Quantifying the frequency and diversity of spliced HBV mRNAs in HIV-HBV co-infection and their role in modulating viral transcription and host immune responses

超过3.16亿人患有慢性乙肝，多达四分之一的艾滋病毒携带者 有乙肝病毒(乙肝病毒)混合感染。艾滋病毒和乙肝病毒混合感染增加了乙肝病毒复制水平，肝脏 疾病发展和患肝细胞癌的风险。乙肝病毒持久化的关键是核-- 驻留的、长寿的共价闭合环状DNA(CccDNA)，编码所有病毒蛋白。核(T)化物 类似疗法(NUC)通过阻断HBVDNA的逆转录而有效地降低血清中的HBVDNA 前基因组RNA(PgRNA)。最近，NUC治疗与cccDNA转录沉默有关。 导致肝脏中的pgrna水平和血清中的hbv rna水平降低，但cccDNA的数量 肝脏保持稳定。致力于开发治愈乙肝病毒的方法，重点是去除或长期控制cccDNA 活性，后者产生一种功能性治愈。另一种治疗肿瘤的方法是聚乙二醇化干扰素-α(聚乙二醇化干扰素)。 干扰素)，一种有效的抗病毒疗法，功能治愈率相对较高(7%)，定义为持久的 血清乙肝表面抗原(HBs)丢失。一项对受试者实施聚乙二醇化干扰素前后的跟踪研究发现 治疗无应答者的剪接HBVDNA(SpHBVDNA)占总HBVDNA的比例升高， 支持sp乙肝病毒在调节干扰素应答中的作用。有20个已鉴定的spHBV转录本 来源于pgRNA，它的一个子集编码非规范的乙肝病毒蛋白。有限数量的研究已经 证实spHBV2的表达干扰了干扰素的反应信号，并可能改变了cccDNA 抄写。鉴于它们在调节宿主免疫反应和病毒转录方面的假定作用，我们 建议将spHBV单感染和HIV-HBV二重感染进行比较。结合使用一部小说 多探针多重液滴数字聚合酶链式反应直接测序，我们将表征在HIV-HBVCo. 在MACS/WIHS联合队列研究中，个体的血清感染和单一乙肝病毒感染。我们 预计与单一感染相比，合并感染时spHBVRNA在总HBVRNA中的含量会有所增加。 此外，我们还将模拟HIV-HBVe混合感染中spHBv在NUC期间的衰变，并比较spHBVe 在同一个人的肝脏和血液中表达。此外，我们还将对肝细胞进行深度测序 乙肝病毒高转录与低转录在HIV-HBV二联感染中的辅助研究 网络，关注先天反应途径基因。然后我们将过度表达候选spHBVe 可能影响HepG2-NTCP细胞的干扰素应答。相反，我们将使用siRNA来击倒候选基因 检测乙肝病毒感染的HepG2-NTCP细胞的干扰素反应。在以下环境中使用此模型 ，我们将测量cccDNA转录、spHBv表达和先天感染之间的相互作用 发信号。这项研究将是艾滋病毒和乙肝病毒混合感染中spHBVs的第一个表征，目标是 寻找新的治疗靶点，特异性地影响慢性乙肝患者的cccDNA转录。

Over 316 million people are living with chronic hepatitis B and as many as one quarter of people living with HIV have hepatitis B virus (HBV) co-infection. HIV-HBV co-infection increases levels of HBV replication, liver disease development, and the risk of hepatocellular carcinoma. The key to HBV persistence is nucleus- resident, long-lived covalently closed circular DNA (cccDNA) coding for all viral proteins. Nucleos(t)ide analogue therapy (NUC) effectively reduces HBV DNA in the serum by halting reverse transcription of pregenomic RNA (pgRNA). Recently, NUC therapy has been associated with cccDNA transcriptional silencing leading to reduced pgRNA levels in the liver and HBV RNA in the serum, however cccDNA quantities across the liver remain stable. Efforts to develop a cure for HBV focus on the removal or long term control of cccDNA activity, the latter yielding a functional cure. An alternative treatment to NUC is pegylated interferon-α (PEG- IFN), a potent antiviral therapy with comparatively greater rates of functional cure (7%), defined as a durable loss of serum HBV surface antigen (HBsAg). A study following participants before and after PEG-IFN found that treatment non-responders had elevated proportions of spliced HBV (spHBV) DNA from total HBV DNA, supporting a role of spHBV in modulating IFN responsiveness. There are 20 identified spHBV transcripts derived from pgRNA, a subset of which encode noncanonical HBV proteins. A limited number of studies have demonstrated that spHBV expression disrupts IFN response signaling and possibly alters cccDNA transcription. Given their putative role in modulating the host immune response and viral transcription, we propose to compare spHBV in HBV mono-infection and HIV-HBV co-infection. Combining use of a novel multiprobe multiplex droplet digital PCR with direct sequencing, we will characterize spHBV in HIV-HBV co- infection and HBV monoinfection in serum from individuals in the MACS/WIHS Combined Cohort Study. We expect an enrichment of spHBV RNA within total HBV RNA in co-infection compared to mono-infection. Additionally, we will model the decay of spHBV during NUC in HIV-HBV co-infection and compare spHBV expression in the same person’s liver and blood. Additionally, we will perform deep sequencing of hepatocytes with high vs low HBV transcription in an HIV-HBV coinfection ancillary study of the Hepatitis B Research Network, focusing on innate response pathway genes. We will then overexpress candidate spHBV that are likely to affect IFN responses in HepG2-NTCP cells. Conversely, we will use siRNA to knockdown candidate spHBV and measure IFN responses in an HBV infected HepG2-NTCP cells. Using this model in the context of HBV infection, we will measure the interplay between cccDNA transcription, spHBV expression, and innate signaling. This study will be the first characterization of spHBV in HIV-HBV co-infection and has the goal of identifying novel therapeutic targets specifically affecting cccDNA transcription in CHB.