【作者】 李建华；

【导师】 袁正宏； 顾建新； 谢幼华；

【作者基本信息】 复旦大学 ， 病原生物学， 2010， 博士

【摘要】 乙型肝炎病毒(Hepatitis B virus, HBV)属于嗜肝DNA病毒科(Hepadnaviradae),具有独特的基因组结构及生物学特性。由HBV感染引起的乙型肝炎是严重危害人类健康的疾病。目前,临床上针对HBV感染的抗病毒药物主要为核苷类似物和干扰素-α(Interferon-α, IFN-α)。IFN-α的抗HBV的机制以及相关的干扰素刺激基因(IFN-stimulated genes, IS Gs)的生物学功能尚未完全阐明。我们和其他实验室以前的研究结果显示,髓样细胞分化蛋白88(Myeloid differentiation primary response protein 88, MyD88)的表达能被干扰素诱导,并且在肝肿瘤细胞中能通过激活NF-κB抑制HBV的复制。与此同时,HBV编码的聚合酶蛋白通过抑制stat1的入核下调MyD88启动子活性从而抑制干扰素诱导的MyD88的表达。这些结果提示,MyD88可能在干扰素抑制HBV复制中发挥关键作用。但是MyD88的抗病毒机制仍不甚明了,有必要进一步研究其详细的作用机制。为了进一步验证MyD88的抗病毒作用,首先用MyD88重组腺病毒(Ad-MyD88)感染有稳定HBV复制的HepG2.2.15细胞,然后用Northern-blot和Southern-blot检测病毒RNA和病毒核心颗粒相关DNA水平。结果显示,与对照相比,Ad-MyD88感染同等程度地降低了病毒RNA和核心颗粒相关DNA的水平,提示MyD88抗病毒作用的一级靶位点可能是病毒RNA,这一点随后用只能进行病毒RNA的转录而不能进行病毒基因组复制的pCIdA-HBV所证实。此外在通过小鼠尾静脉高压注射HBV复制质粒建立的急性感染模型中,MyD88也具有与在体外相似的抗病毒作用。为了探讨是否MyD88在生理条件下也能发挥抗病毒作用,将MyD88 siRNA转入Huh7细胞,然后测定IFN-α的抗病毒活性。结果显示,在MyD88 knock-down细胞中,HBV对IFN-α的抗病毒作用有了一定的抵抗能力,提示MyD88在IFN-α抑制HBV复制中发挥一定作用。MyD88能直接下调病毒RNA水平,这可能发生在转录水平,也可能发生在转录后水平。结果显示,MyD88对由HBV启动子和增强子调控的荧光素酶的活性没有显著影响,但是能抑制受CMV启动子启动的pregenomic RNA的水平。这些结果提示,MyD88下调病毒RNA水平是一转录后水平事件。为了进一步研究MyD88转录后下调pregenomic RNA的机制,利用Tet-off系统研究MyD88对pregenomic RNA稳定性的影响。结果显示MyD88可以降低pregenomic RNA的稳定性。进一步通过核浆分离显示,MyD88降低了pregenomicRNA在胞浆中的稳定性,但是对胞核中pregenomic RNA的稳定性没有影响。RNA干扰实验表明,MyD88对pregenomic RNA在胞浆中的降解是通过细胞中的两条mRNA降解通路即5’-3’mRNA降解通路和3’-5’mRNA降解通路进行。细胞蛋白La可与HBV pregenomic RNA特异结合促进pregenomic RNA的稳定性。但是本研究显示,MyD88诱导pregenomic RNA的降解不依赖于La蛋白与pregenomic RNA的相互作用。进一步通过mapping分析发现HBV(1804-2454)和HBV(1151-1684)是MyD88的应答序列。随后以pregenomic RNA为背景,构建HBV(1804-2454)和HBV(1151-1684)的缺失突变体,然后测定它们对MyD88的敏感性。结果显示,HBV(1151-1684)的缺失突变体仍然保留着对MyD88的应答能力,而HBV(1804-2454)的缺失突变体丧失了对MyD88的敏感性,表明位于HBV pregenomic RNA 5’末端的HBV(1804-2454)序列为MyD88诱导pegenomic RNA降解的一个关键的顺式作用序列。RNA序列HBV(1151-1684)位于HBV pregenomic RNA和preS/S RNAs的重叠区域,因此它可能选择性赋予了preS/S RNAs对MyD88的敏感性。结果显示,RNA序列HBV(1151-1684)选择性介导了HBV preS/S RNA的降解,并且这种降解主要发生在核内。有趣的是RNA序列HBV(1151-1684)与HBV转录后调控元件(posttranscriptional regulatory element, PRE)完全重叠。HBVPRE选择性介导preS2/S RNAs出核,不能出核的RNA最后以一种未知的机制在核中降解。因此MyD88导致preS/S RNAs在核中降解可能是由于MyD88干扰了PRE介导的preS/S RNAs出核所导致。通过CAT活性分析显示,MyD88可以降低PRE介导的CAT活性,并且不是由于MyD88直接导致其在核中降解所造成,因为在PRE出核抑制蛋白NES(-)RanBP1共表达的条件下,MyD88不能进一步抑制CAT的活性,而PRE的核输出因子多嘧啶茎结合蛋白(polypyrimidne tract-binding protein, PTB)的表达几乎完全恢复了PRE促进出核的功能。随后通过核浆分离用Northern-blot证实了CAT活性分析的结果。因此,从以上结果可以得出MyD88抑制了PRE介导的preS2/S RNAs出核。为了进一步研究MyD88抑制PRE介导的preS/S RNAs出核机制,检测了PTB的表达水平。结果显示MyD88可以同等程度地下调PRE核输出因子PTB的蛋白水平和mRNA水平,提示MyD88对PTB表达的抑制发生在PTB mRNA水平。进一步检测MyD88对PTB mRNA稳定性的影响发现,MyD88对PTBmRNA稳定性没有明显影响,提示MyD88对PTB表达水平的抑制是一转录水平事件。总之,本研究进一步探讨了MyD88的抗病毒作用和机制,对该机制的阐明将有助于新的治疗乙型肝炎的药物的开发。更多还原

【Abstract】 The hepatitis B virus (HBV) belongs to the family of Hepadnaviradae with a unique genome structure and life cycle. Hepatitis B is the most common serious liver infection in the world. Current antiviral therapies involve the use of nucleoside analogs and interferon-alpha (IFN-a). Importantly, the precise antiviral mechanism of IFN-a and the biological functions of many IFN-stimulated genes (ISGs) have not been fully elucidated. We and others have shown that the expression of Myeloid differentiation primary response protein 88 (MyD88) can be induced by