轮状病毒与宿主细胞相互作用的研究
摘要
轮状病毒（Rotavirus，RV）属于呼肠孤病毒属，是一种无包膜的二十面体形状病毒。RV衣壳由三层同心包裹的结构蛋白构成，核心含有一个由11个双链RNA片段构成的基因组，后者编码12个病毒蛋白，包括6个结构蛋白(VP1-4,6,7)和6个非结构蛋白(NSP1-6)。轮状病毒具有严格的细胞嗜性，只能有效感染胃肠道上皮及肾上皮细胞。人和动物的婴幼个体对轮状病毒普遍易感。然而，随着年龄的增大，他们对RV的易感性也显著降低。产生这种变化的原因可能是在个体的生长过程中，针对RV的某些宿主限制性细胞因子出现了表达。目前许多研究表明一些宿主蛋白例如APOBEC3G、Lv1、Ref1及EWI-2wint具有明显的抑制病毒感染的作用，而这些宿主限制性因子的表达可增加宿主对病毒的抵抗能力，降低宿主的易感性。然而，在RV感染中，类似的具有抑制RV感染的宿主蛋白却少有发现。如同在其它很多的病毒感染，在RV感染中，宿主蛋白的合成受到了广泛的抑制。但是有研究发现，一些宿主蛋白如整合素α2β1、β2及伴侣蛋白Grp78、Grp94在RV感染过程中表达上调，并在RV的复制过程中发挥重要作用。这些结果表明RV能够选择性促进某些宿主蛋白的表达以保证病毒感染的顺利进行。相反，宿主细胞是否同样可以通过上调一些抗RV的宿主蛋白的表达以抑制RV的感染呢？
RV感染是导致全球范围内5岁以下婴幼儿急性脱水腹泻的主要原因。RV腹泻发生的确切的分子机制到目前为止仍然没有定论。最初RV腹泻被认为是由于小肠绒毛顶部肠上皮细胞被RV感染后生理功能受到破坏引起的。一些体外实验研究表明RV感染可以引起肠上皮细胞的凋亡和坏死。在对RV腹泻动物模型的研究中进一步发现RV感染可以导致小肠粘膜发生病理改变及功能异常，其程度的轻重取决于RV病毒株的毒力。然而，这种机制难以解释为什么在RV腹泻中常常发现肠道粘膜只有轻微的组织病理学改变且没有肠上皮细胞的破坏的现象。其它一些研究发现，RV感染后肠上皮细胞刷状缘相关的一些酶如蔗糖酶异麦芽糖酶（SI）、乳糖酶-根皮苷水解酶，二肽基肽酶IV（DPPIV）、Na+-D-葡萄糖转运体1（SGLT1）以及Na+L-丙氨酸转运体的表达及活性的出现损害，由此推测RV感染导致肠上皮细胞对葡萄糖和氨基酸的消化吸收能力减弱可能是RV感染引起腹泻发生的一种可能的机制。但是这些研究都未能给出一个被广泛接受的RV感染引起腹泻发生的具体的分子机制。
近年来，蛋白质组技术被广泛应用于病毒感染的研究之中，这对揭示病毒感染的病理生理机制，深化我们对病毒-宿主相互作用的认识发挥了巨大作用。在本研究中，我们通过蛋白质组技术来发现新的RV宿主限制性因子以及探究RV腹泻的新机制。本研究分为两部分，第一部分采用二维凝胶电泳(2-DE)等技术发现新的RV宿主限制因子，观察宿主细胞的天然免疫机制是如何应对RV感染的；第二部分则运用一种先进的定量蛋白质组技术SILAC来研究RV感染肠上皮细胞引发腹泻的分子机制，观察RV是如何破坏宿主的天然免疫屏障从而致病的。本研究将会加深对RV感染及RV与宿主细胞相互作用分子机制的认识，为RV腹泻的预防和治疗提供实验依据。
第一部分：新的RV宿主限制性因子发现
首先，我们确定了RV感染的合适时间和MOI以获得最好的感染效果和细胞活力，这对随后的二维凝胶电泳分析十分重要。为进行二维凝胶电泳样本的制备，我们将MA104培养在直径10cm的组织培养皿中，当细胞完全汇合时，用用纯化的人轮状病毒Wa株感染（moi=3），同时用等量的DMEM作为对照感染。细胞感染12后用冷0.9%NaCl冲洗感染的细胞，蛋白裂解液提取细胞总蛋白。采用13cm ReadyStrip IPG胶条(非线性,pI3-10)在IPGphor电泳系统上进行蛋白质的等电聚焦。等电聚焦结束后，将胶条置于预先制好的12.5%聚丙烯酰胺凝胶上垂直电泳以进行多肽分离。电泳结束后，用胶体蓝对凝胶进行染色，并采用GS800扫描仪对染色后的凝胶进行扫描，用PDQuest6.1软件进行图像分析。我们选择RV感染后表达显著上调50%以上的蛋白点，通过MALDI-TOF-MS进行蛋白序列鉴定。结果显示在人RV Wa株感染的MA104细胞中共发现24个蛋白点的表达显著上调50%以上，质谱成功鉴定了其中16个蛋白点。在成功鉴定的蛋白点中，11个为宿主蛋白（10种），5个点为病毒蛋白（4种）。我们随机选取了一些宿主蛋白如ACP1、ALDOA、CYPA和KRT20对它们的表达采用Western blot和qRT-PCR等方法进一步验证。
