Characterization of silently HBV-infected hepatocytes in HIV co-infection

慢性B型肝炎（CH B）影响着全球近4亿人，每年约有100万人死于 肝病和肝细胞癌。HIV-1/B型肝炎病毒（HBV）合并感染很常见。HIV-1 增加CHB的肝病进展，肝病是HIV-1死亡的主要原因 接受抗逆转录病毒治疗（ART）的感染者。因此，需要治疗CHB。虽然双重活跃 ART（DAART）控制HBV和HIV-1病毒血症，它不能治愈CHB，因为它不能根除稳定的 HBV的共价闭合环状DNA（cccDNA）形式，复制的模板，来自肝细胞。我们 初步数据表明，一些肝细胞含有cccDNA，但具有有限的病毒转录（没有 前基因组RNA（pgRNA）），我们将其定义为转录“沉默”HBV感染的肝细胞（tsHBiH）。 这些细胞可能在功能上暂时“治愈”，但也可能是重新激活的细胞。理解 转录沉默的机制可以阐明细胞内的过程，可以利用转换 转录活性HBV感染的肝细胞（taHBiH）转化为tsHBiH，并可能导致功能性治愈。 我们建议通过用单细胞激光捕获法解剖单个肝细胞来比较tsHBiH和taHBiH 显微切割（scLCM）从一个已建立的队列的21个HIV-HBV合并感染的人谁有长期的， 在DAART期间的两个时间点用存档的肝组织用DAART长期控制HBV。在目标1中， scLCM和数字液滴PCR（ddPCR）将用于定量数千例HBV复制形式， 来自我们组群的单个肝细胞以确定tsHBiH或taHBiH的比例。这一目标进一步 测试HIV-1相关的免疫失调如何影响这些细胞的负担。在aim 2中，我们测试 细胞内先天免疫分子通过定量细胞内先天免疫mRNA来维持tsHBiH。我们 还测试替代机制，例如缺陷病毒或cccDNA或其他病毒的表观遗传沉默 因素在目的3中，我们使用成对的肝活检来比较tsHBiH和taHBiH的下降率，并测试 tsHBiH是否持续更长时间。我们还确定了HIV-1相关的免疫失调是否会影响下降率。 该提案的创新方面包括scLCM和ddPCR。我们的研究将首次提供 cccDNA和pgRNA在人类单个肝细胞中的表达，并揭示HBV转录的潜在机制。 相关性 HBV是世界范围内肝脏疾病的主要原因，也是HIV-1感染者的重要合并感染。 个体药物可以控制慢性B型肝炎，但由于不影响稳定的 主要肝细胞（肝细胞）中的遗传病毒物质。我们的目标是了解肝细胞， 转录沉默感染（感染但不复制病毒）和沉默的机制， 研究来自HIV-HBV共感染者的数千个肝细胞的HBV病毒形式。我们还将 确定HIV-1免疫失调如何影响沉默感染肝细胞的比例。

Chronic hepatitis B (CHB) affects nearly 400 million people worldwide, with ~1 million annual deaths due to liver disease and hepatocellular carcinoma. HIV-1/hepatitis B virus (HBV) co-infection is common. HIV-1 increases liver disease progression from CHB with liver disease being a leading cause of mortality in HIV-1 infected persons taking antiretroviral therapy (ART). Thus, a cure for CHB is needed. Although dually active ART (DAART) controls HBV and HIV-1 viremia, it cannot cure CHB because it does not eradicate the stable covalently closed circular DNA (cccDNA) form of HBV, the template for replication, from the hepatocyte. Our preliminary data demonstrate that some hepatocytes contain cccDNA but have limited viral transcription (no pregenomic RNA (pgRNA)), which we define as transcriptionally `silent' HBV-infected hepatocytes (tsHBiH). These cells may be functionally `cured' temporarily, but may also be the cells that reactivate. Understanding mechanisms of transcriptional silencing can elucidate intracellular processes that can be exploited to convert transcriptionally active HBV-infected hepatocytes (taHBiH) to tsHBiH and potentially lead to a functional cure. We propose comparing tsHBiH and taHBiH by dissecting single hepatocytes with single-cell laser capture microdissection (scLCM) from an established cohort of 21 HIV-HBV co-infected people who have long- standing control of HBV with DAART with archived liver tissues at two time points during DAART. In aim 1, scLCM and digital droplet PCR (ddPCR) will be used to quantify HBV replicative forms in thousands of individual hepatocytes from our cohort to determine the proportion that are tsHBiH or taHBiH. This aim further tests how HIV-1 associated immune dysregulation affects the burden of these cells. In aim 2, we test if intracellular innate immune molecules maintain tsHBiH by quantifying intracellular innate immune mRNAs. We also test alternative mechanisms such as defective virus or epigenetic silencing of cccDNA, or other viral factors. In aim 3, we use the paired liver biopsies to compare the decline rates of tsHBiH and taHBiH and test whether tsHBiH persist longer. We also determine if HIV-1 related immune dysregulation affects decline rates. Innovative aspects of this proposal include scLCM and ddPCR. Our research will provide the first quantitations of cccDNA and pgRNA in single hepatocytes in humans, and reveal mechanisms underlying HBV transcription. Relevance HBV is the leading cause of liver disease worldwide and is an important co-infection in HIV-1 infected individuals. Medicines can control chronic hepatitis B but are lifelong because they do not affect the stable genetic viral material in the principal liver cell, the hepatocyte. We aim to understand hepatocytes that are transcriptionally silently-infected (infected but not replicating virus) and the mechanisms of silencing by studying HBV viral forms from thousands of hepatocytes from HIV-HBV co-infected people. We will also determine how HIV-1 immune dysregulation affects the proportion of silently-infected hepatocytes.