Rab8和肌球蛋白Vc参与2型登革病毒感染的研究
摘要
登革病毒(dengue virus,DV)属于黄病毒家族(Flaviviridae),是一种有包膜的单链RNA病毒,它包括四种不同的血清型,分别为DV1、DV2、DV3和DV4。登革病毒感染引起的症状较为复杂,从没有明显或者是仅有轻度症状的登革热(classical dengue fever,DF),到病势凶险伴有多种综合症状的登革出血热和登革休克综合征(dengue hemorrhagic fever/dengue shock syndrome,DHF/DSS)。在过去的几十年中,该病毒以埃及伊蚊(Aedes aegypti)和白纹伊蚊(A. albopictus)为媒介昆虫,在全世界热带和亚热带地区大面积流行,据世界卫生组织报道,每年全世界有近1亿人感染DV,其中以与我国接壤的东南亚地区流行最为严重。DV感染已经成为一个越来越严重的公共卫生问题,它正时刻威胁着人类的健康。但遗憾的是,迄今为止DF和DHF/DSS的致病机制尚未完全阐明。
在对DV感染的神经元、蚊、非洲绿猴肾等多种细胞的超显微观察中,研究人员发现病毒颗粒主要分布于粗面内质网腔、来源于粗面内质网的囊泡以及高尔基体区域。在正确组装病毒的核衣壳并获得包膜和其它附属结构后,一部分病毒被转运到高尔基体中逐步成熟并最终通过胞吐途径释放到细胞外。作为严格的胞内寄生物,DV利用宿主本身的合成和运输系统完成自身的感染过程。因而,寻找可能参与病毒大分子生物合成、病毒颗粒成熟和运输过程的宿主因子的研究备受关注。因为这些问题的阐明不仅有助于我们理解DV的感染过程,同时可能为进一步研制抗DV药物提供新的靶点。
Rab蛋白是Ras超家族中的一类,也是一类低分子量的GTP结合蛋白(大约20-29 kDa),它们负责调控真核细胞的囊泡运输等过程。其中,Rab8分布于反面高尔基体网络、囊泡和细胞膜,主要调控从高尔基体到细胞膜的囊泡运输。它参与了多种胞内合成蛋白的运输和分泌,比如说黑色素体依赖于肌动蛋白(actin)的运输等。在Rab8缺陷的小鼠中,小肠表面的一些肽和载体蛋白错误地定位到胞内的溶酶体,导致小肠对营养的吸收功能受到严重影响。因此,Rab8参与了细胞膜和膜蛋白的生物合成和运输过程,其异常功能状态不仅影响胞吐过程还会影响到胞吞过程。所以我们假设:一方面,因为DV颗粒在内质网到高尔基体区域成熟,所以病毒很可能会利用同样在这一区域的Rab8来完成其组装和运输,实现其胞吐释放的过程;另一方面,DV进入细胞必须的DV受体或者其他与DV内吞相关的细胞成分也需要依靠Rab8实现正确的分泌和定位。
肌动蛋白所介导的囊泡运输主要依赖于Rab8的活性,所以Rab8有可能参与调控囊泡在细胞骨架上运输的马达蛋白的招募过程。肌球蛋白Ⅴ(MyosinⅤ)是介导肌动蛋白运输最有效的一类马达蛋白,其中,肌球蛋白Ⅴc(MyosinⅤc,Myo5c)是2002年发现的该家族中的新成员。研究发现,Myo5c可以和某种带有Rab8的囊泡共存;当其负突变——Myo5c tail在胞内表达时,转铁蛋白堆积在一些细胞区室中,提示Myo5c可能参与人体内某些重要组织和器官中肌动蛋白介导的膜运输过程。而我们在以前的研究中也发现肌动蛋白参与DV2感染的过程。因此推测:Myo5c作为一种肌动蛋白的马达蛋白,可能直接或通过Rab8参与DV2感染过程。
虽然肝脏并不是DV感染的主要靶器官,但是在一些DV感染的临床病例以及尸检中都证实,有病毒抗原存在于肝脏细胞和Kupffer氏细胞,并伴随有肝功能异常。而且还有报道表明,DV可以在HepG2细胞系中复制,随后形成有侵染能力的病毒颗粒会被释放到培养液上清当中。故本文以HepG2细胞为研究对象,探讨Rab8和Myo5c在DV感染中的作用。
本研究的主要结果和结论如下:
一、Rab8截短体蛋白的原核表达、纯化及其抗体制备
首先,采用RT-PCR方法从HepG2细胞总RNA中T-A克隆了人Rab8基因,构建了pMD19T-Rab8质粒,针对Rab8蛋白的C端122个氨基酸设计一对亚克隆引物,以pMD19T-Rab8质粒为模板,用PCR扩增和BamHⅠ/HindⅢ双酶切方法将Rab8-122的DNA片段定向亚克隆到原核表达载体中,构建了原核表达质粒pQE31-Rab8-122。该质粒经双酶切和测序检验构建的准确性。
随后,对含有pQE31-Rab8-122质粒的工程菌诱导表达,1 mM IPTG诱导4 h即可获得以包涵体形式表达的目标蛋白。用Ni亲和层析、pH梯度洗脱和透析的方法纯化目标蛋白。用抗His抗体可以检测到Western blot反应膜上的目标蛋白,其大小约为15 kDa,与预期大小一致。
最后,用获得的Rab8截短体蛋白免疫动物制备抗Rab8多克隆抗体。经ELISA检测,制备的抗血清效价达1∶20000。Western blot实验证明,1∶500稀释的自制兔抗Rab8抗体,能够与诱导表达的6×His-Rab8-122蛋白以及HepG2细胞中分子量约为24 kDa的内源性Rab8蛋白发生特异的抗原抗体反应。Rab8抗血清的成功制备为后续研究奠定了基础。
二、Myo5c截短体蛋白的原核表达、纯化及其抗体制备
首先,根据Myo5c特异性的α-螺旋区设计引物,采用RT-PCR方法从人胃黏膜组织总RNA中获得目标片段,用BamHⅠ/ SalⅠ双酶切方法将Myo5c-297片段的DNA编码序列定向克隆到原核表达载体,构建了原核表达质粒pQE31-Myo5c-297。该质粒经双酶切和测序检验构建的准确性。
用1 mM IPTG对含有pQE31- Myo5c-297质粒的工程菌诱导表达。获得的目标蛋,用Ni亲和层析、pH梯度洗脱和透析的方法纯化。用抗His抗体可以检测到Western blot反应膜上的目标蛋白,其大小约为42 kDa,与预期大小一致。
最后,用获得的Myo5c截短体蛋白免疫动物制备抗Myo5c多克隆抗体。经ELISA检测,制备的抗血清效价达1∶12800。Western blot实验证明,1∶500稀释的自制小鼠抗Myo5c抗体,能够与诱导表达的6×His-Myo5c-297蛋白以及HepG2细胞中分子量约为203 kDa的内源性Myo5c蛋白发生特异的抗原抗体反应。1:200稀释的Myo5c抗血清还能检测人结肠黏膜切片中Myo5c抗原。Myo5c抗血清的成功制备为后续研究奠定了基础。
三、Rab8参与2型登革病毒复制周期
1、Rab8和DV2在HepG2细胞内高度共存DV2感染HepG2细胞,胞内的Rab8分布并没有出现非常显著的改变,仍然分布于核周到细胞膜之间的区域。但是在胞内点状分布的DV2抗原位置也有明显相同点状的Rab8抗原存在。用激光共聚焦分析这些点状结构,发现Rab8与DV2高度共存,其共存率大于90 %。这提示,Rab8可能参与DV2感染细胞的过程。
2、稳定表达Rab8正、负突变体HepG2细胞的建立
为进一步研究Rab8在DV复制周期中的作用,我们利用脂质体法分别将pcDNA3.1、pcDNA3.1-Rab8Q67L和pcDNA3.1-Rab8T22N超纯质粒转染至HepG2细胞,经过筛选,成功地获得具有G418抗性的细胞株,分别命名为HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN ,经流式细胞计数法鉴定Rab8正、负突变体在HepG2Rab8AM和HepG2Rab8DN细胞中的表达,证明所构建的细胞株可用于后续实验研究。
3、表达Rab8正、负突变体导致DV2阳性细胞数减少
免疫荧光染色发现:DV2感染HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞后24 h,大约有60%的HepG2p3.1细胞表现为病毒抗原阳性,而HepG2Rab8AM和HepG2Rab8DN细胞中则最多有20 %- 30 %的细胞呈现为病毒抗原阳性。这说明,表达Rab8正、负突变体影响到DV2感染细胞的过程。
4、表达Rab8正、负突变体导致培养上清中的子代病毒的释放减少利用空斑实验,在DV感染后不同时间点检测HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞培养上清中病毒滴度发现:在感染后8 h,三者所产生的子代病毒没有显著变化;但在感染后24和48 h,以HepG2p3.1细胞为对照,HepG2Rab8AM细胞(表达Rab8正突变体Rab8Q67L)培养上清中子代病毒分别减少了97.0 %和82.2 %;同样HepG2Rab8DN细胞(表达Rab8负突变体Rab8T22N的)培养上清中子代病毒分别减少77.2 %和53.3 %。这表明Rab8正、负突变体的表达能够抑制子代DV的释放;且较之于Rab8负突变体, Rab8正突变体作用更为显著。
5、表达Rab8正、负突变体抑制胞内侵染性病毒颗粒的产生
同样利用空斑实验,在感染后8、24和48 h,检测HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞内病毒滴度发现:在感染后8 h,三者胞内合成的侵染性病毒颗粒没有显著变化;但在感染后24和48 h,HepG2p3.1细胞胞内的DV2颗粒随着感染时间的延长而迅速增加;与之相比较,表达Rab8正突变体Rab8Q67L分别使胞内病毒滴度降低了96.4 %和78.8 %;表达Rab8负突变体Rab8T22N也分别使胞内病毒滴度降低了86.4 %和80.2%。这表明Rab8正、负突变体的表达,能够抑制细胞内侵染性病毒颗粒的产生,Rab8可能是HepG2细胞中参与DV2成熟过程的重要宿主因子。
6、表达Rab8正、负突变体能够抑制病毒RNA复制
为进一步阐明表达Rab8正、负突变体使细胞内DV2产生减少的原因,在DV2感染后不同时相点,利用real-time RT-PCR方法,检测了HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞中DV2 NS1基因(RNA)的相对水平。我们发现感染后24和48 h,表达Rab8正突变体Rab8Q67L分别使胞内病毒RNA降低了51 %和28.5 %;表达Rab8负突变体Rab8T22N也分别使胞内病毒RNA降低了33 %和36 %。结果表明,表达Rab8正、负突变体可在一定程度下调病毒RNA复制。但是与相同时间点细胞内的病毒滴度相比较,病毒滴度的减少远比病毒RNA的减少显著,这提示表达Rab8正、负突变体可能干扰了病毒的组装,而对病毒复制的影响相对较少。
7、表达Rab8正、负突变体抑制病毒进入HepG2细胞
利用病毒进入的实验方法,检测了进入HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞的病毒颗粒数量发现:与对照相比较,表达Rab8正突变体Rab8Q67L抑制81.3 %病毒进入,表达Rab8负突变体Rab8T22N可抑制79.7%病毒进入细胞。结果表明,Rab8参与了DV2进入HepG2细胞的过程。
四、Myo5c参与2型登革病毒释放
1、Myo5c tail稳定表达细胞株的建立
首先构建了Myo5c tail真核表达质粒pCI-Myo5c-tail,以pCI-neo空载体为对照,用脂质体法转染HepG2细胞,利用筛选培养基获得具有G418抗性的细胞株,分别命名为HepG2pCI和HepG2Myo5c-tail。Western Bolt检验结果显示,用鼠抗Myo5c抗血清可以检测到HepG2pCI和HepG2Myo5c-tail细胞中内源性的Myo5c蛋白,大小约为203 kDa,而在HepG2Myo5c-tail细胞中除了可以检测到内源性的Myo5c蛋白外还可以检测到转染pCI-Myo5c-tail质粒表达的Myo5c tail蛋白,大小约为93 kDa。说明HepG2Myo5c-tail细胞能够稳定表达Myo5c tail,所构建的HepG2pCI和HepG2Myo5c-tail细胞可以用于后续实验研究。
2、HepG2细胞中Myo5c可能与Rab8存在相互关联间接免疫荧光双染色法结果表明,Rab8和Myo5c均分布于核周到细胞膜区域,
共聚焦分析两者共存率在局部区域高达90 %。说明在HepG2细胞中Myo5c与Rab8高度共存。
用流式细胞免疫染色计数法检测发现,HepG2Myo5c-tail细胞中内源性Rab8表达量相对于HepG2pCI细胞增加近一倍。提示Myo5c tail的表达,可能导致HepG2细胞内源性Myo5c功能受到抑制,胞内Rab8的量出现代偿性增加。
3、Myo5c间接调控病毒释放间接免疫荧光双染色结果显示,在感染后24 h,Myo5c和DV2抗原虽然均出现在核周和胞浆内,但共聚焦分析表明两者共存率很低,提示Myo5c和DV2之间可能不存在直接的相互作用关系。
利用空斑实验,检测感染后不同时相点HepG2pCI和HepG2Myo5c-tail细胞细胞内外病毒滴度的变化发现:与对照HepG2pCI细胞比较,在感染后8、24和48 h,HepG2Myo5c-tail细胞培养上清中子代病毒分别减少了32.5 %、39.4%和47.3 %,而细胞内病毒滴度无明显变化。表明表达具有负突变效应的Myo5c tail,在一定程度上抑制了子代病毒的释放,但对细胞内DV2的产生影响较小。
Dengue virus (DV) is an enveloped, single-stranded RNA virus belonging to the family Flaviviridae. The viruses are comprised of four distinct serotypes, DV1 through DV4. DV infection causes a wide range of symptoms from an unapparent or mild disease (dengue fever, DF) to severe, life-threatening complications (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). This virus has spread throughout the tropical and subtropical regions worldwide over the past several decades by two mosquito species: Aedes aegypti and A. albopictus. According to the WHO’s reports, almost 10 million of DV cases occurs annually, most of which take place in Southeast Asia. And recently its infection has reemerged as a more and more severe threat against human health. However, the pathogenesis about DF and DHF/DSS remains unclear.