其次，为了明确CYPA在RV感染中的作用，我们先进行了免疫荧光实验。结果发现在RV感染MA104细胞后，CYPA与RV非结构蛋白NSP5出现显著共定位，提示CYPA被招募到RV病毒粒质。继而我们通过免疫共沉淀实验发现， RV结构蛋白VP2与CYPA能够相互共沉淀，表明这两种蛋白质可能存在直接或间接的相互作用。在此基础上为了进一步明确CYPA是否对RV感染有抑制或促进作用，我们在宿主细胞内过表达野生型CYPA及其脯氨酰顺反异构酶功能缺失突变体CYPA/R55A，或干扰宿主细胞内源性CYPA的表达，观察这些处理对RV感染的影响。结果显示，过表达野生型CYPA显著降低RV复制，而过表达CYPA/R55A会显著促进RV复制。降低内源性CYPA表达同样显著促进RV的复制。这些结果显示CYPA可以通过其脯氨酰顺反异构酶活性抑制RV复制。同时，过表达CYPA及其酶功能缺失突变体CYPA/R55A均能降低宿主对RV的易感性。进一步研究发现CYPA还可以通过促进宿主细胞IFN-I反应来抑制RV感染，这种功能不依赖于其脯氨酰顺反异构酶活性但是可能依赖于宿主细胞JNK信号路径的激活。
最后，由于新生BALB/c小鼠对RV非常易感, RV感染容易产生腹泻症状。随着日龄的增大(>2周) BALB/c小鼠对RV易感性显著降低。为了明确小肠上皮细胞CYPA的表达是否与BALB/c小鼠对RV易感性的变化相关，我们比较了不同日龄段BALB/c小鼠小肠上皮细胞中CYPA的表达，发现在新生小鼠(5日龄) CYPA仅在小肠绒毛底部，主要是在肠隐窝处上皮细胞中有少量表达，而在RV容易感染到的小肠绒毛上部的上皮细胞中几乎不见CYPA的表达。相反，在稍大的BALB/c小鼠(15日龄)的小肠绒毛，不管是绒毛顶端还是基底部，其上皮细胞中CYPA均有丰富的表达。这些结果表明，新生BALB/c小鼠小肠绒毛上段上皮细胞中CYPA表达缺失可能是导致其对RV易感的重要原因。
第二部分：RV感染肠上皮细胞引发腹泻新机制的发现
首先，我们用人轮状病毒(Wa株)感染人肠上皮细胞Caco-2，同时在培养基中加入100μg/ml胰蛋白酶，以此模拟肠道消化性的环境，观察肠腔内消化性环境对RV感染肠上皮细胞后果的可能影响。实验发现，在消化性环境中RV的感染会导致肠上皮细胞快速脱落。台盼蓝染色表明脱落的细胞活性良好，流式细胞术结果显示这些脱落的细胞在脱落时没有出现明显的凋亡或坏死。在非消化性环境下肠上皮细胞只会在RV感染的末期才出现明显的脱落，此时脱落细胞几乎全部凋亡及坏死。结果提示，体内RV感染中，被感染的肠上皮细胞可能还没有来的及凋亡或坏死或其它明显功能损害就已经从基底膜脱落，彻底失去功能。我们进一步检测了小肠内不同段肠腔内的胰蛋白酶的活性，结果显示在BABL/c小鼠幼仔的空肠中、下段和回肠的上段胰蛋白酶活性最高。此外，通过结晶紫对BABL/c肠上皮细胞进行活体染色发现，RV感染引起的肠上皮细胞脱落在上述肠段中最为明显。我们的结果提示RV感染导致肠上皮细胞在肠道消化性环境中快速脱落可能才是RV腹泻产生的根本原因。
其次，为了寻找消化性环境中RV感染引肠上皮细胞快速脱落的直接原因，我们采用了一种定量蛋白质组学方法-细胞培养稳定同位素氨基酸标记（SILAC）来分析RV感染后Caco-2细胞蛋白表达谱的变化。结果显示，RV感染显著削弱了与细胞间的粘附相关蛋白以及细胞与细胞外基质的粘附相关的蛋白质的表达，如E-caherin, Plakoglobin,Desmoplakin,层粘连蛋白B2,纤维连接蛋白1, Plectin1, Cingulin以及ZO-1等。Western blot及qRT-PCR进一步证实了这些蛋白质在RV感染后显著下降。免疫荧光实验进一步验证了RV感染引起细胞间连接包括紧密连接，粘附连接以及桥粒连接的严重损坏。进一步通过结晶紫染色发现，RV感染显著破坏肠上皮细胞屏障功能，增大细胞间隙，导致胰蛋白酶向细胞底部严重渗漏，从而破坏细胞-细胞外基质粘附，最终引起上皮细胞脱落。
最后，为了明确RV感染导致细胞粘附功能破坏从而引起肠上皮细胞脱落的的相关信号机制，我们对RV感染中可能涉及到的激酶和磷酸酶与细胞脱落之间的关系进行了研究。我们发现抑制酪氨酸蛋白激酶可以显著抑制消化性环境中RV感染引起的肠上皮细胞的脱落，抑制蛋白酪氨酸磷酸酶则显著促进RV感染及感染的肠上皮细胞的脱落。抑制蛋白酶磷酸2A（PP2A）同样显著促进RV感染引起肠上皮细胞的脱落。此外，免疫印迹法显示RV感染引起PP2A两个主要结构亚单位PPP2R1A和PPP2R1B表达减少。RV感染可以引起细胞胞浆内Ca2+浓度升高，我们结果显示，通过离子霉素和毒胡萝卜素增大胞浆Ca2+浓度加速RV感染引起的细胞脱落。然而，使用BAMPT-AM和U73122抑制胞浆内Ca2+增加却不能阻止消化性环境中RV感染引起的肠上皮细胞的快速脱落。这表明RV感染引起胞浆Ca2+的增加只是导致宿主细胞的脱落促进因素而非决定因素。
