Hepatitis B virus core protein interacts with host RNA binding protein SRSF10 to inhibit HBV RNA production

Hepatitis B virus core protein interacts with host RNA binding protein SRSF10 to inhibit HBV RNA production. A number of studies suggest that HBV core protein (HBc) may have important regulatory functions in the nucleus of infected liver cells. In order to further reveal its specific functions, the study used proteomics to analyze the host factors that interact with HBc in HepaRG cells infected with HBV.

The results showed that the interactome of HBc is mainly composed of RNA-binding proteins (RBPs) involved in mRNA metabolism. Among them, SRSF10 is an RBP that mainly regulates alternative splicing (AS) through phosphorylation-dependent pathways, controls stress and DNA damage responses, and virus replication.

Through a series of studies including knocking down SRSF10 and using SRSF10 phosphorylation inhibitor (1C8), it was found that SRSF10 can be used as a restriction factor to regulate HBV RNA levels, and its dephosphorylation state may have antiviral effects. Surprisingly, neither knockdown of SRSF10 nor 1C8 treatment can change the spliced ​​form of HBV RNA, but can only negatively regulate the level of nascent HBV RNA.

In summary, this study suggests that HBc can interact with a variety of RBPs in the nucleus of HBV-infected cells to regulate viral RNA metabolism, confirming SRSF10, a new anti-HBV limiting factor, which is an important factor for the future development of new targeted hosts. Anti-virus strategies provide ideas.

HBV is a hepatotropic DNA virus with an envelope. It enters hepatocytes by combining with sodium taurocholate co-transporting polypeptide (NTCP). After the process of decoating and uncoating, it exists in the form of relaxed circular DNA (rcDNA). The 3.2 kb size of viral DNA is transported to the nucleus, and the process of converting rcDNA into covalently closed circular DNA (cccDNA) is completed in the nucleus of the host cell.

cccDNA exists in the cell nucleus in the form of episomes and acts as a viral genome. It is transcribed into 5 RNA sizes, which are 3.5 kb (pre-core RNA and pgRNA), 2.4 kb, 2.1 kb and 0.7 kb RNA, which can encode HBeAg , Core protein (HBc), viral polymerase (P protein), three surface proteins (S-, M- and L-HBsAg) and X protein (HBx).

The virus forms a new progeny virus through the process of pgRNA and P protein binding and wrapping into the capsid composed of HBc. PgRNA in the capsid is reverse transcribed into rcDNA, and then the envelope is obtained and secreted outside the cell, or directly enters the nucleus to supplement the cccDNA pool. More and more studies have found that there are multiple HBV RNA splice variants in patient serum and liver tissue. These splice variants are not necessary for virus replication, but may play a role in the pathogenesis of HBV.

HBc is the only structural molecule that constitutes the capsid of the HBV virus, consisting of 183 amino acids, including the N-terminal domain (NTD, aa 1-140) necessary for capsid assembly and the non-essential C-terminal domain ( C-terminal domain, CTD, aa 150-183). CTD contains motifs that control the entry and exit of HBc into and out of the cell nucleus, and has DNA/RNA binding and molecular chaperone activities.

HBc assembles the nucleocapsid in the form of a homodimer. The assembly of the capsid starts from the trimerization of low-speed assembly into a dimer, followed by high-speed assembly into a icosahedral protein capsid. The encapsulation of the P-pgRNA complex occurs during the CTD-mediated capsid assembly of HBc, and it is also a key step in regulating the reverse transcription of pgRNA into rcDNA.

Many studies have found that HBc not only functions as a structural molecule to assemble into a capsid, but also has the ability to regulate the establishment and maintenance of viral infections.

First, HBc can enter the nucleus when the virus infection is established and when the cccDNA pool is supplemented, and exists in the nucleus as a dimer or oligomer, or even as a capsid-like structure;

Secondly, in early studies, researchers observed that HBc can bind to cccDNA, thereby regulating nucleosome positioning. The association between HBc and cccDNA has been further confirmed in in vivo and in vitro studies and is related to active transcription status;

Finally, HBc was found to bind to the promoter regions of a variety of intracellular genes. In summary, the existing research results strongly suggest that the role of HBc in the nucleus during the life cycle of the HBV virus remains to be further studied.

