Viral hijacking of the TENT4-ZCCHC14 complex protects viral RNAs via mixed tailing [TAIL-seq]

TENT4 enzymes generate “mixed tails” on mRNAs by adding adenosines with intermittent non-adenosine residues, which protect mRNAs from deadenylation. Here we discover the extensive mixed tailing in the transcripts of two distinct DNA viruses, hepatitis B virus (HBV) and human cytomegalovirus (HCMV), and their striking similarity in the mechanism to exploit the TENT4-ZCCHC14 complex. TAIL-seq analyses of HBV and HCMV RNAs revealed that TENT4A and TENT4B are responsible for the mixed tailing and protection of viral poly(A) tails. By fCLIP-seq on HBV-producing cells, we found that the HBV posttranscriptional regulatory element (PRE) is the primary site of the TENT4 interactome. A hairpin with a CNGGN-type pentaloop in the PRE is critical for TENT4-dependent regulation. Unexpectedly, HCMV also utilizes a similar pentaloop hairpin, an interesting example of convergent evolution. We further found that the CNGGN pentaloop is recognized by the SAM domain-containing ZCCHC14 protein which in turn recruits TENT4 to stabilize viral transcripts. Altogether, our study reveals the action mechanism of PRE that has been widely used to enhance gene expression, and introduces the TENT4-ZCCHC14 complex as the key host factor and a potential target for antiviral therapeutics. HepG2.2.15 cells and HFF cells were knocked down of control or TENT4

TENT4酶（TENT4 enzymes）可通过在腺苷酸残基之间插入非腺苷酸残基，在信使RNA（mRNA）上形成“混合尾”，从而保护mRNA免于脱腺苷酸化（deadenylation）。本研究首次发现，乙型肝炎病毒（HBV）与人巨细胞病毒（HCMV）这两种不同DNA病毒的转录本中存在广泛的混合尾修饰现象，且二者在利用TENT4-ZCCHC14复合物（TENT4-ZCCHC14 complex）的机制上具有显著相似性。通过对HBV与HCMV的RNA进行尾端测序（TAIL-seq）分析，我们证实TENT4A与TENT4B负责介导病毒多聚腺苷酸尾（poly(A) tails）的混合尾修饰与保护作用。针对产HBV细胞的甲醛交联免疫沉淀测序（fCLIP-seq）结果显示，HBV转录后调控元件（PRE）是TENT4互作组的主要结合位点。PRE中带有CNGN型五环（pentaloop）的发夹结构，对TENT4依赖型调控至关重要。令人意外的是，HCMV同样采用了类似的五环发夹结构，这是一个典型的趋同进化（convergent evolution）案例。我们进一步研究发现，CNGN型五环可被含SAM结构域（SAM domain）的ZCCHC14蛋白识别，后者可招募TENT4以稳定病毒转录本。综上，本研究阐明了长期被用于增强基因表达的PRE的作用机制，并揭示TENT4-ZCCHC14复合物作为关键宿主因子，有望成为抗病毒治疗的潜在靶点。本研究中，我们对HepG2.2.15细胞与HFF细胞分别进行了对照敲低或TENT4靶向敲低处理。