Some ultrastructural data has been reported that DV virions are observed in the lumen of the rough endoplasmic reticulum (rER), rER-derived vesicles and the Golgi region not only in infected mosquito and Vero cells but also in neurons. After nucleocapsids are assembled and acquire their envelopes and associated structures, a part of virus particles are transferred to the Golgi system for maturation and are delivered from the cell by exocytosis. As it parasitizes in cells, DV is supposed to utilize the living cell’s exocytosis pathway to release from hosts. Thus, which regulator of cellular transports would be involved in the maturation of the newly biosynthesized virions? The answer would provide more details on life cycle of DV and maybe provide further insight into controlling DF and DHF/DSS and therapy.
Rab proteins are a Ras-related family of low-molecular weight monomeric GTP-binding proteins (~20 to 29 kDa), which are key regulators of vesicular transports within eukaryotic cells. The Rab8 GTPase localizes predominantly on the trans-Golgi network (TGN) region and transport vesicles and regulates vesicular traffics from Golgi to plasma membrane. It is involved in transports and secretion of many proteins, for example the actin-based movement of melanosomes. Recently, some reports refine our understanding on Rab8. Apical peptidases and transporters abnormally localize to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes lead to a marked reduction in the absorption rate of nutrients in the small intestine. In general, Rab8 is involved in biosynthesis and traffics of plasma membrane and some membrane proteins and its abnormal function have great effect on not only exocytosis but also endocytosis. Therefore, we suppose that DV2 virions which mature at the region from ER to Golgi may employ the exocytosis pathways associated with Rab8 to be released from host cells, on the other hand, DV receptors or components involved in endocytic pathways for DV may also need Rab8 to be properly secreted or located on plasma membrane.
For actin-mediated movement of Rab8-positive vesicles are largely dependent on Rab8 activity, Rab8 would be ideal to regulate specific recruitment of motor proteins to defined vesicles. MyosinⅤc (Myo5c) is a novel member of classⅤmyosins, which class is the most efficient and processive in actin-mediated transport, and selectively co-localizes with a membrane compartment containing Rab8. When Myo5c tail, as a dominant negative mutant of Myo5c, is expressed in cells, transferrin accumulates in some cellular compartment, suggesting that Myo5c is involved in transferrin trafficking. Thus Myo5c may drive actin-mediated membrane trafficking pathway in many physiologically crucial tissues of the human body. Because we have confirmed the contribution of actin during DV2 infection, we suppose whether Myo5c, which is a motor of actin and interacts with Rab8, would be also involved in this infection process.
Although the liver is not a major target organ, the involvement of liver cells in pathogenesis of DV infection has been indicated by the abnormal liver function, pathological findings, and detection of viral antigen in Hepatocytes and Kupffer’s cells at biopsies. It is reported that DV can replicate in a human hepatocarcinoma cell line, HepG2, and infectious particles are released into the culture medium. In this paper, HepG2 cells were used to study the effects of Rab8 and Myo5c on DV2 infection.
Results and conclusions:
1. Preparation of anti-Rab8 polyclonal antibody
Rab8 gene was TA-cloned from HepG2 cells by RT-PCR and the prokaryotic expression plasmid, pQE31-Rab8-122, was constructed with the gene segment encoding 122 amino acids of Rab8 in C terminal. After expression in E. coil and purification by Ni affinity chromatograph, truncated Rab8 protein, which was a 15 kDa fusion protein containing 6×His in N terminal, was used to immunize rabbits to obtain the antiserum. The anti-Rab8 polyclonal antibody showed highly titer and specificity, and could recognize endogenous Rab8 antigens in HepG2 cells. And it might support the experiments on the involvement of Rab8 in viral infection.
2. Preparation of anti-myo5c polyclonal antibody
pQE31-Myo5c-297 was constructed with the DNA fragment encoding 297 amino acids of myo5c inα-helical coiled-coil tail domain. After expression and purification, truncated myo5c protein, which was a 42 kDa fusion protein containing 6×His in N terminal, was used to immunize Balb/c mouse to obtain the antiserum. The anti-myo5c polyclonal antibody showed highly titer and specificity, and could recognize endogenous myo5c antigens in HepG2 cells and human gastric mucosa. And it might support the experiments on the involvement of myo5c in viral infection.
3. Involvement of Rab8 in life cycle of DV2
A. Highly co-localization of Rab8 with DV2 in HepG2 cells.
After infection, although there were no visible changes in distribution pattern of endogenous Rab8 antigen, which predominantly localized in the cytoplasm from the perinuclear region to plasma membrane in HepG2 cells, some obvious puncta-like structure of Rab8 was revealed in the cytoplasma. Rab8 showed highly co-localization with DV2 antigen and the rate of their co-localization was up to about 90% in the puncta-like components analyzed by confocal laser microscope. This result may suggest close association of Rab8 and DV2.
B. Establishment of HepG2 cells stably expressing Rab8 mutants.
HepG2 cells were transfected with plasmids of pcDNA3.1, pcDNA3.1-Rab8Q67L and pcDNA3.1-Rab8T22N by lipofectamine followed by G418 selection. HepG2p3.1, HepG2Rab8AM and HepG2Rab8DN cells were obtained and then assessed by flow cytometric analysis. The result showed that HepG2Rab8AM cells stably expressed Rab8Q67L, a constitutively active mutant of Rab8, and HepG2Rab8DN cells stably expressed Rab8T22N, a dominant negative mutant of Rab8.
C. The process of DV2 infection is inhibited by expressing Rab8 mutants.
More than 60 % HepG2p3.1 cells were infected by DV2 at 24 h p.i., while only about 20-30% DV2 antigen-positive cells were observed in HepG2Rab8AM or HepG2Rab8DN cells. The result by immuno-staining assay indicates that the process of DV2 infection should be impacted by interrupting the function of Rab8 in cells.
D. Supernatant progeny virions are reduced by expressing Rab8 mutants.
The supernatant virus released from HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells at 8, 24 and 48 h p.i. were detected by plaque assay. Expression of Rab8Q67L reduced virus released from HepG2Rab8AM cells by 97.0 % and 82.2 % at 24 and 48 h p.i. respectively, and expression of Rab8T22N reduced virus released from HepG2Rab8DN cells by 77.2 % and 53.3 % at 24 and 48 h p.i. respectively. Thus, our results indicate that Rab8 is most likely to be necessary for DV2 release or production and expression of Rab8Q67L has stronger inhibitory effects than expression of Rab8T22N.