To this end, the author conducted a proteomic analysis of the interacting proteins of HBc in the nucleus of human liver cells. The results show that HBc mainly interacts with RNA-binding protein (RBP) networks related to various post-transcriptional processes. The authors found that SRSF10 in these RBPs can inhibit the synthesis and accumulation of HBV RNA, which provides a new perspective for the development of new antiviral drugs.

01 HBc interacting factors in the nucleus of differentiated hepatocytes are mainly host RNA binding proteins

In order to gain insight into the regulatory function of HBc, the author tried to identify its interacting proteins in the nucleus of human liver cells.

To this end, the authors used tetracycline-induced differentiated hepatocytes (dHepaRG-tr-ST-HBc) that can express N-terminally fused streptavidin-binding peptide (ST) HBc (Figure 1A). The ST-HBc fusion protein is localized in the nucleus of liver cells and assembled into a capsid-like structure in the same form as wild-type (wt) HBc. The ST-HBc/host-factor complex was purified from the nuclear extract through an affinity column with streptavidin-binding peptide (Figure 1B and Figure 1C).

The negative control is provided by dHepaRG-tr cells expressing wt HBc, but because the HBc expressed does not have any label, it cannot bind to the affinity column. In addition, in order to eliminate the purification of the indirectly bound protein due to the mediation of DNA/RNA, the author used the cell lysate after nuclease digestion to purify the ST-HBc complex.

Under each condition (nuclease +/-), three different batches of dHepaRG were used to separately purify ST-HBc related proteins three times, and the eluted proteins were subjected to mass spectrometry (MS)-based label-free quantitative proteomics analysis.

The analysis results identified 60 and 45 proteins significantly related to HBc (P-value <0.01 and fold change> 4). It is worth noting that 38 of these factors are common with or without nuclease, which proves the reliability of their identification (Figure 1D).

Figure 1. Identification of HBc interacting proteins in the nucleus of dHepaRG

Gene ontology (GO) analysis of HBc interacting factors showed that about 50% of the factors significantly related to HBc under nuclease treatment or no treatment were nucleic acid binding proteins, belonging to the RBP family.

In the presence of nucleases, the most abundant protein species (Q-value: 1.8 x 10-29) identified are related to the RNA post-transcription process, especially splicing-related factors (Figure 2A). The second most relevant category (Q-value: 4.4×10-14) is related to ribosomal proteins.

The similarity of the interacting molecules obtained with and without nuclease indicates that most of these interactions occur without nucleic acid mediation, or they form a dense complex, so that the DNA/RNA is protected. Free from nuclease digestion.

Since the main protein category obtained by GO analysis corresponds to the RBP involved in the splicing process, the author will focus on the protein corresponding to this function, as well as the protein identified under the condition of nuclease or not (i.e. used in Figure 1D) 11 proteins in bold). The interactomics of 11 RBPs showed that they have a high degree of interconnection, and several of their primary interacting proteins were also found in the HBc co-purified factors (Figure 2B).

Figure 2. Interactomics analysis of intranuclear HBc

Analysis of the relative abundance of these RBPs showed that in the HBc complex, SRSF10 is the most abundant RBP, followed by RBMX, SRSF1, SRSF5 and TRA2B (Figure 3A). Western blot analysis confirmed the presence of SRSF10, RBMX, DDX17, SRSF2, and TRA2B in the ST-HBc purified complex, as well as two other non-RBP factors PARP1 and DNAJB2 (Figures 3B and 3C). In contrast, Western blot could not confirm the presence of SRSF1 (Figure 3B). The reason for the lack of detection is currently unknown, but it may be due to the poor sensitivity of the antibody used.

Figure 3. Verification of HBc nuclear interacting proteins

02 HBc interacts with multiple SRSF10 isomers

SRSF10 is a member of the splicing factor SR protein family, and is the most abundant cellular protein found in this study to interact with HBc in the nucleus. Like other members of the SR family, SRSF10 consists of an amino-terminal RNA recognition motif (RRM) and a C-terminal arginine/serine enrichment domain (RS) responsible for binding to other RBPs. SRSF10 has two RS structures. Domain, RS1 and RS2.