综上所述，本研究发现CYPA是一种新的抗RV感染的宿主限制性因子。CYPA通过本身的脯氨酰顺反异构酶活性抑制RV的复制，其在肠上皮细胞中的表达可能与宿主对RV的易感性密切相关。CYPA同时还可以通过介导宿主细胞IFN-I反应来抑制RV感染。CYPA的这种功能不依赖于其脯氨酰顺反异构酶活性，而可能依赖于宿主细胞中JNK信号通路的激活。另一方面，我们发现在消化性环境中RV感染会导致肠上皮细胞快速脱落，这种现象可能才是RV腹泻产生的真正原因。而造成细胞脱落的原因与RV感染引起肠上皮细胞粘附功能及屏障功能的破坏，胰蛋白酶渗透到细胞基底部破坏细胞与细胞外基质的粘附密切相关。在这个过程中，RV感染引起的酪氨酸蛋白激酶的激活以及PP2A的抑制起到了关键作用，而RV感染引起的Ca2+的内流是加重而非决定因素。
Rotavirus (RV), a member of the family Reoviridae, is a non-enveloped icosahedral viruswhose capsid is formed by three concentric layers of structural proteins and contains agenome made up of11segments of double-stranded RNA which encode12viral proteinsincluding6structural proteins (VP1-4,6,7) and6non-structural proteins (NSP1-6). RV hasstrict cell tropism and only efficiently infects epithelial cells of gastrointestinal tract andkidney. Infants of human and animals are generally susceptible to RV infection; however, asthey grow older their susceptibility is greatly reduced. The reason for this change could bedue to the expression of some host restrictive factors of RV in enterocytes during growth. Infact, some host proteins such as APOBEC3G, Lv1, Ref1and EWI-2wint have been reported toplay a repressive role in viral infections. The expressions of such host restrictive factors aresupposed to contribute to host resistance to viral infections. However, as few such host factorsthat could protect host against RV infection were identified, the exact reason remains unclear.As many other viral infections, RV infection also elicits a global shutoff of host proteinsynthesis. However, several studies revealed that some host proteins such as α2β1and β2integrins, Grp78and Grp94were up-regulated and required for successful virus reproduction.Therefore, it is possible that RV is able to promote the expression of certain host proteins forefficient viral replication. On the other hand, it is wondering that whether host cells could alsoelicit the expression of some self-proteins to protect themselves against RV infection.