E. Productions of infectious virions are inhibited by expressing Rab8 mutants.
The intracellular virus titers of HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells at 8, 24 and 48 h p.i. were assayed by plaque assay. Expression of Rab8Q67L reduced production of infectious virus in HepG2Rab8AM cells by 96.4 % and 78.8% at 24 and 48 h p.i. respectively, while expression of Rab8T22N reduced production of infectious virus in HepG2Rab8DN cells by 86.4 % and 80.2 % at 24 and 48 h p.i. respectively. Our data indicate that expression of Rab8 mutants significantly inhibits the production of progeny virions and Rab8 may be an important host factor for the formation of infectious DV2 in HepG2 cells.
F. Viral RNA replication was down-regulated by a different level.
To further understand the interruption of DV2 production by expressing Rab8 mutants, the replication levels of viral RNA in HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells were detected by real-time RT-PCR. We found expression of Rab8Q67L inhibited DV2 replication in HepG2Rab8AM cells by 55.3 % and 28.5% at 24 and 48 h p.i., respectively. The similar results were obtained in HepG2Rab8DN cells, and expression of Rab8T22N inhibited replication by 33 % and 36 % at 24 and 48 h p.i., respectively. Those data indicate that viral replication is down-regulated by expressing Rab8 mutants. While as compared with intracellular titers at the same time points, levels of viral RNA decrease not as significantly as the production of infectious virions, suggesting that viral assembly, not replication, is most likely to be interrupted by expression of Rab8 mutants.
G. Expressions of Rab8 mutants inhibit the viral entry into HepG2 cell.
The amounts of DV2 entry into HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells were detected by viral entry assay. The results showed that expression of Rab8T22N reduced DV2 entry into HepG2Rab8DN cells by 79.7 % and expression of Rab8Q67L even reduced viral entry into HepG2Rab8AM by 83.1 %, compared with those in HepG2p3.1 cells. The data show the inhibition of DV2 entry into HepG2 cells by expressing Rab8 mutants, suggesting the involvement of Rab8 in DV2 entry into cells.
4. Modulation of DV2 release by Myo5c
A. Establishment of HepG2 cells stably expressing Myo5c tail.
HepG2 cells were transfected with plasmids of pCI-neo and pCI-Myo5c-tail by lipofectamine. HepG2pCI and HepG2Myo5c-tail cells were obtained by G418 selection and then assessed by Western Bolt. The rabbit anti-Myo5c pAb specifically reacted with both the endogenous Myo5c proteins (203 kDa) in HepG2pCI and with Myo5c-tail protein (93 kDa) expressed in HepG2Myo5c-tail cells. The result showed that HepG2Myo5c-tail cells stably expressed Myo5c tail.
B. Association of Rab8 with Myo5c in HepG2 cells.
By double staining analysis, both Rab8 and Myo5c localized in the cytoplasm from the perinuclear region to plasma membrane in HepG2 cells, and about 90% co-localization of Myo5c with Rab8 was confirmed in HepG2 cells by confocal laser microscope, suggesting the highly co-localization of Rab8 with Myo5c.
By flow cytometric analysis, Rab8 was up-regulated by expressing Myo5c tail in HepG2Myo5c-tail cells, which indicated that compensated increase of Rab8 was induced by functional inhibition of Myo5c.
C. Myo5c indirectly modulates the release of DV2.
The result of double staining showed that although both Myo5c and DV2 localized in the cytoplasm from the perinuclear region to plasma membrane in infected HepG2 cells at 24 h p.i., the rate of their co-localization was poor. Thus, there is rare direct interaction of Myo5c with DV2.
Virus titers of supernatants and cell fraction from HepG2pCI and HepG2Myo5c-tail cells were assayed by plaque assay. As compared with the control, expression of Myo5c tail reduced progeny virus release by 32.5 %, 39.4 % and 47.3 % at 8, 24 and 48 h p.i., respectively. However, viral titers in HepG2Myo5c-tail cells fraction only showed a tendency of slightly decreasing. The data indicate that Myo5c indirectly modulate the release of DV2 from HepG2 cells but not the formation of infectious virions.
在对DV感染的神经元、蚊、非洲绿猴肾等多种细胞的超显微观察中,研究人员发现病毒颗粒主要分布于粗面内质网腔、来源于粗面内质网的囊泡以及高尔基体区域。在正确组装病毒的核衣壳并获得包膜和其它附属结构后,一部分病毒被转运到高尔基体中逐步成熟并最终通过胞吐途径释放到细胞外。作为严格的胞内寄生物,DV利用宿主本身的合成和运输系统完成自身的感染过程。因而,寻找可能参与病毒大分子生物合成、病毒颗粒成熟和运输过程的宿主因子的研究备受关注。因为这些问题的阐明不仅有助于我们理解DV的感染过程,同时可能为进一步研制抗DV药物提供新的靶点。
Rab蛋白是Ras超家族中的一类,也是一类低分子量的GTP结合蛋白(大约20-29 kDa),它们负责调控真核细胞的囊泡运输等过程。其中,Rab8分布于反面高尔基体网络、囊泡和细胞膜,主要调控从高尔基体到细胞膜的囊泡运输。它参与了多种胞内合成蛋白的运输和分泌,比如说黑色素体依赖于肌动蛋白(actin)的运输等。在Rab8缺陷的小鼠中,小肠表面的一些肽和载体蛋白错误地定位到胞内的溶酶体,导致小肠对营养的吸收功能受到严重影响。因此,Rab8参与了细胞膜和膜蛋白的生物合成和运输过程,其异常功能状态不仅影响胞吐过程还会影响到胞吞过程。所以我们假设:一方面,因为DV颗粒在内质网到高尔基体区域成熟,所以病毒很可能会利用同样在这一区域的Rab8来完成其组装和运输,实现其胞吐释放的过程;另一方面,DV进入细胞必须的DV受体或者其他与DV内吞相关的细胞成分也需要依靠Rab8实现正确的分泌和定位。
肌动蛋白所介导的囊泡运输主要依赖于Rab8的活性,所以Rab8有可能参与调控囊泡在细胞骨架上运输的马达蛋白的招募过程。肌球蛋白Ⅴ(MyosinⅤ)是介导肌动蛋白运输最有效的一类马达蛋白,其中,肌球蛋白Ⅴc(MyosinⅤc,Myo5c)是2002年发现的该家族中的新成员。研究发现,Myo5c可以和某种带有Rab8的囊泡共存;当其负突变——Myo5c tail在胞内表达时,转铁蛋白堆积在一些细胞区室中,提示Myo5c可能参与人体内某些重要组织和器官中肌动蛋白介导的膜运输过程。而我们在以前的研究中也发现肌动蛋白参与DV2感染的过程。因此推测:Myo5c作为一种肌动蛋白的马达蛋白,可能直接或通过Rab8参与DV2感染过程。
虽然肝脏并不是DV感染的主要靶器官,但是在一些DV感染的临床病例以及尸检中都证实,有病毒抗原存在于肝脏细胞和Kupffer氏细胞,并伴随有肝功能异常。而且还有报道表明,DV可以在HepG2细胞系中复制,随后形成有侵染能力的病毒颗粒会被释放到培养液上清当中。故本文以HepG2细胞为研究对象,探讨Rab8和Myo5c在DV感染中的作用。
本研究的主要结果和结论如下:
一、Rab8截短体蛋白的原核表达、纯化及其抗体制备
首先,采用RT-PCR方法从HepG2细胞总RNA中T-A克隆了人Rab8基因,构建了pMD19T-Rab8质粒,针对Rab8蛋白的C端122个氨基酸设计一对亚克隆引物,以pMD19T-Rab8质粒为模板,用PCR扩增和BamHⅠ/HindⅢ双酶切方法将Rab8-122的DNA片段定向亚克隆到原核表达载体中,构建了原核表达质粒pQE31-Rab8-122。该质粒经双酶切和测序检验构建的准确性。
随后,对含有pQE31-Rab8-122质粒的工程菌诱导表达,1 mM IPTG诱导4 h即可获得以包涵体形式表达的目标蛋白。用Ni亲和层析、pH梯度洗脱和透析的方法纯化目标蛋白。用抗His抗体可以检测到Western blot反应膜上的目标蛋白,其大小约为15 kDa,与预期大小一致。
最后,用获得的Rab8截短体蛋白免疫动物制备抗Rab8多克隆抗体。经ELISA检测,制备的抗血清效价达1∶20000。Western blot实验证明,1∶500稀释的自制兔抗Rab8抗体,能够与诱导表达的6×His-Rab8-122蛋白以及HepG2细胞中分子量约为24 kDa的内源性Rab8蛋白发生特异的抗原抗体反应。Rab8抗血清的成功制备为后续研究奠定了基础。
二、Myo5c截短体蛋白的原核表达、纯化及其抗体制备
首先,根据Myo5c特异性的α-螺旋区设计引物,采用RT-PCR方法从人胃黏膜组织总RNA中获得目标片段,用BamHⅠ/ SalⅠ双酶切方法将Myo5c-297片段的DNA编码序列定向克隆到原核表达载体,构建了原核表达质粒pQE31-Myo5c-297。该质粒经双酶切和测序检验构建的准确性。
用1 mM IPTG对含有pQE31- Myo5c-297质粒的工程菌诱导表达。获得的目标蛋,用Ni亲和层析、pH梯度洗脱和透析的方法纯化。用抗His抗体可以检测到Western blot反应膜上的目标蛋白,其大小约为42 kDa,与预期大小一致。
最后,用获得的Myo5c截短体蛋白免疫动物制备抗Myo5c多克隆抗体。经ELISA检测,制备的抗血清效价达1∶12800。Western blot实验证明,1∶500稀释的自制小鼠抗Myo5c抗体,能够与诱导表达的6×His-Myo5c-297蛋白以及HepG2细胞中分子量约为203 kDa的内源性Myo5c蛋白发生特异的抗原抗体反应。1:200稀释的Myo5c抗血清还能检测人结肠黏膜切片中Myo5c抗原。Myo5c抗血清的成功制备为后续研究奠定了基础。
三、Rab8参与2型登革病毒复制周期
1、Rab8和DV2在HepG2细胞内高度共存DV2感染HepG2细胞,胞内的Rab8分布并没有出现非常显著的改变,仍然分布于核周到细胞膜之间的区域。但是在胞内点状分布的DV2抗原位置也有明显相同点状的Rab8抗原存在。用激光共聚焦分析这些点状结构,发现Rab8与DV2高度共存,其共存率大于90 %。这提示,Rab8可能参与DV2感染细胞的过程。
2、稳定表达Rab8正、负突变体HepG2细胞的建立
为进一步研究Rab8在DV复制周期中的作用,我们利用脂质体法分别将pcDNA3.1、pcDNA3.1-Rab8Q67L和pcDNA3.1-Rab8T22N超纯质粒转染至HepG2细胞,经过筛选,成功地获得具有G418抗性的细胞株,分别命名为HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN ,经流式细胞计数法鉴定Rab8正、负突变体在HepG2Rab8AM和HepG2Rab8DN细胞中的表达,证明所构建的细胞株可用于后续实验研究。
3、表达Rab8正、负突变体导致DV2阳性细胞数减少
免疫荧光染色发现:DV2感染HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞后24 h,大约有60%的HepG2p3.1细胞表现为病毒抗原阳性,而HepG2Rab8AM和HepG2Rab8DN细胞中则最多有20 %- 30 %的细胞呈现为病毒抗原阳性。这说明,表达Rab8正、负突变体影响到DV2感染细胞的过程。
