在自然感染鸭乙型肝炎病毒的cccDNA扩增效率是由病毒分泌效

StephenW

PLoS One. 2015 Dec 29;10(12):e0145465. doi: 10.1371/journal.pone.0145465. eCollection 2015.
Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency.
Zhang YY1.
Author information

    1HBVtech, Germantown, Maryland, United States of America.

Abstract

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.

PMID:
    26713436
    [PubMed - in process]

Free full text

Discussion

The purpose of this study was to investigate cccDNA amplification in a cell culture system, which resembles natural hepadnaviral infection in which the synthesis of envelope proteins was not experimentally ablated and the experimental manipulation was kept at minimum.

The previous studies identified the envelope proteins as a regulatory molecule for cccDNA amplification [12, 15–18] in the mutation based system. However, it remains unknown how cccDNA amplification is regulated during natural infection. In humans, chronic HBV infection produces and maintains a huge pool of envelope proteins, the majority of which are presented as HBsAg subviral particles that also contain both M and L proteins in addition to S major component. The ratio of HBsAg subviral particle to HBV virion is about 1000–10,000:1 [21] and a stable serum HBsAg level can be as high as 10,000 ng/ml [39]. The presence of high HBsAg level makes regulation of HBsAg level inefficient. It raises a question whether HBsAg or other viral molecules including virions are used to regulate cccDNA amplification in natural HBV infection. This study demonstrates that virion secretion efficiency, but not cellular envelope proteins level, is used to regulate cccDNA amplification efficiency in natural hepadnaviral infection.

In infected cells with higher cccDNA amplification, we found that the relative virus secretion efficiency was significantly lower, while the converse was true in infected cells with lower cccDNA amplification. The direct impact of changed virus secretion efficiency is exerted on the intracellular rcDNA level. Reduced virus secretion leads to increased rcDNA accumulation in the cells while increased virus secretion leads to reduced intracellular accumulation of rcDNA.

We detected a higher portion of free nucleocapsids that were captured by the anti-DHBc antibody in the cells with higher cccDNA amplification efficiency, suggesting more available nucleocapsids for the intracellular pathway once the secretion efficiency is down-regulated. Our results also suggest that the intracellular rcDNA level is an important factor determining the destination of the synthesized rcDNA and occupies the central position in regulating cccDNA amplification. The higher intracellular rcDNA level the more rcDNA molecules are available for cccDNA amplification. The role of the intracellular rcDNA level in regulating cccDNA level is consistent with the observed impact of nucleos(t)ide analogues (NA) on cccDNA level in treated patients. The NAs do not directly inhibit the cccDNA synthesis, but they do impact the cccDNA level through lowering intracellular rcDNA level in inhibiting DNA replication. Approximately 100 fold and 98% reduction of intracellular viral DNA in the NA-treated livers was detected, respectively, which was accompanied by approximately 100 fold and 84% reduction of cccDNA level, respectively [23, 40]. Another study showed that median intrahepatic total HBV DNA and cccDNA were decreased by 2.2 and 2.4log10 at the end of 48 weeks of antiviral therapy, respectively [24].

We also found that the intracellular DHBsAg accumulation was higher in cells with higher accumulation of rcDNA and cccDNA amplification, and cells with lower cccDNA amplification had lower rcDNA and DHBsAg accumulation (Fig 4C). This finding that is opposite to the inverse relationship between the envelope proteins and cccDNA amplification level observed in the mutation based system, suggests that the cellular envelope proteins concentration is not the direct target of regulation, but the virion level is in WT infected cells. Nonetheless, this study provides a new clue to studying cccDNA regulation in natural infection and a simple approach to prepare two DHBV serum inocula that are identical in all aspects except cccDNA amplification.

Initially, we thought that DHBV virions were damaged at 4 C storage. However, when a small volume (150 μl) of normal duck serum was added to the treated inoculum infected cells, both replicative intermediates and cccDNA were restored to the comparable levels detected in the standard inoculum infected cells. This observation led to the conclusion that the functionality of virions was not significantly compromised at 4C. A possible reason for reduced cccDNA amplification was that serum component(s) that may involve in down-regulating the efficiency of virion secretion, was damaged or degraded at 4C storage, because an inclusion of small volume of the normal duck serum can restore the cccDNA amplification efficiency.

