这项新研究提供了有关乙型肝炎病毒如何建立慢性感染的详

StephenW

New study provides detailed view of how hepatitis B virus establishes chronic infection

The research offers potential strategies for development of new treatments

Princeton University

Research News
IMAGE

IMAGE: Human liver cell lines (green, with blue nuclei) infected with HBV express an HBV protein (in red) in this image captured by immunofluorescence microscopy. view more

Credit: Image by Stephanie Maya, Princeton University

Researchers at Princeton have determined how five cellular proteins contribute to an essential step in the life cycle of hepatitis B virus (HBV). The article describing these findings appeared March 11, 2021 in the journal Nature Communications.

Viruses have been with us, shaping our lives, societies and economies for millennia. While some viruses rapidly explode onto the world stage, others smolder in our communities for decades, shattering lives but making few headlines. Hepatitis B virus hasn't caused any nationwide lockdowns or stock market crashes because it is slow to spread from person to person and is rarely immediately fatal. It is nonetheless incredibly damaging because it can establish lifelong chronic infection with profound consequences for its victims.

"An estimated two billion people have been exposed to HBV, of whom 250-400 million are chronically infected," said Alexander Ploss, associate professor of molecular biology at Princeton University, and senior author on the study. "Currently, there is no cure for chronic HBV infection, and patients need to be on a lifelong antiviral regimen. Approximately 887,000 individuals die each year from HBV-related liver diseases or liver cancer."

Ploss and his team are striving to understand HBV's life cycle in hopes of finding a way to prevent the virus from establishing damaging chronic infections.

"Central to HBV replication is the formation of covalently closed circular DNA (cccDNA) from relaxed circular DNA (rcDNA) which is carried into the host cell by the virus during the initial infection," Ploss said. "We have recently demonstrated that HBV relies on five host proteins - namely PCNA, RFC complex, POLδ, FEN-1, and LIG1 - that are necessary and sufficient for this conversion step."

As its name implies, rcDNA is a loop of DNA. DNA is a molecule made up of nucleotides arranged in linear fashion along paired, complementary strands. The sequence of nucleotides on one strand encodes the instructions for making a protein while the other strand is its mirror image. Whereas human cellular DNA contains over 20,000 genes, HBV's DNA genome only contains four. None of the viral proteins made from these genes is required for the conversion of rcDNA to cccDNA. Instead, the virus coopts cellular proteins to accomplish this and other steps of viral replication.

A key feature of HBV rcDNA is that each of its two strands contains a gap in its nucleotide sequence. One strand, called the plus strand, has a gap that is considerably larger than and offset from the gap on the other, minus strand. Cells perceive gaps in DNA as damage that needs to be filled in and repaired. The cellular proteins that carry out DNA repair can't tell the difference between viral DNA and cellular DNA, so they set to work "repairing" rcDNA as soon as it arrives in the nucleus. This repair process converts rcDNA into an intact circle of double-stranded DNA (that is, cccDNA) that can be maintained in the cell's nucleus.

Postdoctoral fellow Lei Wei wanted to understand how this repair process takes place in detail. To investigate this, he developed a method to monitor the process of repair taking place on rcDNA. He then identified what steps are involved in repair of each individual strand, tracked the order in which they are completed, and determined which cellular proteins are needed for each step.

The experiments showed that conversion of the plus strand into a continuous circle happens rapidly and requires all five of the cellular proteins working in concert. In contrast, repair of the minus strand requires only two of the five proteins (FEN-1 and LIG1) but is slower because there is a viral protein attached to one end of the minus strand that must be removed before the nucleotide gap can be sealed.

"In this paper, Wei and Ploss provide a compelling story in elucidating the cellular machinery that is essential for converting the incoming HBV genome into the cccDNA," said Dr. T. Jake Liang, a National Institutes of Health Distinguished Investigator in the Liver Diseases Branch who is an expert on HBV and related viruses. "This work offers not only important insights into the biochemical pathway of cccDNA biogenesis but also potential strategies to target cccDNA for therapeutic development."

Furthering that goal, the Princeton researchers showed that two compounds targeting cellular proteins could disrupt rcDNA conversion to cccDNA in test tubes. Wei and Ploss are hopeful that future studies will identify drugs that work in the human body.

"Our findings, biochemical approaches, and the novel reagents that we generated and engineered, open the door to providing an in depth understanding how this major human virus establishes persistence in host cells," Ploss said.

###

This work was supported in part by grants from the National Institutes of Health, the American Cancer Society, the Burroughs Wellcome Fund, the New Jersey Commission on Cancer Research, and Princeton University.