4、表达Rab8正、负突变体导致培养上清中的子代病毒的释放减少利用空斑实验,在DV感染后不同时间点检测HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞培养上清中病毒滴度发现:在感染后8 h,三者所产生的子代病毒没有显著变化;但在感染后24和48 h,以HepG2p3.1细胞为对照,HepG2Rab8AM细胞(表达Rab8正突变体Rab8Q67L)培养上清中子代病毒分别减少了97.0 %和82.2 %;同样HepG2Rab8DN细胞(表达Rab8负突变体Rab8T22N的)培养上清中子代病毒分别减少77.2 %和53.3 %。这表明Rab8正、负突变体的表达能够抑制子代DV的释放;且较之于Rab8负突变体, Rab8正突变体作用更为显著。
5、表达Rab8正、负突变体抑制胞内侵染性病毒颗粒的产生
同样利用空斑实验,在感染后8、24和48 h,检测HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞内病毒滴度发现:在感染后8 h,三者胞内合成的侵染性病毒颗粒没有显著变化;但在感染后24和48 h,HepG2p3.1细胞胞内的DV2颗粒随着感染时间的延长而迅速增加;与之相比较,表达Rab8正突变体Rab8Q67L分别使胞内病毒滴度降低了96.4 %和78.8 %;表达Rab8负突变体Rab8T22N也分别使胞内病毒滴度降低了86.4 %和80.2%。这表明Rab8正、负突变体的表达,能够抑制细胞内侵染性病毒颗粒的产生,Rab8可能是HepG2细胞中参与DV2成熟过程的重要宿主因子。
6、表达Rab8正、负突变体能够抑制病毒RNA复制
为进一步阐明表达Rab8正、负突变体使细胞内DV2产生减少的原因,在DV2感染后不同时相点,利用real-time RT-PCR方法,检测了HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞中DV2 NS1基因(RNA)的相对水平。我们发现感染后24和48 h,表达Rab8正突变体Rab8Q67L分别使胞内病毒RNA降低了51 %和28.5 %;表达Rab8负突变体Rab8T22N也分别使胞内病毒RNA降低了33 %和36 %。结果表明,表达Rab8正、负突变体可在一定程度下调病毒RNA复制。但是与相同时间点细胞内的病毒滴度相比较,病毒滴度的减少远比病毒RNA的减少显著,这提示表达Rab8正、负突变体可能干扰了病毒的组装,而对病毒复制的影响相对较少。
7、表达Rab8正、负突变体抑制病毒进入HepG2细胞
利用病毒进入的实验方法,检测了进入HepG2p3.1、HepG2Rab8AM和HepG2Rab8DN细胞的病毒颗粒数量发现:与对照相比较,表达Rab8正突变体Rab8Q67L抑制81.3 %病毒进入,表达Rab8负突变体Rab8T22N可抑制79.7%病毒进入细胞。结果表明,Rab8参与了DV2进入HepG2细胞的过程。
四、Myo5c参与2型登革病毒释放
1、Myo5c tail稳定表达细胞株的建立
首先构建了Myo5c tail真核表达质粒pCI-Myo5c-tail,以pCI-neo空载体为对照,用脂质体法转染HepG2细胞,利用筛选培养基获得具有G418抗性的细胞株,分别命名为HepG2pCI和HepG2Myo5c-tail。Western Bolt检验结果显示,用鼠抗Myo5c抗血清可以检测到HepG2pCI和HepG2Myo5c-tail细胞中内源性的Myo5c蛋白,大小约为203 kDa,而在HepG2Myo5c-tail细胞中除了可以检测到内源性的Myo5c蛋白外还可以检测到转染pCI-Myo5c-tail质粒表达的Myo5c tail蛋白,大小约为93 kDa。说明HepG2Myo5c-tail细胞能够稳定表达Myo5c tail,所构建的HepG2pCI和HepG2Myo5c-tail细胞可以用于后续实验研究。
2、HepG2细胞中Myo5c可能与Rab8存在相互关联间接免疫荧光双染色法结果表明,Rab8和Myo5c均分布于核周到细胞膜区域,
共聚焦分析两者共存率在局部区域高达90 %。说明在HepG2细胞中Myo5c与Rab8高度共存。
用流式细胞免疫染色计数法检测发现,HepG2Myo5c-tail细胞中内源性Rab8表达量相对于HepG2pCI细胞增加近一倍。提示Myo5c tail的表达,可能导致HepG2细胞内源性Myo5c功能受到抑制,胞内Rab8的量出现代偿性增加。
3、Myo5c间接调控病毒释放间接免疫荧光双染色结果显示,在感染后24 h,Myo5c和DV2抗原虽然均出现在核周和胞浆内,但共聚焦分析表明两者共存率很低,提示Myo5c和DV2之间可能不存在直接的相互作用关系。
利用空斑实验,检测感染后不同时相点HepG2pCI和HepG2Myo5c-tail细胞细胞内外病毒滴度的变化发现:与对照HepG2pCI细胞比较,在感染后8、24和48 h,HepG2Myo5c-tail细胞培养上清中子代病毒分别减少了32.5 %、39.4%和47.3 %,而细胞内病毒滴度无明显变化。表明表达具有负突变效应的Myo5c tail,在一定程度上抑制了子代病毒的释放,但对细胞内DV2的产生影响较小。
Dengue virus (DV) is an enveloped, single-stranded RNA virus belonging to the family Flaviviridae. The viruses are comprised of four distinct serotypes, DV1 through DV4. DV infection causes a wide range of symptoms from an unapparent or mild disease (dengue fever, DF) to severe, life-threatening complications (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). This virus has spread throughout the tropical and subtropical regions worldwide over the past several decades by two mosquito species: Aedes aegypti and A. albopictus. According to the WHO’s reports, almost 10 million of DV cases occurs annually, most of which take place in Southeast Asia. And recently its infection has reemerged as a more and more severe threat against human health. However, the pathogenesis about DF and DHF/DSS remains unclear.
Some ultrastructural data has been reported that DV virions are observed in the lumen of the rough endoplasmic reticulum (rER), rER-derived vesicles and the Golgi region not only in infected mosquito and Vero cells but also in neurons. After nucleocapsids are assembled and acquire their envelopes and associated structures, a part of virus particles are transferred to the Golgi system for maturation and are delivered from the cell by exocytosis. As it parasitizes in cells, DV is supposed to utilize the living cell’s exocytosis pathway to release from hosts. Thus, which regulator of cellular transports would be involved in the maturation of the newly biosynthesized virions? The answer would provide more details on life cycle of DV and maybe provide further insight into controlling DF and DHF/DSS and therapy.
Rab proteins are a Ras-related family of low-molecular weight monomeric GTP-binding proteins (~20 to 29 kDa), which are key regulators of vesicular transports within eukaryotic cells. The Rab8 GTPase localizes predominantly on the trans-Golgi network (TGN) region and transport vesicles and regulates vesicular traffics from Golgi to plasma membrane. It is involved in transports and secretion of many proteins, for example the actin-based movement of melanosomes. Recently, some reports refine our understanding on Rab8. Apical peptidases and transporters abnormally localize to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes lead to a marked reduction in the absorption rate of nutrients in the small intestine. In general, Rab8 is involved in biosynthesis and traffics of plasma membrane and some membrane proteins and its abnormal function have great effect on not only exocytosis but also endocytosis. Therefore, we suppose that DV2 virions which mature at the region from ER to Golgi may employ the exocytosis pathways associated with Rab8 to be released from host cells, on the other hand, DV receptors or components involved in endocytic pathways for DV may also need Rab8 to be properly secreted or located on plasma membrane.
For actin-mediated movement of Rab8-positive vesicles are largely dependent on Rab8 activity, Rab8 would be ideal to regulate specific recruitment of motor proteins to defined vesicles. MyosinⅤc (Myo5c) is a novel member of classⅤmyosins, which class is the most efficient and processive in actin-mediated transport, and selectively co-localizes with a membrane compartment containing Rab8. When Myo5c tail, as a dominant negative mutant of Myo5c, is expressed in cells, transferrin accumulates in some cellular compartment, suggesting that Myo5c is involved in transferrin trafficking. Thus Myo5c may drive actin-mediated membrane trafficking pathway in many physiologically crucial tissues of the human body. Because we have confirmed the contribution of actin during DV2 infection, we suppose whether Myo5c, which is a motor of actin and interacts with Rab8, would be also involved in this infection process.
Although the liver is not a major target organ, the involvement of liver cells in pathogenesis of DV infection has been indicated by the abnormal liver function, pathological findings, and detection of viral antigen in Hepatocytes and Kupffer’s cells at biopsies. It is reported that DV can replicate in a human hepatocarcinoma cell line, HepG2, and infectious particles are released into the culture medium. In this paper, HepG2 cells were used to study the effects of Rab8 and Myo5c on DV2 infection.
Results and conclusions:
1. Preparation of anti-Rab8 polyclonal antibody
Rab8 gene was TA-cloned from HepG2 cells by RT-PCR and the prokaryotic expression plasmid, pQE31-Rab8-122, was constructed with the gene segment encoding 122 amino acids of Rab8 in C terminal. After expression in E. coil and purification by Ni affinity chromatograph, truncated Rab8 protein, which was a 15 kDa fusion protein containing 6×His in N terminal, was used to immunize rabbits to obtain the antiserum. The anti-Rab8 polyclonal antibody showed highly titer and specificity, and could recognize endogenous Rab8 antigens in HepG2 cells. And it might support the experiments on the involvement of Rab8 in viral infection.