IFN-a, and that MyD88 has an antiviral activity against HBV in hepatoma cells that is mediated by nuclear factor-kappaB (NF-κB) activation. To counteract its inhibition, the HBV polymerase dampens activation of the MyD88 promoter by blocking nuclear translocation of Statl, thereby reducing IFN-a-inducible MyD88 expression, further suggesting a critical role for MyD88 in antiviral activity against HBV. The aim of the present study was to further investigate the antiviral activity of MyD88 and the mechanism of action.To determine the effect of MyD88 on established HBV replication, a cell line stably transformed with replicating HBV genomic DNA, HepG2.2.15, was infected with adenovirus expressing MyD88 (Ad-MyD88). The amount of viral RNA and core particle-associated DNA were determined by northern and southern blot analyses, respectively. The results showed that viral DNA levels were decreased to the same extent as viral RNA levels by Ad-MyD88 infection, suggesting that the main primary antiviral target of MyD88 was most likely the viral RNA, which was verified using using the pCIdA-HBV construct, which is capable of viral gene expression and incapable of viral geneome replication. In addition, consistent with in vitro results, MyD88 significantly reduced the levels of viral core particle-associated DNA and RNA in a mouse model of acute HBV infection established using hydrodynamic-based transfection.To investigate the antiviral activity of MyD88 in physiological condition, Huh7 cells were transfected with siRNA targeting MyD88, and the antiviral activity of IFN-a was tested. The results showed that HBV showed resistant to IFN-a treatment in the MyD88 knock-down cells to some extent, suggesting that MyD88 plays an active antiviral role in the IFN-a-mediated inhibition of HBV replication.To determine whether this inhibition occurs transcriptionally or posttranscriptionally, we first employed reporter plasmids in which the luciferase reporter gene was under control of HBV promoters/enhancers. The results showed that MyD88 had little inhibitory effect on the activity of the viral promoters/enhancers. But MyD88 inhibited the expression of pregenomic RNA from pCMV-HBV in which the transcription of viral pregenomic RNA is under control of CMV promoter. All these results suggest that MyD88 posttranscriptionally reduces the levels of HBV RNA.Because the inhibition of pregenomic RNA expression is a posttranscriptional event, we investigated whether the decrease in RNA was due to an accelerated turnover rate of the pregenomic RNA using the Tet-off system. The results showed that MyD88 accelerated the decay of the pregenomic RNA. Furthermore, cytoplasmic and nuclear fractionation analysis showed that MyD88-induced decay of the pregnomic RNA occurred in the cytoplasm, and not in the nucleus. In addition, we blocked the 5’-3’and 3’-5’mRNA decay pathways by siRNAs and found that the two mRNA decay pathways were required for the MyD88-induced decay of viral pregenomic RNA.It has been reported that the La protein contributes to HBV pregenomic RNA stability through specific binding to the viral RNA. In this study, we showed that the MyD88-induced decay of viral pregenomic RNA was independent of