DHX9 interacts with APOBEC3B and attenuates the anti-HBV effect of APOBEC3B

Hepatitis B virus (HBV) is a partially double-stranded DNA virus that replicates by reverse transcription. We previously demonstrated that the host restriction factor-APOBEC3B (A3B) inhibited HBV replication which was dependent on its deaminase activity during reverse transcription. However, the host factors involved in the process of regulating the anti-HBV function of A3B are less known. In this research, to obtain a comprehensive understanding of the interaction networks of A3B, we conducted coimmunoprecipitation and mass spectrometry to identify A3B-interacting proteins in the presence of HBV. By this approach, we determined that DExD/H-box helicase 9 (DHX9) suppressed the anti-HBV effect of A3B, and this suppression was dependent on their interaction. Although DHX9 did not affect the deamination activity of A3B <i>in vitro</i> assay or the viral DNA editing of A3B in HepG2-NTCP cells that support HBV infection, it inhibited the binding of A3B with pgRNA. These data suggest that DHX9 can interact with A3B and attenuate the anti-HBV efficacy of A3B. <b>Abbreviations:</b> 3D-PCR: differential DNA denaturation PCR; APOBEC3: apolipoprotein B mRNA-editing catalytic polypeptide 3; cccDNA: covalently closed circular DNA; co-IP: coimmunoprecipitation; DDX: DExD-box RNA helicases; HBc: HBV core protein; HBV: hepatitis B virus; HepAD38: HepG2 cell line stably transfected with HBV DNA; HepG2-NTCP: HepG2 cell line stably transfected with Na+/taurocholate cotransporter polypeptide; Huh7: human hepatoma cell line; pgRNA: pregenomic RNA; PPI: protein–protein interactions; RC DNA: relaxed circular DNA.

乙型肝炎病毒（Hepatitis B virus, HBV）是一种经逆转录过程复制的部分双链DNA病毒。本团队此前已证实，宿主限制因子载脂蛋白B mRNA编辑催化多肽3B（APOBEC3B, A3B）可通过其逆转录过程中的脱氨酶活性抑制HBV复制。然而，目前对于调控A3B抗HBV功能的宿主因子仍知之甚少。
本研究为全面解析A3B的相互作用网络，在HBV存在的细胞模型中通过免疫共沉淀（coimmunoprecipitation, co-IP）结合质谱技术筛选与A3B互作的蛋白。通过该方法，我们证实DExD/H盒解旋酶9（DExD/H-box helicase 9, DHX9）可通过与A3B的相互作用削弱其抗HBV效应。尽管在支持HBV感染的HepG2-NTCP细胞模型以及体外（in vitro）实验中，DHX9并未影响A3B的脱氨酶活性与病毒DNA编辑能力，但其可抑制A3B与前基因组RNA（pregenomic RNA, pgRNA）的结合。上述结果表明，DHX9可通过与A3B相互作用，削弱A3B的抗HBV功效。
**缩写说明：**
3D-PCR：差异DNA变性PCR（differential DNA denaturation PCR）
APOBEC3：载脂蛋白B mRNA编辑催化多肽3（apolipoprotein B mRNA-editing catalytic polypeptide 3）
cccDNA：共价闭合环状DNA（covalently closed circular DNA）
co-IP：免疫共沉淀（coimmunoprecipitation）
DDX：DExD盒RNA解旋酶家族（DExD-box RNA helicases）
HBc：HBV核心蛋白（HBV core protein）
HBV：乙型肝炎病毒（Hepatitis B virus）
HepAD38：稳定转染HBV DNA的HepG2细胞系
HepG2-NTCP：稳定表达钠-牛磺胆酸共转运多肽的HepG2细胞系
Huh7：人肝癌细胞系
pgRNA：前基因组RNA（pregenomic RNA）
PPI：蛋白质-蛋白质相互作用（protein–protein interactions）
RC DNA：松弛环状DNA（relaxed circular DNA）