2. Preparation of anti-myo5c polyclonal antibody
pQE31-Myo5c-297 was constructed with the DNA fragment encoding 297 amino acids of myo5c inα-helical coiled-coil tail domain. After expression and purification, truncated myo5c protein, which was a 42 kDa fusion protein containing 6×His in N terminal, was used to immunize Balb/c mouse to obtain the antiserum. The anti-myo5c polyclonal antibody showed highly titer and specificity, and could recognize endogenous myo5c antigens in HepG2 cells and human gastric mucosa. And it might support the experiments on the involvement of myo5c in viral infection.
3. Involvement of Rab8 in life cycle of DV2
A. Highly co-localization of Rab8 with DV2 in HepG2 cells.
After infection, although there were no visible changes in distribution pattern of endogenous Rab8 antigen, which predominantly localized in the cytoplasm from the perinuclear region to plasma membrane in HepG2 cells, some obvious puncta-like structure of Rab8 was revealed in the cytoplasma. Rab8 showed highly co-localization with DV2 antigen and the rate of their co-localization was up to about 90% in the puncta-like components analyzed by confocal laser microscope. This result may suggest close association of Rab8 and DV2.
B. Establishment of HepG2 cells stably expressing Rab8 mutants.
HepG2 cells were transfected with plasmids of pcDNA3.1, pcDNA3.1-Rab8Q67L and pcDNA3.1-Rab8T22N by lipofectamine followed by G418 selection. HepG2p3.1, HepG2Rab8AM and HepG2Rab8DN cells were obtained and then assessed by flow cytometric analysis. The result showed that HepG2Rab8AM cells stably expressed Rab8Q67L, a constitutively active mutant of Rab8, and HepG2Rab8DN cells stably expressed Rab8T22N, a dominant negative mutant of Rab8.
C. The process of DV2 infection is inhibited by expressing Rab8 mutants.
More than 60 % HepG2p3.1 cells were infected by DV2 at 24 h p.i., while only about 20-30% DV2 antigen-positive cells were observed in HepG2Rab8AM or HepG2Rab8DN cells. The result by immuno-staining assay indicates that the process of DV2 infection should be impacted by interrupting the function of Rab8 in cells.
D. Supernatant progeny virions are reduced by expressing Rab8 mutants.
The supernatant virus released from HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells at 8, 24 and 48 h p.i. were detected by plaque assay. Expression of Rab8Q67L reduced virus released from HepG2Rab8AM cells by 97.0 % and 82.2 % at 24 and 48 h p.i. respectively, and expression of Rab8T22N reduced virus released from HepG2Rab8DN cells by 77.2 % and 53.3 % at 24 and 48 h p.i. respectively. Thus, our results indicate that Rab8 is most likely to be necessary for DV2 release or production and expression of Rab8Q67L has stronger inhibitory effects than expression of Rab8T22N.
E. Productions of infectious virions are inhibited by expressing Rab8 mutants.
The intracellular virus titers of HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells at 8, 24 and 48 h p.i. were assayed by plaque assay. Expression of Rab8Q67L reduced production of infectious virus in HepG2Rab8AM cells by 96.4 % and 78.8% at 24 and 48 h p.i. respectively, while expression of Rab8T22N reduced production of infectious virus in HepG2Rab8DN cells by 86.4 % and 80.2 % at 24 and 48 h p.i. respectively. Our data indicate that expression of Rab8 mutants significantly inhibits the production of progeny virions and Rab8 may be an important host factor for the formation of infectious DV2 in HepG2 cells.
F. Viral RNA replication was down-regulated by a different level.
To further understand the interruption of DV2 production by expressing Rab8 mutants, the replication levels of viral RNA in HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells were detected by real-time RT-PCR. We found expression of Rab8Q67L inhibited DV2 replication in HepG2Rab8AM cells by 55.3 % and 28.5% at 24 and 48 h p.i., respectively. The similar results were obtained in HepG2Rab8DN cells, and expression of Rab8T22N inhibited replication by 33 % and 36 % at 24 and 48 h p.i., respectively. Those data indicate that viral replication is down-regulated by expressing Rab8 mutants. While as compared with intracellular titers at the same time points, levels of viral RNA decrease not as significantly as the production of infectious virions, suggesting that viral assembly, not replication, is most likely to be interrupted by expression of Rab8 mutants.
G. Expressions of Rab8 mutants inhibit the viral entry into HepG2 cell.
The amounts of DV2 entry into HepG2Rab8AM, HepG2Rab8DN and HepG2p3.1 cells were detected by viral entry assay. The results showed that expression of Rab8T22N reduced DV2 entry into HepG2Rab8DN cells by 79.7 % and expression of Rab8Q67L even reduced viral entry into HepG2Rab8AM by 83.1 %, compared with those in HepG2p3.1 cells. The data show the inhibition of DV2 entry into HepG2 cells by expressing Rab8 mutants, suggesting the involvement of Rab8 in DV2 entry into cells.
4. Modulation of DV2 release by Myo5c
A. Establishment of HepG2 cells stably expressing Myo5c tail.
HepG2 cells were transfected with plasmids of pCI-neo and pCI-Myo5c-tail by lipofectamine. HepG2pCI and HepG2Myo5c-tail cells were obtained by G418 selection and then assessed by Western Bolt. The rabbit anti-Myo5c pAb specifically reacted with both the endogenous Myo5c proteins (203 kDa) in HepG2pCI and with Myo5c-tail protein (93 kDa) expressed in HepG2Myo5c-tail cells. The result showed that HepG2Myo5c-tail cells stably expressed Myo5c tail.
B. Association of Rab8 with Myo5c in HepG2 cells.
By double staining analysis, both Rab8 and Myo5c localized in the cytoplasm from the perinuclear region to plasma membrane in HepG2 cells, and about 90% co-localization of Myo5c with Rab8 was confirmed in HepG2 cells by confocal laser microscope, suggesting the highly co-localization of Rab8 with Myo5c.
By flow cytometric analysis, Rab8 was up-regulated by expressing Myo5c tail in HepG2Myo5c-tail cells, which indicated that compensated increase of Rab8 was induced by functional inhibition of Myo5c.
C. Myo5c indirectly modulates the release of DV2.
The result of double staining showed that although both Myo5c and DV2 localized in the cytoplasm from the perinuclear region to plasma membrane in infected HepG2 cells at 24 h p.i., the rate of their co-localization was poor. Thus, there is rare direct interaction of Myo5c with DV2.
Virus titers of supernatants and cell fraction from HepG2pCI and HepG2Myo5c-tail cells were assayed by plaque assay. As compared with the control, expression of Myo5c tail reduced progeny virus release by 32.5 %, 39.4 % and 47.3 % at 8, 24 and 48 h p.i., respectively. However, viral titers in HepG2Myo5c-tail cells fraction only showed a tendency of slightly decreasing. The data indicate that Myo5c indirectly modulate the release of DV2 from HepG2 cells but not the formation of infectious virions.
引文
1. Clyde K, Kyle JL and Harris E. Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. J Virol, 2006, 80: 11418-11431.
2. An J, Zhou DS, Kawasaki K, et al. The pathogenesis of spinal cord involvement in dengue virus infection. Virchows Arch, 2003, 442: 472-481.
3. Barth OM. Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis. J Submicrosc Cytol Pathol, 1999, 31: 407-412.
4. Takai Y, Sasaki T and Matozaki T. Small GTP-binding proteins. Physiol Rev, 2001,81: 153-208.
5. Huber LA, Pimplikar S, Parton RG, et al. Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol, 1993, 123: 35-45.
6. Chabrillat ML, Wilhelm C, Wasmeier C, et al. Rab8 regulates the actin-based movement of melanosomes. Mol Biol Cell, 2005, 16: 1640-1650.
7. Sato T, Mushiake S, Kato Y, et al. The Rab8 GTPase regulates apical protein localization in intestinal cells. Nature, 2007, 488: 366-369.
8. Per?nen J, Auvinen P, Virta H, et al. Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. J Cell Biol, 1996, 135: 153-167.
9. Rodriguez OC and Cheney RE. Human myosin-Ⅴc is a novel classⅤmyosin expressed in epithelial cells. J Cell Biol, 2002, 115: 991-1004.
10.王嘉丽,陈炜,万颖杰等.登革病毒感染后ECV304单层细胞通透性与微丝关系的研究.中国人兽共患病杂志, 2005, 21: 953–957.
11. Bhamarapravati N, Tuchinda P and Boonyapaknavik V. Pathology of Thailand haemorrhagic fever: a study of 100 autopsy cases. Ann Trop Med Parasitol, 1967, 61: 500-510.
12. Burke T. Dengue haemorrhagic fever: a pathological study. Trans R Soc Trop Med Hyg, 1968, 62: 682-692.
13. Kuo CH, Tai DI, Chang-Chien CS, et al. Liver biochemical tests and dengue fever. AmJ Trop Med Hyg, 1992, 47: 265-270.
14. Marianneau P, Cardona A, Edelman L, et al. Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death. J Virol, 1997, 71: 3244-3249.
15. Marianneau P, Megret F, Olivier R, et al. Dengue 1 virus binding to human hepatoma HepG2 and simian Vero cell surfaces differs. J Gen Virol, 1996, 77: 2547-2554.
16. Rosen L, Khin MM and U T. Recovery of virus from the liver of children with fatal dengue: reflections on the pathogenesis of the disease and its possible analogy with that of yellow fever. Res Virol, 1989, 140: 351-360.
17. Chavrier P, Vingron M, Sander C, et al. Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol Cell Biol, 1990, 10: 6578-6585.
18. Wilson AL, Erdman RA, Castellano F, et al. Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases. Biochem J, 1998, 333: 497-504.
19. Taylor KA. Regulation and recycling of myosinⅤ. Curr Opin Cell Biol, 2007, 19:67-74.
20.司徒镇强,吴军政主编.细胞培养.西安:世界图书出版社.
21. Karniguian A, Zahraoui A, and Tavitian A. Identification of small GTP-binding rab proteins in human platelets: Thrombin-induced phosphorylation of rab3B, rab6, and rab8 proteins. Proc Natl Acad Sci USA, 1993, 90: 7647-7651.
22. Deretic D, Huber LA, Ransom N, et al. Rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. J Cell Sci, 1995, 108; 215-224.
23. Ren M, Zeng J, Lemos-Chiarandini C, et al. In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase. Proc Natl Acad Sci USA, 1996, 93: 5151-5155.