The two isomers of SRSF10 are 37 kDa and 20 kDa, respectively, and the smaller isomer lacks the C-terminal RS2 domain. In the infected cell culture model, the interaction of HBc and SRSF10 has been extensively studied by immunoprecipitation method, but this method has not been detected in HBV-infected dHepaRG in vitro or freshly isolated primary cultured human hepatocyte (PHH) system The interaction between HBc and SRSF10 may be due to the relatively low level of infection in these models.

In order to determine the interaction between HBc and SRSF10, the authors used hepatocytes from mice (HuHep mice) transplanted with human hepatocytes and infected with HBV to perform co-immunoprecipitation (co-IP) analysis. The HBV replication level of this model is very high. high. Using these samples for HBc IP, a single band was detected with two different anti-SRSF10 antibodies, but it did not correspond to the two main SRSF10 subtypes of 37 kDa and 20 kDa detected in the input, but at 35kD and Change between 25 kD (Figure 3D).

The protein band between 35 kDa and 25 kDa in the gel band was analyzed by mass spectrometry, and 6 different peptide fragments were identified, covering the first 100 aa of SRSF10 (Figure 3E). This analysis confirmed the presence of SRSF10. In addition to SRSF10, seven other proteins previously identified in the ST-HBc elution fraction were also detected, five of which are SR proteins (Figure 1D).

It is worth noting that the co-IP using dHepaRG-HBc or ST-HBc nuclear extracts also detected this new SRSF10 subtype and other traditional SRSF10 subtypes. At present, there are at least 9 different SRSF10 subtypes, all of which contain RRM and RS1 domains, which may be produced by alternative splicing of mRNA.

This new low expression subtype is about 30-32 kDa, which may correspond to a 217aa protein form containing RRM and RS1 domains and a shorter RS2 domain.

These results indicate that there is an interaction between HBc and SRSF10 and other previously identified SR proteins in HBV-infected PHH. This also indicates that HBc may be associated with different SRSF10 subtypes, further indicating the potential importance of this interaction to the virus life cycle.

03 SRSF10 regulates HBV RNA levels

Next, the author studied the effect of SRSF10 knock-down (KD) on HBV infection. In this study, the siRNAs used for KD SRSF10 are located in the RRM and RS1 coding sequences, respectively, so they can target all SRSF10 subtypes that share these regions, including those that are not observed in Western blot.

After confirming that siRNA knockdown of SRSF10 did not affect NTCP levels in PHH, indicating that HBV internalization was not affected (Figure 4A-4C), the authors found that SRSF10 knockdown significantly increased the accumulation of total HBV RNA and pgRNA, but did not affect cccDNA Level (Figure 4D). Similar results were also observed in dHepaRG cells, but in this cell model, cccDNA levels were moderately but significantly increased.

Northern blot also confirmed the effect of SRSF10 KD on HBV RNA and confirmed the increase of three observable HBV RNA molecules. In sharp contrast, the KD of RBMX has the opposite effect on HBV replication, and the levels of all viral indicators are reduced, including cccDNA (Figure 4E and 4F, S3). These results indicate that RBP related to HBc plays a unique role in the life cycle of HBV.

In order to determine whether SRSF10 KD has a similar effect on established HBV infection, the authors also conducted a KD experiment of transfected siRNA 7 days after infection, at which time the virus replication has reached a plateau. In dHepaRG cells, the increase of HBV RNA after SRSF10 KD can be observed.

Consistent with what was observed in cells transfected with siRNA before infection, SRSF10 KD also increased cccDNA levels after infection, indicating that in this cell line, in addition to its effect on HBV RNA, SRSF10 may also regulate the circulation of cccDNA and/ Or stability.

Figure 4. The effect of SRSF10 or RBMX KD on HBV replication in PHH cells

Finally, in order to study whether the effect of SRSF10 on HBV RNA is dependent on HBc, the authors used AAV vectors to introduce wt or mutant HBV genomes that cannot produce HBc into hepatocytes, and compared their replication levels without SRSF10 (Figure S6A).