Our data suggest that the virion and DHBsAg secretion efficiency from infected cells is subjected to regulation by an unknown serum factor, which may bind to the cellular membrane and negatively affect secretion of virions and subviral particles (Fig 4C) though the mechanism for such down-regulation is not known this time. The capacity to restore cccDNA amplification varies with different normal duck serum added (Fig 3B). Thus regulation of cccDNA amplification in WT infected cells involves both the host and viral molecules. The excessive accumulation of envelope proteins in the cells is reported in clinical HBV infection [41]. For instance, the Ground-Glass cells can be detected in the livers of patients with chronic HBV infection. The cytopathic appearance of Ground Glass cell results from excessive accumulation of viral envelope proteins[42], which can be caused by increased synthesis of envelope proteins, but not accompanied by corresponding increase of secretion efficiency or accompanied by reduced secretion efficiency. Either scenario implicates the involvement of host factors.

Understanding of regulation of cellular membrane secretion efficiency is not only important for HBV secretion and cccDNA amplification, but also is valuable for illustrating why biological molecules like fat or bile acids can be excessively accumulated in the hepatocytes, which cause fatty liver disease or cholestasis. The serum factors involved in regulating the virus secretion can be identified with proteomic analysis in the future. We believe that publication of this study will facilitate collective efforts in the field to identify this serum factor.
Conclusions

In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. The regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.
Acknowledgments

StephenW

公共科学图书馆之一。 2015年12月29日; 10（12）：e0145465。 DOI：10.1371 / journal.pone.0145465。 eCollection 2015年。
在自然感染鸭乙型肝炎病毒的cccDNA扩增效率是由病毒分泌效率调控。
张YY1。
作者信息

    1HBVtech，日耳曼，马里兰，美利坚合众国。

抽象

以往的突变基础的研究表明，烧蚀合成导致升高的嗜肝病毒共价闭合环状DNA（cccDNA的）扩增病毒包膜蛋白的，但它仍然是未知的，因为缺乏研究系统如何cccDNA的放大被调节在天然嗜肝病毒感染。在这项研究中，我们报告一个简单的程序来制备两个完全相同的鸭乙型肝炎病毒接种物，但它们具有在cccDNA的扩增在感染的细胞培养10-100倍的差异。我们表明，感染的细胞具有较高的cccDNA扩增显著减少病毒分泌效率，其导致放松环状DNA（rcDNA）和DHBsAg在细胞中的更高的积累。感染的细胞具有较低的cccDNA扩增显著增加病毒分泌效率，导致降低细胞内rcDNA和DHBsAg积累。与在基础实验系统的突变而产生的结果相反，cccDNA的扩增在天然嗜肝病毒感染的调节绕过蜂窝包膜蛋白浓度的直接调节，而是调制病毒分泌效率，最终影响细胞内rcDNA浓度，一个重要的决定因素合成rcDNA的感染细胞的目的地。

结论：
    26713436
    [考研 - 过程]

免费全文

讨论

本研究的目的是调查在细胞培养系统，它类似于天然嗜肝病毒感染，其中包膜蛋白的合成未经实验烧蚀和实验操作保持在最低限度cccDNA的扩增。

在以往的研究中确定的包膜蛋白作为的cccDNA的突变为基础的系统的扩增[12，15-18]调控分子。但是，它仍然未知如何cccDNA的放大是自然感染过程中调节。在人类中，慢性HBV感染产生和维持一个庞大的包膜蛋白的池，其中大部分被作为HBsAg的亚病毒颗粒还包含M和L蛋白，除了至S主要成分。 HBsAg的亚病毒粒子对HBV病毒粒子的比例为约1000-10,000：1 [21]和一个稳定的血清HBsAg水平可高达10,000纳克/毫升[39]。高层次乙肝表面抗原的存在使得乙肝表面抗原水平的调控效率低下。它提出了一个问题，乙肝表面抗原或其他病毒分子，包括病毒粒子是否被用于调节天然HBV感染cccDNA的放大。这项研究表明，病毒颗粒分泌效率，但不是细胞包膜蛋白的水平，用于调节天然嗜肝病毒感染的cccDNA扩增效率。

在感染的细胞具有较高的cccDNA扩增，我们发现，相对病毒分泌效率显​​著降低，而相反的是在受感染的细胞具有较低的cccDNA扩增真。的改变的病毒分泌效率的直接影响作用在细胞内rcDNA水平。减少病毒分泌导致增加rcDNA积​​累在细胞内而增强的病毒分泌导致降低细胞内rcDNA的积累。