24. Gerges NZ, Backos DS and Esteban JA. Local control of AMPA receptor trafficking at the postsynaptic terminal by a small GTPase of the Rab family. J Biol Chem, 2004, 279: 43870–43878.
25. Uno T, Nakao A and Katsurauma C. Phosphorylation of Rab Proteins from the Brain of Bombyx mori. Arch Insect Bioch Phys, 2004, 57: 68-77.
26. Hattula K, Furuhjelm J, Arffman A, et al. A Rab8-specific GDP/GTP exchange factoris involved in actin remodeling and polarized membrane transport. Mol Cell Biol, 2002, 13: 3268-3280.
27. Ang AL, F?lsch H, Koivisto UM, et al. The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells. J Cell Biol, 2003, 163: 339-350.
28. Bujard H,Gentz R,Lanzer M,et al. A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol, 1987,155: 416-433.
29. Stuber D,Bannwarth W,Pink JR,et al. New B cell epitopes in the plasmodium falciparum malaria circumsporozoite pritein. Eur J Immunol, 1990,20: 819-824.
30.叶菁,陈广生,黄亚渝等.人MAGE-1基因的克隆、原核表达及抗体的制备.免疫学杂志, 2004,20:259-263.
31. J.萨姆布鲁克等著,金冬雁译.分子克隆实验指南(第二版).北京:科学出版社,1992:855-857.
32. Sellers JR and Veigel C. Walking with myosinⅤ. Curr Opin Cell Biol, 2006, 18: 68-73.
33. Gross SP, Tuma MC, Deacon SW, et al. Interactions and regulation of molecular motors in Xenopus melanophores. J Cell Biol, 2002, 156: 855-865.
34. Levi V, Gelfand VI, Serpinskaya AS, et al. Melanosomes transported by myosin-V in Xenopus melanophores perform slow 35 nm steps. Biophys J, 2006, 90: 107-109.
35. Kural C, Serpinskaya AS, Chou YH, et al. Tracking melanosomes inside a cell to study molecular motors and their interaction. Proc Natl Acad Sci USA, 2007, 104: 5378-5382.
36. Komori Y, Iwane AH and Yanagida T. MyosinⅤmakes two brownian 90 degrees rotations per 36-nm step. Nat Struct Mol Biol, 2007, 14: 968-973.
37. Cappello G, Pierobon P, Symonds C, et al. MyosinⅤstepping mechanism. Proc Natl Acad Sci USA, 2007, 104(39): 15328-15333.
38. Chen YT, Holcomb C, and Moore HH. Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag. Proc. Natl. Acad. Sci. USA, 1993, 90: 6508-6512.
39. Huber LA, Hoop MJ, Dupree P, et al. Protein transport to the dendritic plasma 73membrane of cultured neurons is regulated by rab8p. J Cell Biol, 1993, 123: 47-55.
40. Hung SL, Lee PL, Chen HW, et al. Analysis of the steps involved in dengue virus entry into host cells. Virology, 1999, 257:156-167.
41. Morrison TB, Weiss JJ and Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998, 24: 954-962.
42. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt Method. Methods, 2001, 25: 402-408.
43. Hattula K, Furuhjelm J, Tikkanen J, et al. Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. J Cell Sci, 2006, 119: 4866-4877.
44. Seabra MC, Mules EH, and Hume AN. Rab GTPases, intracellular traffic and disease. Trends Mol Med, 2002, 8: 23-30.
45. Murray JL, Mavrakis M, McDonald NJ, et al. Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol, 2005, 79: 11742-11751.
46. Stadler K, Allison SL, Schalich J, et al. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol, 1997, 71: 8475-8481.
47. Heinz FX and Allison SL. Flavivirus structure and membrane fusion. Adv Virus Res, 2003, 59: 63-97.
48. Guirakhoo F, Bolin RA and Roehrig JT. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology, 1992, 191: 921-931.
49. Guirakhoo F, Heinz FX, Mandl CW, et al. Fusion activity of flaviviruses: comparison of mature and immature (prM containing) tick-borne encephalitis virions. J Gen Virol, 1991, 72: 1323-1329.
50. Zhang Y, Corver J, Chipman PR, et al. Structures of immature flavivirus particles. EMBO J, 2003, 22: 2604-2613.
51. Keelapang P, Sriburi R, Supasa S, et al. Alterations of prM cleavage and virus export in prM junction chimeric dengue viruses. J Virol, 2004, 78: 2367-2381.
52. Huber LA, Dupree P and Dotti CG. A Deficiency of the Small GTPase rab8 InhibitsMembrane Traffic in Developing Neurons. Mol Cell Biol, 1995, 15: 918-924.
53. Moritz OL, Tam BM, Hurd LL,et al. Mutant rab8 impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus Rods. Mol Biol Cell, 2001, 12: 2341-2351.
54. Sieczkarski SB and Whittaker GR. Dissecting virus entry via endocytosis. J Gen Virol, 2002, 83: 1535-1545.
55. Hattula K, Furuhjelm J, Tikkanen J, et al. Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. J Cell Sci, 2006, 119: 4866-4877.
56. de Marco N, Buono M, Troise F, et al. Optineurin Increases Cell Survival and Translocates to the Nucleus in a Rab8-dependent Manner upon an Apoptotic Stimulus. J Biol Chem, 2006, 281: 16147-16156.
57. Sieczkarski SB and Whittaker GR. Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic, 2003, 4: 333-343.
58. Vidricaire G and Tremblay MJ. Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells. J Immunol. 2005, 175: 6517-6530.
59. Krishnan MN, Sukumaran B, Pal U, et al. Rab 5 is required for the cellular entry of dengue and West Nile viruses. J Virol, 2007, 81: 4881-4885.
60. Vonderheit A and Helenius A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol, 2005, 3: 1225-1238.
61. Jordens I, Marsman M, Kuijl C,et al. Rab proteins, connecting transport and vesicle fusion. Traffic, 2005, 6: 1070-1077.
62. de Renzis S, Sonnichsen B and Zerial M. Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes. Nat Cell Biol, 2002, 4: 124-133.
63. Schimmoller F, Simon I and Pfeffer SR. Rab GTPases, directors of vesicle docking. J Biol Chem, 1998, 273: 22161-22164.
64. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science, 1998, 279: 519-526.
65. Vale RD. The molecular motor toolbox for intracellular transport. Cell, 2003, 112: 467-480.
66. Tuxworth RI and Titus MA. Unconventional myosins: anchors in the membrane traffic relay. Traffic, 2000, 1: 11-18.
67. Mehta AD, Rock RS, Rief M, et al. MyosinⅤis a processive actin-based motor. Nature, 1999, 400: 590-593..
68. Rief M, Rock RS, Mehta AD, et al. MyosinⅤstepping kinetics: a molecular model for processivity. Proc Natl Acad Sci USA, 2000, 97: 9482-9486.
69. Pastural E, Barrat FJ, Dufourcq-Lagelouse R, et al. Griscelli desease maps to chromosome 15q21 and is associated with mutations in the myosin-Ⅴa gene. Nat Genet, 16: 289-292.
70. Menasche G, Pastural E, Feldman J, et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet 2000, 25: 173-176.
71. Hume AN, Collinson LM, Rapak A, et al. Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J Cell Biol, 2001, 152: 795-808.
72. Hammer JA and Wu XS. Rabs grab motors: defining the connections between Rab GTPases and motor proteins. Curr Opin Cell Biol, 2002, 14: 69-75.
73. Lapierre LA, Kumar R, Hales CM, et al. MyosinⅤb is associated with plasma membrane recycling systems. Mol Biol Cell, 2001, 12: 1843-1857.
74. Rodman JS and Wandinger-Ness A. Rab GTPases coordinate endocytosis. J Cell Sci, 2000, 113: 183-192.
75. Zhang L, Peeples ME, Boucher RC, et al. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol, 2002, 76: 5654-5666.
76. Brock SC, Goldenring JR and Crowe JE. Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells. Proc Natl Acad Sci USA, 2003, 100: 15143-15148.
1. Seabra MC, Mules EH, and Hume AN. Rab GTPases, intracellular traffic and disease. Trends Mol Med, 2002, 8: 23-30.
2. Stenmerk H and Olkkonen VM. The Rab GTPase family.Genome Biol, 2001. 2: 3007.1-3007.7.
3. Tuvim MJ, Adachi R, Hoffenberg S, et al. Traffic control: rab GTPases and the regulation of interotganellar transport. News Physio Sci, 2001, 16: 56-61.
4. Martinez O and Goud B. Rab proteins. Biochim Biophys Acta, 1998, 1404: 101-112.
5. Calero M, Chen CZ, Zhu W, et al. Dual prenylation is required for Rab protein localization and function. Mol Biol Cell, 2003, 14: 1852-1867.
6. Takai Y, Sasaki T and Matozaki T. Small GTP-binding proteins. Physiol Rev, 2001,81: 153-208.
7. Nuoffer C, Davidson HW, Matteson J, et al. A GDP-bound of Rabl inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments. J Cell Bio1, 1994, 125: 225-237.
8. Riederer MA, Soldati T, Shapiro AD, et al. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Gell Bio1, 1994, 125: 573-582.
9. Jones S, Jedd G, Kahn RA, et al. Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers. Genetics, 1999, 152: 1543-1556.
10. Echard A, Jollivet F, Martinez O, et al. Interaction of a Golgi-associated kinesin-like protein with Rab6. Science, 1998, 279: 580-585.
11. Waters MG and Pfeffer SR. Membrane tethering in intracellular transport. Curr Opin Cell Biol, 1999, 11: 453-459.
12. Ungermann C, Price A and Wickner W. A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking. Pros Natl Acad Sci USA, 2000, 97: 8889-8891.
13. Cao X and Barlowe C. Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes. J Cell Biol, 2000, 149: 55-66.
14. McBride HM, Rybin V, MurpHy C, et al. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. Cell, 1999, 98: 377-386.
15. Overmeyer JH and Maltese WA. Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein. J Biol Chem, 1992, 267: 22686-22692.
16. Nielsen E, Severin F, Backer JM, et al. Rab5 regulates motility of early endosomes on microtubules. Nat Cell Biol, 1999, 1: 376–382
17. Mannova P and Forstova J. Mouse polyomavirus utilizes recycling endosomes for a traffic pathway independent of COPI vesicle transport. J Virol, 2003, 77: 1672-1681.
