAS protein changed similar (Figure 2C and D).

Figure 2 Hepatitis B virus-miR-3 inhibits cGAS protein expression by binding cGAS 3'-untranslated region binding. A: Schematic of the fluorescent reporter construct designed for the study; B: Relative luciferase activity in HepG2 cells transfected with pmirGLO cGAS wild-type or mutant fluorescent plasmids, and HBV-miR-3 agomir; C: Gradual decline in luciferase activity induced by HBV-miR-3 agomir after 72 hours; D: Dose-dependent repression of cGAS protein mediated by hepatitis B virus-miR-3 agomir after 72 hours. Each experiment was repeated three times. Data are presented as mean ± SD. P < 0.01. WT: Wild-type; MUT: Mutant; NC: Negative control.

HBV-miR-3 down-regulated cGAS protein expression decreased Sting phosphorylation and IFN production

HBV-miR-3 downregulation reduced the expression of the cGAS protein, resulting in decreased Sting phosphorylation and IFN-β production. Three models were used to confirm these findings: HepG2 cells vs HepG2.2.15 cells (Figure 3A), a cell culture model involving HBV infection in HepG2-NTCP cells (Figure 3B), and transfection of varying concentrations of HBV plasmids into HepG2 cells (Figure 3C). Notably, a consistent trend suggests that HBV-miR-3 influences the phosphorylation of the Sting protein rather than its expression. Salmon sperm DNA stimulation of HepG2 cells led to a significant increase in the cGAS-Sting pathway compared to untreated HepG2 cells (Figure 3D). This resulted in elevated levels of cGAS, enhanced Sting phosphorylation, IFN-β production, while Sting expression remained unchanged. This suggests that salmon sperm DNA promotes IFN-β expression through the cGAS-Sting pathway. We investigated the inhibitory effect of HBV-miR-3 on the cGAS-Sting pathway by adding different concentrations (2 nM, 4 nM, and 8 nM) to HepG2 cells stimulated with salmon sperm DNA. The study indicated that HBV-miR-3 agomir decreases cGAS levels, which leads to decreased production of P-Sting and IFN-β. Furthermore, the inhibitory action of HBV-miR-3 agomir has a dose-dependent response.

Figure 3 Impact of hepatitis B virus-miR-3 on Sting phosphorylation and IFN-β production. A: CGAS protein expression, Sting phosphorylation, and IFN-β production in HepG2.2.15 cells compared to HepG2 cells; B: CGAS protein expression, Sting phosphorylation, and IFN-β production in HepG2-NTCP cells treated with hepatitis B virus (HBV) from HepG2.2.15 cell supernatant; C: CGAS protein expression, Sting phosphorylation, and IFN-β production in HepG2-NTCP cells treated with HBV from HepG2.2.15 cell supernatant; D: Co-treatment with HBV-miR-3 agonist and salmon sperm DNA increased cGAS protein expression, Sting phosphorylation, and IFN-β production in HepG2 cells. Each experiment was repeated three times. Data are presented as mean ± SD.

HBV-miR-3 antagomir reverses the effects of HBV-miR-3 on the cGAS-Sting pathway

First, we confirmed that the HBV-miR-3 antagomir worked as intended. Figure 4A shows that the inhibitor can effectively restore the reduced fluorescence activity caused by HBV-miR-3 agomir suppression. Next, we noticed that HBV-miR-3 antagomir could increase the production of the cGAS-Sting pathway in HepG2 cells transfected with pHBV1.3 (Figure 4B). Following treatment with varying doses of HBV-miR-3 antagomir, the cGAS-Sting pathway was increased in HepG2.2.15 cells (Figure 4C).

Figure 4 Hepatitis B virus-miR-3 antagomir resued cGAS-Sting pathway activity. A: Effect of 72-hour HBV-miR-3 antagomir treatment on the cGAS-Sting pathway in HepG2 cells transfected with various dosages of pHBV1.3; B: Expression of the cGAS-Sting pathway in HepG2.2.15 cells after a 72-hour treatment with HBV-miR-3 antagomir; C: Expression of the cGAS-Sting pathway in HepG2-NTCP cells infected with hepatitis B virus from HepG2.2.15 supernatant after 72 hours of HBV-miR-3 inhibitor treatment. Each experiment was conducted three times. Data are presented as mean ± SD.

