NEDD4-binding protein 1 suppresses HBV replication by degrading pgRNA

Chronic infection with hepatitis B virus (HBV) places patients at increased risk for liver cirrhosis and hepatocellular carcinoma. Although nucleos(t)ide analogs are mainly used for the treatment of HBV, they require long-term administration and may lead to the emergence of drug resistant mutants. Therefore, to identify targets for the development of novel anti-HBV drugs, we screened for HBV-suppressive host factors using a plasmid expression library of RNA-binding proteins (RBPs). We screened 132 RBPs using an expression plasmid library by measuring HBV relaxed circular DNA (rcDNA) levels in hepatocellular carcinoma. After we identified one RBP which suppressed rcDNA, the domain-deficient mutants were generated to determine the region contributing to the anti-HBV effect. Also, we measured pregenomic RNA (pgRNA) and covalently closed circular DNA (cccDNA) level in the RBP transfected cells and confirmed the degradation of pgRNA by Northern blotting. Our screen identified NEDD4-binding protein 1 (N4BP1) having an anti-HBV effect. In hepatocellular carcinoma cell lines transfected or infected with HBV, overexpression of N4BP1 decreased rcDNA levels while knockdown or knockout of N4BP1 expression rescued rcDNA levels. We found that both the KH-like and RNase domains of N4BP1 were required for the protein’s anti-HBV effect. N4BP1 suppressed HBV replication by promoting pgRNA, PreS1 and PreS2/S degradation. In summary, we found that N4BP1 is a newly identified host factor able to counteract HBV production by reducing HBV RNA levels. Author summary There is still a large number of HBV-infected people in the world today because of no curative treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to identify new host factors which inhibit HBV replication. As a result, we found that NEDD4-binding protein 1 (N4BP1) expression suppresses rcDNA production by promoting the degradation of pregenomic RNA, 2.4kb and 2.1kb HBV RNA. Furthermore, KH-like domain or RNase domain of N4BP1 were involved in this anti-HBV effect. In addition, the N4BP1 levels were lower in HCC resection samples of exacerbated patients, suggesting that individual N4BP1 levels might be related to HCC progression. This novel factor can potentially become a key to new HBV treatments.

levels while knockdown or knockout of N4BP1 expression rescued rcDNA levels. We found that both 48 the KH-like and RNase domains of N4BP1 were required for the protein's anti-HBV effect. N4BP1 49 suppressed HBV replication by promoting pgRNA, PreS1 and PreS2/S degradation. In summary, we 50 found that N4BP1 is a newly identified host factor able to counteract HBV production by reducing 51 HBV RNA levels.

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Author summary

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There is still a large number of HBV-infected people in the world today because of no curative 55 treatment for HBV infection. In this study, we focused on and screened RNA-binding proteins to 164 are needed to suppress HBV replication, we constructed deletion mutants lacking either the KH-like 165 (ΔKH; deletion of amino acids 59-143) or the RNase domain (ΔRNase; deletion of amino acids 617-166 769).

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The plasmid encoding wild-type or mutant N4BP1 was respectively co-transfected into naïve 168 Huh7 cells with the plasmid containing the 1.3-fold-overlength genome of HBV genotype C and the 169 rcDNA levels measured. The expression of the deletion mutants was confirmed by WB using an HA-170 tagged antibody (Fig 5B,upper). The rcDNA level in N4BP1 overexpressing cells was significantly 171 reduced, while the rcDNA levels in the ΔKH-or ΔRNase-expressing cells were comparable to those 172 in the control cells (Fig 5B, lower).

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Residue D623 in the RNase domain of N4BP1 ( Fig 5A) has been shown to be important for the 174 protein's RNase activity [20]. A D623N substitution causes loss of RNase activity without changing 175 the structure of N4BP1. Co-transfecting the Huh7 cells with a plasmid expressing this N4BP1 D623 176 mutant and the HBV overlength genome also did not lead to a decrease in rcDNA levels ( Fig 5C).