18. Sieczkarski SB and Whittaker GR. Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic, 2003, 4: 333-343.
19. Enouf V, Chwetzoff S, Trugnan G, et al. Interactions of rotavirus VP4 spike protein with the endosomal protein Rab5 and the prenylated Rab acceptor PRA1. J Virol, 2003, 77: 7041-7047.
20. Vidricaire G and Tremblay MJ. Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells. J Immunol. 2005, 175: 6517-6530.
21. Querbes W, O'Hara BA, Williams G, et al. Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands. J Virol, 2006, 80: 9402-9413.
22. Krishnan MN, Sukumaran B, Pal U, et al. Rab 5 is required for the cellular entry of dengue and West Nile viruses. J Virol, 2007, 81: 4881-4885
23. Colpitts TM, Moore AC, Kolokoltsov AA, et al. Venezuelan equine encephalitis virus infection of mosquito cells requires acidification as well as mosquito homologs of the endocytic proteins Rab5 and Rab7. Virology, 2007, 369: 78-91.
24. Coyne CB, Shen L, Turner JR, et al. Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5. Cell Host Microbe, 2007, 2: 181-192.
25. Feng Y, Press B, and Wandinger-Ness A. Rab7: an important regulator of late endocytic membrane traffic. J Cell Biol, 1995, 131: 1435–1452.
26. Vonderheit A and Helenius A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol, 2005, 3: 1225-1238.
27. Ding W, Zhang LN, Yeaman C, et al. rAAV2 traffics through both the late and the recycling endosomes in a dose-dependent fashion. Mol Ther, 2006, 13: 671-682.
28. Brass AL, Dykxhoorn DM, Benita Y, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science, 2008, 319: 921-926.
29. Sklan EH, Serrano RL, Einav S, et al. TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication. J Biol Chem, 2007, 282: 36354-36361. .
30. Varthakavi V, Smith RM, Martin KL, et al. The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of HIV-1 particle release. Traffic, 2006, 7: 298-307.
31. Murray JL, Mavrakis M, McDonald NJ, et al. Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol, 2005, 79: 11742-11751.
32. Sfakianos JN and Hunter E. M-PMV capsid transport is mediated by Env/Gag interactions at the pericentriolar recycling endosome. Traffic, 2003, 4: 671-680.
33. Liebl D, Difato F, HorníkováL, et al. Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in Rab11-positive endosomes. J Virol, 2006, 80: 4610-4622.
34. Raamsman MJ, Locker JK, de Hooge A, et al. Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol, 2000, 74: 2333-2342.
35. Homman-Loudiyi M, Hultenby K, Britt W, et al. Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase II. J Virol, 2003, 77: 3191-3203.
36. Sklan EH, Staschke K, Oakes TM, et al. A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication. J Virol, 2007, 81: 11096-11105.
2. An J, Zhou DS, Kawasaki K, et al. The pathogenesis of spinal cord involvement in dengue virus infection. Virchows Arch, 2003, 442: 472-481.
3. Barth OM. Ultrastructural aspects of the dengue virus (flavivirus) particle morphogenesis. J Submicrosc Cytol Pathol, 1999, 31: 407-412.
4. Takai Y, Sasaki T and Matozaki T. Small GTP-binding proteins. Physiol Rev, 2001,81: 153-208.
5. Huber LA, Pimplikar S, Parton RG, et al. Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane. J Cell Biol, 1993, 123: 35-45.
6. Chabrillat ML, Wilhelm C, Wasmeier C, et al. Rab8 regulates the actin-based movement of melanosomes. Mol Biol Cell, 2005, 16: 1640-1650.
7. Sato T, Mushiake S, Kato Y, et al. The Rab8 GTPase regulates apical protein localization in intestinal cells. Nature, 2007, 488: 366-369.
8. Per?nen J, Auvinen P, Virta H, et al. Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts. J Cell Biol, 1996, 135: 153-167.
9. Rodriguez OC and Cheney RE. Human myosin-Ⅴc is a novel classⅤmyosin expressed in epithelial cells. J Cell Biol, 2002, 115: 991-1004.
10.王嘉丽,陈炜,万颖杰等.登革病毒感染后ECV304单层细胞通透性与微丝关系的研究.中国人兽共患病杂志, 2005, 21: 953–957.
11. Bhamarapravati N, Tuchinda P and Boonyapaknavik V. Pathology of Thailand haemorrhagic fever: a study of 100 autopsy cases. Ann Trop Med Parasitol, 1967, 61: 500-510.
12. Burke T. Dengue haemorrhagic fever: a pathological study. Trans R Soc Trop Med Hyg, 1968, 62: 682-692.
13. Kuo CH, Tai DI, Chang-Chien CS, et al. Liver biochemical tests and dengue fever. AmJ Trop Med Hyg, 1992, 47: 265-270.
14. Marianneau P, Cardona A, Edelman L, et al. Dengue virus replication in human hepatoma cells activates NF-kappaB which in turn induces apoptotic cell death. J Virol, 1997, 71: 3244-3249.
15. Marianneau P, Megret F, Olivier R, et al. Dengue 1 virus binding to human hepatoma HepG2 and simian Vero cell surfaces differs. J Gen Virol, 1996, 77: 2547-2554.
16. Rosen L, Khin MM and U T. Recovery of virus from the liver of children with fatal dengue: reflections on the pathogenesis of the disease and its possible analogy with that of yellow fever. Res Virol, 1989, 140: 351-360.
17. Chavrier P, Vingron M, Sander C, et al. Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line. Mol Cell Biol, 1990, 10: 6578-6585.
18. Wilson AL, Erdman RA, Castellano F, et al. Prenylation of Rab8 GTPase by type I and type II geranylgeranyl transferases. Biochem J, 1998, 333: 497-504.
19. Taylor KA. Regulation and recycling of myosinⅤ. Curr Opin Cell Biol, 2007, 19:67-74.
20.司徒镇强,吴军政主编.细胞培养.西安:世界图书出版社.
21. Karniguian A, Zahraoui A, and Tavitian A. Identification of small GTP-binding rab proteins in human platelets: Thrombin-induced phosphorylation of rab3B, rab6, and rab8 proteins. Proc Natl Acad Sci USA, 1993, 90: 7647-7651.
22. Deretic D, Huber LA, Ransom N, et al. Rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis. J Cell Sci, 1995, 108; 215-224.
23. Ren M, Zeng J, Lemos-Chiarandini C, et al. In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase. Proc Natl Acad Sci USA, 1996, 93: 5151-5155.
24. Gerges NZ, Backos DS and Esteban JA. Local control of AMPA receptor trafficking at the postsynaptic terminal by a small GTPase of the Rab family. J Biol Chem, 2004, 279: 43870–43878.
25. Uno T, Nakao A and Katsurauma C. Phosphorylation of Rab Proteins from the Brain of Bombyx mori. Arch Insect Bioch Phys, 2004, 57: 68-77.
26. Hattula K, Furuhjelm J, Arffman A, et al. A Rab8-specific GDP/GTP exchange factoris involved in actin remodeling and polarized membrane transport. Mol Cell Biol, 2002, 13: 3268-3280.
27. Ang AL, F?lsch H, Koivisto UM, et al. The Rab8 GTPase selectively regulates AP-1B–dependent basolateral transport in polarized Madin-Darby canine kidney cells. J Cell Biol, 2003, 163: 339-350.
28. Bujard H,Gentz R,Lanzer M,et al. A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo. Methods Enzymol, 1987,155: 416-433.
29. Stuber D,Bannwarth W,Pink JR,et al. New B cell epitopes in the plasmodium falciparum malaria circumsporozoite pritein. Eur J Immunol, 1990,20: 819-824.
30.叶菁,陈广生,黄亚渝等.人MAGE-1基因的克隆、原核表达及抗体的制备.免疫学杂志, 2004,20:259-263.
31. J.萨姆布鲁克等著,金冬雁译.分子克隆实验指南(第二版).北京:科学出版社,1992:855-857.
32. Sellers JR and Veigel C. Walking with myosinⅤ. Curr Opin Cell Biol, 2006, 18: 68-73.
33. Gross SP, Tuma MC, Deacon SW, et al. Interactions and regulation of molecular motors in Xenopus melanophores. J Cell Biol, 2002, 156: 855-865.
34. Levi V, Gelfand VI, Serpinskaya AS, et al. Melanosomes transported by myosin-V in Xenopus melanophores perform slow 35 nm steps. Biophys J, 2006, 90: 107-109.
35. Kural C, Serpinskaya AS, Chou YH, et al. Tracking melanosomes inside a cell to study molecular motors and their interaction. Proc Natl Acad Sci USA, 2007, 104: 5378-5382.
36. Komori Y, Iwane AH and Yanagida T. MyosinⅤmakes two brownian 90 degrees rotations per 36-nm step. Nat Struct Mol Biol, 2007, 14: 968-973.
37. Cappello G, Pierobon P, Symonds C, et al. MyosinⅤstepping mechanism. Proc Natl Acad Sci USA, 2007, 104(39): 15328-15333.
38. Chen YT, Holcomb C, and Moore HH. Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag. Proc. Natl. Acad. Sci. USA, 1993, 90: 6508-6512.
39. Huber LA, Hoop MJ, Dupree P, et al. Protein transport to the dendritic plasma 73membrane of cultured neurons is regulated by rab8p. J Cell Biol, 1993, 123: 47-55.
40. Hung SL, Lee PL, Chen HW, et al. Analysis of the steps involved in dengue virus entry into host cells. Virology, 1999, 257:156-167.
41. Morrison TB, Weiss JJ and Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998, 24: 954-962.
42. Livak KJ and Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt Method. Methods, 2001, 25: 402-408.
43. Hattula K, Furuhjelm J, Tikkanen J, et al. Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. J Cell Sci, 2006, 119: 4866-4877.
44. Seabra MC, Mules EH, and Hume AN. Rab GTPases, intracellular traffic and disease. Trends Mol Med, 2002, 8: 23-30.
45. Murray JL, Mavrakis M, McDonald NJ, et al. Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol, 2005, 79: 11742-11751.
46. Stadler K, Allison SL, Schalich J, et al. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol, 1997, 71: 8475-8481.
47. Heinz FX and Allison SL. Flavivirus structure and membrane fusion. Adv Virus Res, 2003, 59: 63-97.
48. Guirakhoo F, Bolin RA and Roehrig JT. The Murray Valley encephalitis virus prM protein confers acid resistance to virus particles and alters the expression of epitopes within the R2 domain of E glycoprotein. Virology, 1992, 191: 921-931.