HBV-miR-3 antagomir inhibits HBV replication

HepG2 cells were transfected with pHBV1.3 and HBV-miR-3 antagomir, then HBV DNA, HBV pgRNA, HBsAg protein, and cGAS-Sting-IFN pathway were evaluated (Figure 5A-C). Similar results were obtained when HepG2-NTCP cells infected with HepG2.2.15 supernatant were treated with HBV-miR-3 antagomir (Figure 5D-F).

Figure 5 Hepatitis B virus-miR-3 antagomir inhibited hepatitis B virus replication. A-C: HepG2 cells transfected with pHBV1.3 were treated with an HBV-miR-3 inhibitor for 72 hours, resulting in alterations in hepatitis B virus (HBV) DNA (A), HBV pgRNA (B), and HBsAg (C); D-F: HepG2-NTCP cells were infected with HBV from HepG2.2.15 cell supernatant and treated with an HBV-miR-3 antagomir for 72 hours, resulting in alterations in HBV DNA (D), HBV pgRNA levels (E), and HBsAg (F). Each experiment was repeated three times. The data are presented as mean ± SD. NC: Negative control; HBV: Hepatitis B virus.

DISCUSSION

This study identifies HBV-miR-3 as a novel regulator of cGAS in HBV-infected hepatocytes. We also introduced a potential mechanism of immune evasion in HBV chronic infection: HBV-miR-3 can inhibit IFN-β production, resulting in reduced viral clearance.

Our data suggest that HBV-miR-3 plays a crucial role in mediating immune evasion during chronic HBV infection. Previous studies have mainly focused on the role of HBV-miR-3 in HBV-related HCC and viral replication regulation[14-16]. In 2015, Li et al[17] demonstrated that PPM1A regulates the STING pathway, a crucial component of the innate immune response, suggesting its role in immune modulation during HBV infection. Building on this, in 2020, Chavalit et al[18] showed that HBV miR-3 interacts with PPM1A, promoting HCC development. This underscores a complex network between HBV miR-3, PPM1A, and STING in liver disease and cancer. Earlier research has demonstrated that HBV is capable of suppressing IFN production, thereby reducing viral clearance ability[19]. More recent investigations have further confirmed that this immune evasion effect is primarily associated with the upregulation of HBV-miR-3 in hepatocytes[20]. Therefore, in this study, we employed chronic HBV-infected cell models and utilized HBV-miR-3 mimics (agomir) and inhibitors (antagomir) to investigate the role of HBV-miR-3 in modulating host immune responses. Consistent with previous research, the presence of HBV significantly downregulates the expression of cGAS in hepatocytes[13]. Additionally, the introduction of HBV-miR-3 agomir led to a decrease in cGAS expression, which was rescued by the application of antagomir, resulting in restored cGAS levels. Collectively, these findings highlight HBV-miR-3 as a detrimental factor affecting the host's innate immune response during HBV infection.

This study reveals the role of HBV-miR-3 in HBV immune escape for the first time; different from previous studies focusing on HBV-related liver cancer or replication. The innate immune system determines the outcome of HBV infection through complex interactions with the virus. Innate immune receptors (PRRs), such as cGAS, and RIG[21-23], can recognize PAMPs carried by microorganisms and DAMPs released by necrotic cells, initiating immunity, defense, repair and homeostasis[24]. Among these receptors, the cGAS/Sting pathway is a novel innate immune sensor that can recognize double-stranded DNA/RNA from different sources, including viruses, apoptotic bodies, exosomes, mitochondria, micronuclei and retroelements. In addition, HBV-miR-3 is a miRNA from three HBV transcripts (PreC, PreS1 and PreS2), located at 373-393 nt of the HBV genome[16]. Our findings support previous findings that HBV-miR-3 is only found in HBV-integrated cells. Then, in HepG2 cells transfected with varying amounts of pHBV1.3 plasmid, we looked at the relationship between HBV DNA, HBV pgRNA, and HBV-miR-3 and discovered that they were all positively correlated. IntereStingly, previous studies found that HBV-miR-3 targets HBV pgRNA. Our results are consistent with Gan et al[25], showing that HBV pgRNA and HBV-miR-3 were also positively correlated. This suggests that HBV-miR-3 may be involved in a wider cellular regulatory network. Therefore, its association with HBV pgRNA may go beyond the scope of targeted degradation.

