HDV RNA在細胞核與細胞質中的複製動態

Abstract

丁型肝炎病毒(Hepatitis delta virus, HDV)是一種需要乙型肝炎病毒(Hepatitis B virus, HBV)提供包膜蛋白的負股RNA病毒。由於HDV本身不具備病毒專屬的聚合酶，因此其基因體複製需依賴宿主細胞的RNA聚合酶。與其他負股RNA病毒相似，HDV必須先將其基因體轉錄為messenger RNA (mRNA)，才能產生病毒蛋白；但不同於典型的負股RNA病毒，HDV並不攜帶自身的RNA依賴性RNA聚合酶(RNA dependent RNA polymerase)，而是利用宿主的聚合酶進行轉錄與複製。 雖然HDV一般被認為是在細胞核中進行複製，然而也有部分研究指出其複製中間產物反基因體RNA也會在細胞質中出現，然而，HDV是否能在細胞質中複製，目前尚未被充分研究。本研究旨在探討HDV是否可於細胞質中進行複製，以及其是否可能包裹宿主聚合酶以協助此一過程。透過HDV感染的Huh7-NTCP細胞進行核質分離，結果發現HDV的基因體與反基因體RNA，以及丁型肝炎抗原(HDAg)皆可在細胞質與細胞核中被偵測，顯示HDV在細胞質中可能具備複製能力。為了進一步驗證此假說，我們純化HDV病毒顆粒並進行等密度梯度離心。雖然質譜分析(LC-MS/MS)未能確認RNA polymerase II subunit RPB1與HDV病毒顆粒的結合，但Western blot與Northern blot分析顯示病毒成分與RPB1出現在相同分離層，暗示RPB1可能與HDV存在某種關聯。然而，類似的共分布現象也出現在HBV病毒顆粒與次病毒顆粒中，顯示此種關聯可能僅為非特異性結合。此外，使用抗HBsAg抗體進行的免疫沉澱實驗未能成功拉下病毒蛋白，因此無法進一步證實病毒顆粒與RPB1之間的關聯。 綜上所述，HDV病毒顆粒可能與宿主聚合酶存在關聯，但其特異性及生物學意義仍需更深入探討。

Hepatitis delta virus (HDV) is a negative-strand RNA virus that relies on hepatitis B virus (HBV) for envelope proteins supply. Since HDV lacks its own polymerase, it utilizes the host RNA polymerase for genome replication. Like other negative-strand RNA viruses, HDV must transcribe its genome into messenger RNA (mRNA) to produce viral proteins. However, unlike typical RNA viruses, it uses host polymerases instead of encoding its own. Although HDV is generally considered to replicate in the nucleus, some evidence suggests that its replication intermediate—antigenomic RNA—may also be present in the cytoplasm. However, this aspect of HDV replication has not been thoroughly explored. In this study, we investigated whether HDV replicates in the cytoplasm and whether it encapsulates host polymerases to support this process. Through nuclear and cytoplasmic fractionation of HDV-infected Huh7-NTCP cells, we detected both genomic and antigenomic HDV RNAs, as well as hepatitis delta antigen (HDAg), in both the cytoplasmic and nuclear compartments. These findings suggest the possibility of cytoplasmic HDV replication. To further examine this hypothesis, HDV virions were purified and subjected to isopycnic density gradient centrifugation. Although LC MS/MS failed to confirm the association between RNA polymerase II subunit RPB1 and virions, Western blot and Northern blot analyses revealed that viral components co-fractionated with RNA polymerase II subunit RPB1. This raised the hypothesis that HDV virions may encapsulate host polymerase. However, similar co-localization patterns were also observed in HBV virions and subviral particles, suggesting the possibility of non-specific association. Moreover, immunoprecipitation assays using an anti-HBsAg antibody failed to pull down viral proteins, leaving the association between virions and RNA polymerase II-RPB1 unconfirmed. Together, these results provide biochemical evidence suggesting a potential association between HDV virions and host polymerase, though further studies are needed to determine the specificity and functional relevance of this interaction.