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These results suggest that both the KH-like and RNase domains are critical for the anti-HBV effect of 178 N4BP1.

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Having shown the effect of N4BP1 on rcDNA, we then sought to determine more specifically 11 182 which step of HBV replication N4BP1 suppresses. To that end, we assessed the impact of N4BP1 183 overexpression on cccDNA and pgRNA levels. HepG2-hNTCP C4 cells were infected with HBV and 184 cells were harvested at 14 dpi. While cccDNA levels were not affected by overexpression of N4BP1, 185 pgRNA and rcDNA levels were both significantly reduced (Fig 6A-6C).

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For further verification, we performed the same experiments using PHHs. Adenovirus vectors 187 were used to overexpress N4BP1 in PHHs (Fig 6D), and the cells were then infected with HBV 188 genotype C. As shown in Fig 6E-6G, even in PHHs, cccDNA levels were comparable to control cells, 189 but pgRNA and rcDNA levels were both significantly decreased. Collectively, these results indicate 190 that N4BP1 augmented pgRNA degradation.

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To elucidate the mechanism of pgRNA suppression by N4BP1, we turned to HepAD38.7 cells.

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First, the cells were cultured for 2 days in media without tetracycline to induce HBV replication. Then

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N4BP1 or the domain deletion mutant plasmids were transfected, and tetracycline added at 2 dpt to 194 stop viral replication. Cells were collected starting 2 days after adding tetracycline to measure the 195 pgRNA levels ( Fig 6H). As shown in Fig 6I, Fig 6J).

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It has been reported that N4BP1 expression is increased 3-to 5-fold with IFN-α treatment in

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Jurkat cells and THP-1 cells [20]. To see if this was the case in PHHs and hepatoma cell lines, we 208 treated Huh7, HepG2-hNTCP C4, and PHHs with 1000 U/ml of IFN-α and 100 ng/ml of IFN-λ, which 209 are known to also inhibit HBV replication. Cells were collected at 24 and 48 hours post-drug treatment 210 and N4BP1 was examined by qPCR and WB ( Fig 7A). The ISG15 was included as a positive control.

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As shown in Fig 7B and 7C, levels of N4BP1 mRNA as measured by qPCR were slightly increased 212 in both Huh7 and HepG2-hNTCP C4 cells by IFN-α or IFN-λ. WB also showed that IFN-α or IFN-λ 213 treatments had no effect on N4BP1 protein levels in Huh7 cells and HepG2-hNTCP C4 cells. the step of pgRNA production in the HBV life cycle (Fig 6B, 6F, 6I). We also found that N4BP1

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suppresses not only the 3.5 kb RNA of HBV, but also the 2.4 kb and 2.1 kb RNAs by NB (Fig 6J),

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N4BP1 is known to be an inhibitor of innate immunity and inflammation-mediated cytokine 248 production. NF-κB is an important transcription factor for the elicitation of cytokine responses, and 249 its activation is necessary for many immune responses. N4BP1 interacts with the NF-κB signal 250 essential modulator (NEMO, also known as IκB kinase γ) to suppress Toll-like receptor (TLR)-

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The production of HBV particles (genotype D) was adapted from a previous report [39]. Briefly,

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HepAD38.7 cells were cultured under tetracycline (400 ng/mL) for maintenance. To produce HBV 376 particles, tetracycline was removed and the supernatant collected after 6 and 9 days. The collected 377 supernatant was mixed with 30% polyethylene glycol (PEG) 8000 (Sigma) and incubated overnight 378 at 4℃. Then it was spun at 3,000 rpm for 20 minutes at 4℃ for concentration. Before HBV infection,

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At 2 days post-transduction, the cells were infected with 5,000 genome equivalents (GEq)/cell of HBV

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The following antibodies were used in this study: anti-N4BP1 (Bethyl Laboratories, TX, USA),