Following that, we utilized miRDB (http://mirdb.org/cgi-bin/custom_predict/customDetail.cgi) to perform a computational analysis to identify potential HBV-miR-3 binding sites within the 3'-UTR of cGAS mRNA. These predicted sites require experimental validation. To demonstrate this, dual luciferase reporter plasmids were created and used, as well as experiments with HBV-miR-3 agomirs and antagomir. Notably, the HBV-miR-3 agomir significantly reduced cGAS WT fluorescence activity within the dual-luciferase reporter plasmid system. This dose-dependent reduction was observed. Importantly, HBV-miR-3 antagomir effectively counteracted the dampened fluorescence activity mediated by HBV-miR-3 agomir. These findings strongly support the existence and efficacy of the cGAS-HBV-miR-3 interaction. HBV suppresses cGAS responses through multiple pathways. For example, HBx inhibits DNA sensing signaling by ubiquitination and autophagic degradation of cGAS. Additionally, the HBV polymerase disrupts Sting's K63-linked ubiquitination, effectively blocking the cGAS-Sting pathway. Furthermore, HBV escapes innate immunity by activating HAT1 to acetylate H4K5/H4K12, upregulate miR-181a-5p or KPNA2, and finally inhibit cGAS-Sting/IFN signaling[26]. This paper also discovered a link between miRNA and host immunity, but it is the miRNA of human live cells. This time, our focus is on the interaction between cGAS and HBV-miR-3, which is secreted by HBV as exosomes.

In order to clarify the association between HBV-miR-3 and IFN, we pursued further investigations downstream of the cGAS-Sting pathway. This exploration unveiled that the reduction of cGAS triggered by HBV exerted downstream consequences, culminating in diminished Sting phosphorylation and reduced IFN-β production. Intriguingly, the addition of salmon sperm DNA, or HBV-miR-3 inhibitors prompted a restoration of the cGAS-Sting pathway's activity in the presence of HBV or HBV-miR-3, countering the previous downregulation.

The results indicate that stimulation with salmon sperm DNA (Figure 3D) or treatment with the HBV-miR3 inhibitor (Figure 5) can partially restore the cGAS-Sting pathway previously inhibited by HBV-miR3. Salmon sperm DNA, being double-stranded, is recognized by cGAS or other factors that activate the cGAS-Sting pathway, leading to increased IFN expression and ultimately reducing HBV replication, as reported in the literature[27,28]. Similarly, we discovered that the HBV-miR3 inhibitor relieved HBV's immunosuppressive effects, resulting in lower replication. This was specifically evidenced by a statistically significant reduction in HBV DNA and HBV pgRNA in the supernatant as shown by PCR results, the restoration of the cGAS-Sting pathway in cells as indicated by Western blot analysis, and a reduction in HBsAg levels, increasing the likelihood of HBV clearance. Also, the activation of the cGAS-Sting pathway could potentially help control chronic HBV infection and reduce the incidence of HBV-related liver cancer. Additionally, the immunological control of HBV-miR-3 is likewise quite complex. In a previous clinical retrospective analysis involving 650 individuals, we found that HBV-miR-3 exhibited a strong predictive effect for HBeAg seroconversion in HBeAg-positive patients undergoing IFN-α 2b treatment. Specifically, lower levels of HBV-miR-3 correlated with greater reductions, indicating a higher likelihood of seroconversion[15]. Additionally, studies have shown that post-IFN-α treatment, HBV-miR-3 targets the SOCS5 protein enhancing IFN's antiviral activity[20]. These findings collectively indicate that the role of HBV-miR-3 in immune regulation is not fixed but varies depending on the stage of HBV infection and the presence of exogenous IFN treatment, targeting multiple genes across various cells to exert a comprehensive effect.