The authors used two types of mutant AAV-HBV: the point mutation of ATG (AAVHBVnoHBc), and the mutant (AAVHBVΔHBc) with 406 bases missing between the HBc ATG and the start of the P protein ORF. Transfection of dHepaRG cells with these three vectors can establish HBV infection. Through quantitative HBV RNA and secreted antigen detection (S6B-S6D figure), the results show that AAVHBV noHBc mutant can establish HBV infection, and AAVHBVΔHBc cells are detected by HBsAg.

The KD of SRSF10 increased the levels of all viral indicators in AAVHBVwt transfected cells. It is worth noting that in the absence of HBc, the increase in HBV RNA and secreted antigens detected after SRSF10 KD was significantly reduced, indicating that the antiviral effect of SRSF10 may partly depend on HBc.

In summary, these results indicate that SRSF10 acts as an inhibitory factor to regulate HBV RNA levels.

04 Small molecule inhibitors of SRSF10 phosphorylation can inhibit HBV replication and antigen secretion

Previous studies have shown that the activity of SRSF10 is strictly controlled by phosphorylation, which can regulate its interaction and splicing activity with other RBPs. The dephosphorylation of SRSF10 occurs during heat shock, DNA damage or mitosis. Recent studies have found that compound 1C8 (Figure 5) can inhibit the phosphorylation of SRSF10, especially serine133, and has no effect on any other SR proteins. Studies have confirmed that 1C8 can inhibit the replication of HIV-1, the mechanism may be that it affects the transcription and splicing of HIV-1, leading to abnormal levels of viral proteins required for replication.

Figure 5. The effect of 1C8 on established HBV infection

Through two-dimensional gel electrophoresis, the authors confirmed that 1C8 can induce dephosphorylation of SRSF10 in differentiated human hepatocytes. To explore the effect of 1C8 on HBV replication, the authors first evaluated its effect on HBV-infected dHepaRG cells (Figure 5B). In this case, treatment with 1C8 resulted in a reduction in viral RNA and secreted viral proteins (Figure 5C).

It is worth noting that this phenotype is different from other antiviral compounds. This effect is weaker but still exists in HBV-infected PHH cells (Figure 5D). The dose-dependent analysis of dHepaRG showed that the median effective concentration (EC50) for HBV RNA/secreted DNA and HBsAg/HBeAg were about 10μM and 5μM, respectively, without cytotoxicity.

In subsequent analysis, the authors tried to determine whether 1C8 has the same activity in other HBV genotypes as the D-type used in all previous experiments by the authors. In dHepaRG cells, the authors found that 1C8 can inhibit the replication of C genotype HBV, and the viral RNA and all secreted proteins are significantly reduced (S9A figure).

The preliminary analysis of the other 5 HBV genotypes also showed that 1C8 can significantly reduce the secretion of HBsAg and HBeAg, especially for genotypes G and H, suggesting that its effect may be pan-genotypic.

These results indicate that 1C8 can inhibit HBV replication by reducing HBV RNA levels. The inhibitory effect of 1C8 on HBV RNA is opposite to that observed after SRSF10 KD, indicating that dephosphorylated SRSF10 exerts antiviral activity. In the KD experiment, siRNA transfection resulted in a decrease in dephosphorylated SRSF10, leading to the opposite phenomenon.

05 1C8’s antiviral effect partly depends on SRSF10, which promotes the reduction of HBV RNA level instead of RNA splicing

In order to verify whether the effect of 1C8 on HBV RNA accumulation is related to SRSF10, the authors combined SRSF10 KD and 1C8 treatments to conduct experiments (Figure 6A). According to the proposed model, the inhibitory effect of 1C8 on viral RNA production disappeared after depletion of SRSF10.

As previously observed, treatment with 1C8 or siSRSF10 alone has the opposite effect on HBV RNA levels. It is worth noting that in the cells receiving the two treatments, knocking down SRSF10 can partially rescue the inhibitory effect of 1C8, bringing it to a level similar to that observed in control cells, but not as high as in siSRSF10 transfected cells Detected level (Figure 6B and 6C).