我们检测由抗DHBc抗体在细胞内具有较高的cccDNA扩增效率捕获的游离核壳一个较高部分，提示用于胞内途径，一旦分泌效率更可用的核壳是下调。我们的结果还表明，细胞内rcDNA水平的一个重要的决定因素，合成rcDNA的目的地，并占据在调节cccDNA的放大的中心位置。较高的细胞内水平rcDNA越rcDNA分子可用于cccDNA的扩增。在调节cccDNA的水平细胞内rcDNA水平的作用是与核苷对cccDNA的水平在治疗的患者中所观察到的影响（）类似物（NA）是一致的。在NAS不直接抑制cccDNA的合成，但他们通过降低细胞内rcDNA水平，抑制DNA复制的影响cccDNA的水平。大约100倍和98％减少的细胞内病毒DNA的NA处理的肝脏中检测到，分别，这是伴随着约100倍，84％减少的cccDNA的水平，分别[23，40]。另一项研究表明，平均肝内总HBV DNA和cccDNA的人在，48周抗病毒治疗的分别为[24]年末下降2.2和2.4log10。

我们还发现，细胞内DHBsAg积累在细胞rcDNA的更高的积累和cccDNA的扩增，并具有较低的cccDNA扩增细胞更高了较低rcDNA和DHBsAg积累（图4C）。这个发现，即相反在基于突变系统中观察到的包膜蛋白和cccDNA的放大电平之间的反比关系，表明细胞包膜蛋白浓度没有调节的直接目标，但病毒体水平是在野生型感染的细胞。然而，这项研究提供了新的线索来研究cccDNA的监管自然感染和一个简单的方法来准备两个鸭乙肝病毒血清接种是除了cccDNA的放大各个方面完全相同。

起初，我们认为DHBV病毒粒子被毁坏在4℃下贮存。但是，当正常鸭血清的小体积（150微升）加入到经处理的接种物感染的细胞，无论是复制中间体和cccDNA的被恢复到在标准接种物感染细胞中检测到的水平相当。这一观察结果导致了病毒体的功能并未被显著在4℃损害的结论。一个可能的原因降低cccDNA的扩增是血清组分可能涉及在下调病毒颗粒分泌的效率，被损坏或在4℃贮存降解，因为夹杂物量的正常鸭血清的小可以恢复cccDNA的扩增效率。

我们的数据表明，从感染的细胞的病毒粒子和DHBsAg分泌效率是通过一个未知血清因子，其可以结合到细胞膜和病毒体和亚病毒颗粒（图4C），虽然该机制对这种向下的分泌产生负面影响进行调节监管不知道这一次。恢复cccDNA的放大能力添加了不同的正常鸭血清（图3B）而异。因而cccDNA的扩增在WT感染细胞的调节包括主机和病毒的分子。包膜蛋白在细胞中过度累积报告在临床HBV感染[41]。例如，毛玻璃细胞可以在慢性HBV感染的肝脏中检测到。从病毒包膜蛋白[42]，其可以通过包膜蛋白的合成增加引起，但不伴随相应的分泌效率的增加或伴有减少分泌效率的过度积累的细胞病变的外观磨砂玻璃细胞的结果。无论哪种情形暗示的宿主因子的参与。

理解的细胞膜分泌效率调节的是不仅对HBV分泌和cccDNA的扩增重要的，但也是有价值用于说明为什么生物分子，如脂肪或胆汁酸可以被过度累积在肝细胞，这引起脂肪肝疾病或胆汁淤积。参与调节病毒分泌的血清因素可以识别与在将来蛋白质组学分析。我们认为，公布这项研究将有助于在该领域的集体努力，以查明这一血清因素。
结论

在这项研究中，我们报告一个简单的程序来制备两个完全相同的鸭乙型肝炎病毒接种物，但它们具有在cccDNA的扩增在感染的细胞培养10-100倍的差异。我们表明，感染的细胞具有较高的cccDNA扩增显著减少病毒分泌效率，其导致放松环状DNA（rcDNA）和DHBsAg在细胞中的更高的积累。感染的细胞具有较低的cccDNA扩增显著增加病毒分泌效率，导致降低细胞内rcDNA和DHBsAg积累。 cccDNA的扩增在天然嗜肝病毒感染的调控绕过蜂窝包膜蛋白浓度的直接调节，而是调制病毒分泌效率，最终影响细胞内rcDNA浓度，一个重要的决定因素在感染的细胞中合成的rcDNA的目的地。