49. Guirakhoo F, Heinz FX, Mandl CW, et al. Fusion activity of flaviviruses: comparison of mature and immature (prM containing) tick-borne encephalitis virions. J Gen Virol, 1991, 72: 1323-1329.
50. Zhang Y, Corver J, Chipman PR, et al. Structures of immature flavivirus particles. EMBO J, 2003, 22: 2604-2613.
51. Keelapang P, Sriburi R, Supasa S, et al. Alterations of prM cleavage and virus export in prM junction chimeric dengue viruses. J Virol, 2004, 78: 2367-2381.
52. Huber LA, Dupree P and Dotti CG. A Deficiency of the Small GTPase rab8 InhibitsMembrane Traffic in Developing Neurons. Mol Cell Biol, 1995, 15: 918-924.
53. Moritz OL, Tam BM, Hurd LL,et al. Mutant rab8 impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus Rods. Mol Biol Cell, 2001, 12: 2341-2351.
54. Sieczkarski SB and Whittaker GR. Dissecting virus entry via endocytosis. J Gen Virol, 2002, 83: 1535-1545.
55. Hattula K, Furuhjelm J, Tikkanen J, et al. Characterization of the Rab8-specific membrane traffic route linked to protrusion formation. J Cell Sci, 2006, 119: 4866-4877.
56. de Marco N, Buono M, Troise F, et al. Optineurin Increases Cell Survival and Translocates to the Nucleus in a Rab8-dependent Manner upon an Apoptotic Stimulus. J Biol Chem, 2006, 281: 16147-16156.
57. Sieczkarski SB and Whittaker GR. Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic, 2003, 4: 333-343.
58. Vidricaire G and Tremblay MJ. Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells. J Immunol. 2005, 175: 6517-6530.
59. Krishnan MN, Sukumaran B, Pal U, et al. Rab 5 is required for the cellular entry of dengue and West Nile viruses. J Virol, 2007, 81: 4881-4885.
60. Vonderheit A and Helenius A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol, 2005, 3: 1225-1238.
61. Jordens I, Marsman M, Kuijl C,et al. Rab proteins, connecting transport and vesicle fusion. Traffic, 2005, 6: 1070-1077.
62. de Renzis S, Sonnichsen B and Zerial M. Divalent Rab effectors regulate the sub-compartmental organization and sorting of early endosomes. Nat Cell Biol, 2002, 4: 124-133.
63. Schimmoller F, Simon I and Pfeffer SR. Rab GTPases, directors of vesicle docking. J Biol Chem, 1998, 273: 22161-22164.
64. Hirokawa N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science, 1998, 279: 519-526.
65. Vale RD. The molecular motor toolbox for intracellular transport. Cell, 2003, 112: 467-480.
66. Tuxworth RI and Titus MA. Unconventional myosins: anchors in the membrane traffic relay. Traffic, 2000, 1: 11-18.
67. Mehta AD, Rock RS, Rief M, et al. MyosinⅤis a processive actin-based motor. Nature, 1999, 400: 590-593..
68. Rief M, Rock RS, Mehta AD, et al. MyosinⅤstepping kinetics: a molecular model for processivity. Proc Natl Acad Sci USA, 2000, 97: 9482-9486.
69. Pastural E, Barrat FJ, Dufourcq-Lagelouse R, et al. Griscelli desease maps to chromosome 15q21 and is associated with mutations in the myosin-Ⅴa gene. Nat Genet, 16: 289-292.
70. Menasche G, Pastural E, Feldman J, et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat Genet 2000, 25: 173-176.
71. Hume AN, Collinson LM, Rapak A, et al. Rab27a regulates the peripheral distribution of melanosomes in melanocytes. J Cell Biol, 2001, 152: 795-808.
72. Hammer JA and Wu XS. Rabs grab motors: defining the connections between Rab GTPases and motor proteins. Curr Opin Cell Biol, 2002, 14: 69-75.
73. Lapierre LA, Kumar R, Hales CM, et al. MyosinⅤb is associated with plasma membrane recycling systems. Mol Biol Cell, 2001, 12: 1843-1857.
74. Rodman JS and Wandinger-Ness A. Rab GTPases coordinate endocytosis. J Cell Sci, 2000, 113: 183-192.
75. Zhang L, Peeples ME, Boucher RC, et al. Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology. J Virol, 2002, 76: 5654-5666.
76. Brock SC, Goldenring JR and Crowe JE. Apical recycling systems regulate directional budding of respiratory syncytial virus from polarized epithelial cells. Proc Natl Acad Sci USA, 2003, 100: 15143-15148.
1. Seabra MC, Mules EH, and Hume AN. Rab GTPases, intracellular traffic and disease. Trends Mol Med, 2002, 8: 23-30.
2. Stenmerk H and Olkkonen VM. The Rab GTPase family.Genome Biol, 2001. 2: 3007.1-3007.7.
3. Tuvim MJ, Adachi R, Hoffenberg S, et al. Traffic control: rab GTPases and the regulation of interotganellar transport. News Physio Sci, 2001, 16: 56-61.
4. Martinez O and Goud B. Rab proteins. Biochim Biophys Acta, 1998, 1404: 101-112.
5. Calero M, Chen CZ, Zhu W, et al. Dual prenylation is required for Rab protein localization and function. Mol Biol Cell, 2003, 14: 1852-1867.
6. Takai Y, Sasaki T and Matozaki T. Small GTP-binding proteins. Physiol Rev, 2001,81: 153-208.
7. Nuoffer C, Davidson HW, Matteson J, et al. A GDP-bound of Rabl inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments. J Cell Bio1, 1994, 125: 225-237.
8. Riederer MA, Soldati T, Shapiro AD, et al. Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network. J Gell Bio1, 1994, 125: 573-582.
9. Jones S, Jedd G, Kahn RA, et al. Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers. Genetics, 1999, 152: 1543-1556.
10. Echard A, Jollivet F, Martinez O, et al. Interaction of a Golgi-associated kinesin-like protein with Rab6. Science, 1998, 279: 580-585.
11. Waters MG and Pfeffer SR. Membrane tethering in intracellular transport. Curr Opin Cell Biol, 1999, 11: 453-459.
12. Ungermann C, Price A and Wickner W. A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking. Pros Natl Acad Sci USA, 2000, 97: 8889-8891.
13. Cao X and Barlowe C. Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes. J Cell Biol, 2000, 149: 55-66.
14. McBride HM, Rybin V, MurpHy C, et al. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13. Cell, 1999, 98: 377-386.
15. Overmeyer JH and Maltese WA. Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein. J Biol Chem, 1992, 267: 22686-22692.
16. Nielsen E, Severin F, Backer JM, et al. Rab5 regulates motility of early endosomes on microtubules. Nat Cell Biol, 1999, 1: 376–382
17. Mannova P and Forstova J. Mouse polyomavirus utilizes recycling endosomes for a traffic pathway independent of COPI vesicle transport. J Virol, 2003, 77: 1672-1681.
18. Sieczkarski SB and Whittaker GR. Differential requirements of Rab5 and Rab7 for endocytosis of influenza and other enveloped viruses. Traffic, 2003, 4: 333-343.
19. Enouf V, Chwetzoff S, Trugnan G, et al. Interactions of rotavirus VP4 spike protein with the endosomal protein Rab5 and the prenylated Rab acceptor PRA1. J Virol, 2003, 77: 7041-7047.
20. Vidricaire G and Tremblay MJ. Rab5 and Rab7, but not ARF6, govern the early events of HIV-1 infection in polarized human placental cells. J Immunol. 2005, 175: 6517-6530.
21. Querbes W, O'Hara BA, Williams G, et al. Invasion of host cells by JC virus identifies a novel role for caveolae in endosomal sorting of noncaveolar ligands. J Virol, 2006, 80: 9402-9413.
22. Krishnan MN, Sukumaran B, Pal U, et al. Rab 5 is required for the cellular entry of dengue and West Nile viruses. J Virol, 2007, 81: 4881-4885
23. Colpitts TM, Moore AC, Kolokoltsov AA, et al. Venezuelan equine encephalitis virus infection of mosquito cells requires acidification as well as mosquito homologs of the endocytic proteins Rab5 and Rab7. Virology, 2007, 369: 78-91.
24. Coyne CB, Shen L, Turner JR, et al. Coxsackievirus entry across epithelial tight junctions requires occludin and the small GTPases Rab34 and Rab5. Cell Host Microbe, 2007, 2: 181-192.
25. Feng Y, Press B, and Wandinger-Ness A. Rab7: an important regulator of late endocytic membrane traffic. J Cell Biol, 1995, 131: 1435–1452.
26. Vonderheit A and Helenius A. Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes. PLoS Biol, 2005, 3: 1225-1238.
27. Ding W, Zhang LN, Yeaman C, et al. rAAV2 traffics through both the late and the recycling endosomes in a dose-dependent fashion. Mol Ther, 2006, 13: 671-682.
28. Brass AL, Dykxhoorn DM, Benita Y, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Science, 2008, 319: 921-926.
29. Sklan EH, Serrano RL, Einav S, et al. TBC1D20 is a Rab1 GTPase-activating protein that mediates hepatitis C virus replication. J Biol Chem, 2007, 282: 36354-36361. .
30. Varthakavi V, Smith RM, Martin KL, et al. The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of HIV-1 particle release. Traffic, 2006, 7: 298-307.
31. Murray JL, Mavrakis M, McDonald NJ, et al. Rab9 GTPase is required for replication of human immunodeficiency virus type 1, filoviruses, and measles virus. J Virol, 2005, 79: 11742-11751.
32. Sfakianos JN and Hunter E. M-PMV capsid transport is mediated by Env/Gag interactions at the pericentriolar recycling endosome. Traffic, 2003, 4: 671-680.
33. Liebl D, Difato F, HorníkováL, et al. Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in Rab11-positive endosomes. J Virol, 2006, 80: 4610-4622.
34. Raamsman MJ, Locker JK, de Hooge A, et al. Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol, 2000, 74: 2333-2342.
35. Homman-Loudiyi M, Hultenby K, Britt W, et al. Envelopment of human cytomegalovirus occurs by budding into Golgi-derived vacuole compartments positive for gB, Rab 3, trans-golgi network 46, and mannosidase II. J Virol, 2003, 77: 3191-3203.
36. Sklan EH, Staschke K, Oakes TM, et al. A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication. J Virol, 2007, 81: 11096-11105.