the interaction of La with viral pregenomic RNA. The mapping analysis showed that HBV(1804-2454) and HBV(1151-1684) are MyD88-responsive regions of the pregenomc RNA. To investigate the relative contribution of the two MyD88-responsive sequences to MyD88-induced decay of viral pregenomic RNA, we constructed deletion mutants of these sequences and tested their response to MyD88. Our results showed that the HBV(1151-1684) deletion mutant retained sensitivity to MyD88, while the HBV(1804-2454) deletion mutant was resistant to MyD88. These results define the HBV(1804-2454) region as a crucial cis-regulatory sequence for MyD88-induced decay of viral pregenomic RNA.The HBV(1151-1684) region, which is located in the 3’overlapping region of the pregenomic RNA and preS/S RNAs, was not required for MyD88-induced decay of the pregenomic RNA, we determined whether it selectively conferred sensitivity of preS/S RNAs to MyD88. The results showed that the HBV(1151-1684) region selectively mediated the decay of viral preS/S RNAs. Furthermore, cytoplasmic and nuclear fractionation analysis showed that MyD88-induced decay of the preS/S RNAs occurred in the nucleus, and not in the cytoplasm.Interestingly, we found that the HBV(1151-1684) region overlaps with an RNA cis element termed the posttranscriptional regulatory element (PRE). The PRE selectively mediates the nuclear export of viral preS/S RNAs. In addition, viral preS/S RNAs fail to translocate to the cytoplasm and degrade in the nucleus with an elusive mechanism. We evaluated whether the decay of preS/S RNAs in the nucleus was associated with a deficiency in nuclear transport mediated by the PRE using the pRSV138PRE-CAT construct. The results showed that CAT activity derived from the PRE-containing transcript was significantly decreased by MyD88. In addition, MyD88 did not further diminish CAT expression, when co-expressed with NES(-)RanBP1, an inhibitor of the PRE-mediated nuclear export. Expression of polypyrimidne tract-binding protein (PTB), an export factor for PRE-containing RNA, almost fully restored the function of the PRE. Given that the CAT assays only represent an indirect measure of RNA levels, we also performed northern blot analysis for CAT RNA in the nucleus and cytoplasm. The results showed that the changes in RNA levels are in good agreement with the observed changes in CAT activity. Therefore, we conclude that MyD88 inhibits the nuclear export of HBV preS/S RNAs mediated by the PRE.To uncover the mechanism underlying the impaired PRE function in nuclear export, we evaluated the expression of PTB in MyD88-overexpressing cells. The results showed that MyD88 has an equally inhibitory effect on both PTB protein and mRNA levels, suggesting that PTB mRNA may be the target of MyD88. We investigated the effect of MyD88 on the stability of PTB mRNA. The results showed that the expression of MyD88 could not accelerate the degradation of PTB mRNA. Therefore, MyD88 transcriptionally inhibits the expression of PTB.In conclusion, our results provide further insights into the mechanism of MyD88 antiviral activity. Elucidation of this antiviral pathway may ultimately lead to the development of new therapeutics for HBV infection.更多还原