These results could give a possible explanation for the controversial point: Whether HBV can be monitored by cGAS. That is, HBV inhibits the activation of cGAS-Sting pathway through HBV-miR-3, including Sting phosphorylation and IFN production. This phenomenon is similar to hepatitis E virus (HEV) evasion of immune clearance via HEV-miR-A6 expression. HEV-miR-A6 inhibits IRF3 phosphorylation, thereby inhibiting IFN expression and facilitating viral replication[29]. Additionally, in the case of Epstein-Barr virus (EBV), EBV-miR-BART5 targets the PUMA protein, influencing apoptosis[30], Furthermore, in Kaposi's sarcoma-associated herpesvirus, miR-K5 targets BCLAF1, promotes the virus's latent infection[31]. These findings suggest that, as a result of mutual competition, viruses such as EBV, HEV, and HBV have evolved strategies to suppress the host immune system via miRNA synthesis.

The immune evasion mechanisms of HBV are highly complex, employing multiple strategies to evade the immune system. HBV does not directly destroy hepatocytes but synthesizes large amounts of viral components within them, including HBV DNA, HBsAg, HBeAg, and HBx. It is generally believed that these molecules continuously stimulate the immune system, leading to the suppression and exhaustion of immune cells in the liver. HBsAg negatively affects the normal immune responses of various innate immune cells, including monocytes/macrophages, Kupffer cells, NK cells, dendritic cells, and monocytic myeloid-derived suppressor cells[32]. HBeAg can inhibit the NF-kB pathway and ROS production, weakening LPS-induced NLRP3 inflammasome activation and IL-1β production[33]. HBV-miR-3 inhibition of cGAS represents a more sophisticated approach. It directly interferes with the cellular machinery responsible for recognizing and responding to the presence of the virus, thereby providing a more robust and immediate means of immune evasion. HBV-miR-3 targets cGAS, the primary sensor of cytoplasmic DNA, directly impeding the innate immune response. Consequently, HBV significantly reduces the host's ability to detect and respond to viral DNA. This targeted inhibition is more direct and potentially more effective than other broad-spectrum immune evasion mechanisms. By inhibiting cGAS, HBV-miR-3 helps maintain chronic HBV infection: A sustained low-level immune response insufficient to clear HBV but causing continuous liver inflammation and damage. This delicate balance prevents strong antiviral responses while avoiding complete immune suppression, which could trigger more intense immune reactions.

The chemical nature of the HBV-miR3 inhibitor is an antisense miRNA of HBV-miR3. This aligns with current developments in new HBV drugs, including RNA interference agents (siRNA), antisense RNA, and compounds that activate the innate immune system (such as TLR-3, TLR-8, TLR-9, RIG-1, and MDA5 agonists)[9,28,34], which have similar mechanisms. Compounds that activate the innate immune system, such as TLR-7, TLR-8, and TLR-9 agonists, have achieved some success when combined with other treatments. Comprehensively exploring the immunomodulatory functions of HBV-miRNAs will enhance insights into viral immune evasion mechanisms. Furthermore, such research will be critical in the development of therapeutic approaches for managing HBV-associated immune tolerance. Notably, the functions of HBV-miR-3 influence on the immune may not be limited to the regulation of cGAS expression, which needs further research into potential interactions with other immune molecules. This study has limitations, such as the lack of in vivo experiments, no pathological tissue evidence, and the absence of the use of cGAMP and Sting agonists or inhibitors. Further research is needed to address these limitations.

CONCLUSION

Immune evasion by HBV is facilitated through the action of HBV-miR-3, which inhibits cGAS and suppresses cGAS-Sting-IFN signaling. The observed differential expression of HBV-miR-3 and cGAS provides evidence for their negative correlation. Inhibition of HBV-miR-3 results in enhanced IFN expression, ultimately leading to a reduction in HBV proliferation. These findings collectively establish HBV-miR-3 as a promising therapeutic target for CHB treatment.