This result indicates that the antiviral effect of 1C8 depends on SRSF10. In the cells treated with 1C8 and knocked down SRSF10, the incomplete recovery can be explained by the persistence of low levels of dephosphorylated SRSF10. Another possibility is that 1C8 also targets other cells and/or viral factors involved in antiviral effects.

HBV RNA that plays a role in virus replication is unspliced. However, in research, a variety of spliced ​​HBV mRNA has been detected, which indicates that if there is an active mechanism for splicing, then in chronic infection, this mechanism must be local and// Or invalid.

Among the numerous spliced ​​RNAs of HBV, there are two main forms of splicing that can produce new viral proteins, some of which may be involved in the pathogenesis of the virus. Virus particles containing shorter viral genomes have also been found in current research.

In the author’s previous analysis, the primers used to quantitatively detect HBV RNA (total RNA and pgRNA) were located in the unspliced ​​region of the HBV genome. Therefore, the changes in HBV RNA levels observed after SRSF10 KD or 1C8 treatment may be due to some splicing mutation. To explore this possibility, RNA extracted from transfected hepatocytes was analyzed using RT-qPCR, using primers that specifically detect each spliced ​​and unspliced ​​RNA.

Unexpectedly, there was a widespread increase in all HBV RNA variants in SRSF10 knockdown cells, especially in PHH. Similarly, after 1C8 treatment of HBV-infected dHepaRG cells, both spliced ​​and unspliced ​​viral RNA were significantly reduced. These results indicate that the promotion or antiviral effects of SRSF10 KD or 1C8, respectively, are not related to changes in spliced ​​or unspliced ​​HBV RNA levels.

Both of these treatment methods may have an effect on the synthesis and/or stability of HBV RNA. To verify this conjecture, the author quantified total RNA and newborn HBV RNA after SRSF10 KD or 1C8 treatment. The newly transcribed RNA was labeled with ethynyl uridine (EU) within 2 hours before the sample was collected to quantitatively detect the newborn HBV RNA (Figures 7A and 7B).

As expected, the total HBV RNA in dHepaRG cells increased in knocking down SRSF10 before HBV infection. In contrast, in cells transfected with siSRSF10, the newly transcribed HBV RNA increased, and its level was similar to the observed total RNA level (Figure 7C). The same analysis of 1C8-treated cells showed that the compound also reduced total HBV RNA and newborn HBV RNA (Figure 7D).

In summary, these analysis results indicate that SRSF10 and 1C8 did not change the splicing level of HBV RNA, but played a role by changing the stability of transcription and/or nascent viral RNA.

    
Figure 6. The effect of SRSF10 KD and 1C8 combined treatment on HBV-infected dHepaRG cells

Figure 7. Newborn HBV RNA after SRSF10 KD and 1C8 treatment

Discussion

The analysis of this study found that HBc can interact with a variety of RBPs in the nucleus, and these RBPs are involved in all stages of mRNA metabolism. In the hepatocytes of HBV-infected human liver chimeric mice, some of the above-mentioned protein interactions with HBc can also be found, verifying the findings of cell experiments. Both in vivo and in vitro experiments revealed that these RBPs played a corresponding role in the HBV replication process, and more relevant experiments can be performed in the future for further verification.

This study found that HBc can interact with a variety of RBPs in cells, suggesting that this interaction may be involved in the metabolic process of HBV RNA from multiple steps of transcription and post-transcription through a highly interconnected network of various proteins. HBc has similar characteristics to RBP, and it also has a positively charged carboxyl terminal (CTD) composed of a long arginine/serine-rich domain (RS domain) separated by 7 serine residues.

When HBc is expressed in bacteria, this special CTD shows strong RNA binding ability. It is worth noting that some studies have found that HBc also has the ability to bind DNA and can interact with cccDNA. There is no research to prove that HBc can bind to the virus and/or cellular RNA in the nucleus of HBV-infected liver cells, but like some RBPs, HBc may have the ability to bind to DNA and RNA.

Experimental results based on pgRNA assembly prove that this binding activity may be regulated by phosphorylation of its CTD. Another possibility is that the connection between HBc and cccDNA and/or RNA may be indirectly regulated by RNA molecules and/or their interaction with cellular RBP.