StephenW

这项新研究提供了有关乙型肝炎病毒如何建立慢性感染的详细视图

该研究提供了开发新疗法的潜在策略

普林斯顿大学

研究新闻
图像

图像：感染了HBV的人肝细胞系（绿色，带有蓝色核）在通过免疫荧光显微镜拍摄的该图像中表达HBV蛋白（红色）。查看更多

图片来源：普林斯顿大学Stephanie Maya摄

普林斯顿大学的研究人员已经确定了五种细胞蛋白如何在乙型肝炎病毒（HBV）的生命周期中发挥重要作用。描述这些发现的文章发表在2021年3月11日的《自然通讯》杂志上。

几千年来，病毒与我们息息相关，影响着我们的生活，社会和经济。尽管有些病毒迅速爆油炸到世界舞台上，但另一些病毒在我们的社区中燃烧了几十年，破坏了生命，但很少成为头条新闻。乙肝病毒尚未引起全国范围的封锁或股市崩溃，因为它在人与人之间传播的速度很慢，而且很少立即致命。尽管如此，它却具有令人难以置信的破坏性，因为它可以建立终生的慢性感染，对其受害者造成深远的影响。

普林斯顿大学分子生物学副教授，该研究的高级作者亚历山大·普洛斯说：“估计有20亿人感染了乙肝病毒，其中250-400亿是慢性感染。” “目前，尚无治愈慢性HBV感染的方法，患者需要接受终身抗病毒治疗。每年约有887,000人死于HBV相关的肝病或肝癌。”

Ploss和他的团队正在努力了解HBV的生命周期，希望找到一种方法来防止该病毒建立有害的慢性感染。

“ HBV复制的中心是从松弛的环状DNA（rcDNA）形成共价闭合的环状DNA（cccDNA），该病毒在最初感染时被病毒携带到宿主细胞中，” Ploss说。 “我们最近证明，HBV依赖于五种宿主蛋白​​-即PCNA，RFC复合物，POLδ，FEN-1和LIG1-这对于该转化步骤是必要和充分的。”

顾名思义，rcDNA是一个DNA环。 DNA是由核苷酸组成的分子，该核苷酸沿成对的互补链以线性方式排列。一条链上的核苷酸序列编码制造蛋白质的说明，而另一条链则是其镜像。人的细胞DNA包含20,000多个基因，而HBV的DNA基因组仅包含四个基因。 rcDNA转换为cccDNA不需要由这些基因制成的病毒蛋白。取而代之的是，病毒选择细胞蛋白来完成病毒复制的这一步骤和其他步骤。

HBV rcDNA的关键特征是其两条链的每一条在其核苷酸序列中均包含一个缺口。一个链（称为正链）的间隙比另一个（负链）的间隙大得多，并且与之偏移。细胞将DNA中的缺口视为需要填补和修复的损伤。进行DNA修复的细胞蛋白无法分辨病毒DNA和细胞DNA之间的差异，因此，一旦rcDNA到达细胞核，它们便开始“修复” rcDNA。这种修复过程将rcDNA转换成完整的双链DNA环（即cccDNA），该环可以保留在细胞核中。

博士后研究员雷伟（Ri Wei）希望详细了解修复过程。为了对此进行研究，他开发了一种方法来监测在rcDNA上发生的修复过程。然后，他确定了每一条链的修复涉及哪些步骤，跟踪了完成这些链的顺序，并确定了每个步骤需要哪些细胞蛋白。

实验表明，将正链转换成一个连续的圆会很快发生，并且需要所有五个细胞蛋白协同工作。相比之下，负链的修复仅需要五种蛋白质中的两种（FEN-1和LIG1），但修复速度较慢，因为负链的一端附着有病毒蛋白，在密封核苷酸间隙之前必须将其去除。 。

美国国立卫生研究院肝脏疾病杰出研究者T. Jake Liang博士说：“在本文中，Wei和Ploss提供了一个令人信服的故事，阐明了将传入的HBV基因组转化为cccDNA所必需的细胞机制。”是HBV和相关病毒专家的分支。 “这项工作不仅为cccDNA生物发生的生化途径提供了重要的见识，而且为靶向cccDNA的治疗开发提供了潜在的策略。”

为了实现这一目标，普林斯顿大学的研究人员表明，靶向细胞蛋白的两种化合物可能会破坏试管中的rcDNA转化为cccDNA。 Wei和Ploss希望未来的研究能够识别出在人体中起作用的药物。
“我们的发现，生化方法以及我们产生和设计的新型试剂，为深入了解这种主要人类病毒如何在宿主细胞中建立持久性打开了大门。”

###

这项工作得到了美国国立卫生研究院，美国癌症协会，巴勒斯惠康基金会，新泽西州癌症研究委员会和普林斯顿大学的部分资助。