Among these RBPs that interact with HBc, this study focused on SRSF10, which has the highest content in the HBc complex. Previous studies have found that SRSF10 is a member of the SR protein family and a regulator of variable shearing. It can undergo dephosphorylation under heat shock treatment and can inhibit shearing.

Changes in the dephosphorylation level of SRSF10 can also control its interaction with a variety of RBPs, especially TRA2A, TRA2B, hnRNPK, F and H. Importantly, SRSF10 can affect the variable splicing of multiple transcripts in cells, which are involved in a variety of cell life activities such as stress response, DNA damage response, autophagy and cancer.

During the stress response process, SRSF10 can appear in paraspeckles, nuclear stress bodies and cytoplasmic stress granules. SRSF10 is also involved in the regulation of viral RNA, especially HIV-1.

Like all members of the SR protein family, SRSF10 consists of an N-terminal RNA recognition motif (RRM) and a C-terminal RS region responsible for binding to RBP. There are two common subtypes of SRSF10, the sizes are 37 and 20 kDa, and the smaller SRSF10 lacks the C-terminal region.

Co-immunoprecipitation analysis of human liver chimeric mouse liver infected with HBV found that HBc interacts with a new isoform of SRSF10. The size of this new SRSF10 is about 32 kDa. The analysis was performed using dHepaRG cells expressing HBc. The same result can be found. The specific characteristics of this new subtype of SRSF10 are still unclear, but it may be one of the nine protein variants produced by variable splicing of the pre-mRNA of SRSF10.

The researchers knocked down SRSF10 in differentiated hepatocytes and found that depletion of SRSF10 can cause an increase in viral RNA, protein and secreted DNA levels. This result can be effectively repeated in human primary hepatocytes and dHepaRG cells.

These results suggest that SRSF10 can inhibit the production of viral RNA. Although this study found that SRSF10 can interact with HBc, the role of this interaction in regulating the amount of overall viral RNA remains unclear. Future research should analyze the role of HBc in the occurrence of nuclear viral RNA and how HBc resists the inhibitory effect of SRSF10.

By using the phosphorylation inhibitor 1C8 of SRSF10 to treat HBV-infected dHepaRG cells and human primary hepatocytes, it can be found that the level of viral RNA is significantly reduced, thereby inhibiting HBV replication. This result further suggests the important role of SRSF10 in the HBV life cycle . The above experiments indicate that the dephosphorylated form of SRSF10 inhibits HBV replication.

The phosphorylation of the CTD serine residue of HBc is a very important step in the formation of nucleocapsid in the cytoplasm, which mainly regulates the process of pgRNA assembly and reverse transcription. Further research, if the type of kinases inhibited by 1C8 can be identified, will be of great help to the analysis of its mode of action and the development of new and efficient anti-hepatitis B drugs in the future.

Finally, this study found that, consistent with studies in other viruses, neither SRSF10 nor 1C8 changed the cleavage of HBV RNA. The data suggests that SRSF10 and 1C8 affect HBV replication by acting on newly generated HBV RNA.

SR protein has a variety of functions in the shearing process. In addition to some more classic functions, SR protein can play a role in the transcription, stability and nuclear process of viral RNA metabolism in cells. In addition to pre-mRNA cleavage, some SR proteins have been found to directly or indirectly affect the CTD phosphorylation of RNA polymerase II and stimulate transcription extension.

In the future, we can further study the epigenetic changes in the HBc/SRSF10 cccDNA/RNA binding process under 1C8 treatment, which will help understand the underlying mechanism.

In summary, the study revealed that HBc in the nucleus can bind to RBP in the cell, and found that SRSF10 is a limiting factor in the production of HBV RNA. Therefore, in the future, SRSF10-based antiviral compounds targeting the host can be further evaluated in order to improve the existing anti-HBV treatment regimens.

~~~~ Hepatitis B virus core protein interacts with host RNA binding protein SRSF10 to inhibit HBV RNA production ~~~~~

~~~~ Hepatitis B virus core protein interacts with host RNA binding protein SRSF10 to inhibit HBV RNA production ~~~~~

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