Rotavirus (RV) is the primary cause of acute dehydrating diarrhea worldwide in infantsand young children under5years of age. However, the precise mechanism of the occurrenceof rotavirus diarrhea is still inconclusive until now. Previously, rotavirus diarrhea was thoughtbe due to enterocyte destruction from the top of intestinal villus. Some reports revealed thatrotavirus infection induced apoptosis and necrosis of enterocyts in vitro and extensive studiesin animal models have reported the presence of histopathologic changes and functionalabnormalities in intestinal infected mucosa that varied from mild to severe depending on the rotavirus strain virulence. However, this mechanism cannot explain the facts in which mildhistopathologic changes without enterocyte destruction are usually found in rotavirus diarrhea.Some recent reports provided some other insights into the mechanism of the occurrence ofrotavirus diarrhea. They found a default in the expression and activity of the brush bordermembrane (BBM)-associated enzymes such as sucrase-isomaltase(SI) Lactase-phlorizinhydrolase, dipeptidyl peptidase IV (DPP IV) as well as Na+-D-glucose symport activity(SGLT1), Na+-L-alanine symporter and Na+-L-alanine symporter after rotavirus infection.These reports presumed that the diminished absorptive capacity of glucose and amino acids ofenterocytes might contribute to the occurrence of rotavirus-induced diarrhea. However, thesestudies still failed to exhibit a convinced molecular mechanism of the occurrence of rotavirusdiarrhea.
In recent years proteomic techniques have been widely used in studying viral infectionsand these have given a better understanding of the underlying virus-host interactions thatmight contribute to pathogenesis following viral infection. In this study, we focused ourinterest on identifying novel host restrictive factors to RV infection and new mechanism ofRV diarrhea by proteomic tools. There were two sections of our study. In section I, we were todiscover novel host restrictive factors to RV using2-dimensional gel electrophoresis (2-DE)coupled with mass spectrometry (MS); in section II, we were to explore new mechanism ofRV diarrhea where a more advance proteomic technique SILAC was used. Our study wouldmake a great advance of the knowledge on RV infection and was beneficial to the preventionand therapy of RV diarrhea.
Section I: Discovery of novel host restrictive factors to RV
Firstly, we determined the appropriate moi and time of infection in order to obtain thebest infectivity and cell viability, which was important for later2DE analysis. For2-DEsample preparation, MA104monolayers cultured in tissue culture dishes (100mm×20mm)were infected with purified RV (Wa strain) at a moi of3, or with DMEM as mock infection.At12h p.i., the infected cells were washed with cold0.9%NaCl, lysed in lysis buffer.2-DEanalysis was carried out using13cm ReadyStrip IPG strips (nonlinear, pI3-10). Then theIPG strips were focused by the IPGphor electrophoresis system. After separation in the firstdimension, the strips were then laid atop precast12.5%gels for separation of polypeptides byvertical electrophoresis. The gels were stained using colloidal blue, scanned with a GS800 scanner and analyzed with PDQuest6.1software; Expression intensity ratios larger than1.5(ratioHRV/mock≥1.5) were set as a threshold indicating significant changes (p≤0.05). A totalof24spots were found significantly up-regulated in HRV-infected MA104cells, amongwhich11protein spots representing10host protein and5protein spots representing4viralproteins were successfully identified by MALDI-TOF-MS analysis. Some of host proteinsidentified such as ACP1, ALDOA, CYPA and KRT20were further validated by western blotand qRT-PCR.
Secondly, to clarify the possible roles of CYPA in RV infection, we carried outImmunofluorescence and Co-immunoprecipitation assays. We found that CYPA was recruitedto the viroplasms upon RV infection in MA104cells. RV structural protein VP2and CYPAcould be co-immunoprecipitated with each other, indicating an interaction between these twoproteins. To make sure whether CYPA played an important role in RV infection, weoverexpressed wild type of CYPA and its PPIase negative mutant CYPA/R55A and knockeddown the endogenous CYPA. We found that Overexpressing wild type of CYPA significantlyreduced RV reproduction in MA104cells, while overexpressing CYPA/R55A greatlyenhanced RV reproduction. Knockdown of endogenous CYPA also remarkably facilitated thereproduction of RV. Therefore, it was indicated that CYPA inhibited RV reproduction throughthe PPIase activity. Notably, the fact that the PPIase activity of CYPA was necessary ininhibiting RV reproduction but was unnecessary in reducing host susceptibility to RVsuggested that there were different ways for CYPA to inhibit RV infection. We further foundthat CYPA could inhibit RV infection by promoting host IFN-β response that was independenton its Pease activity but dependent on JNK signaling pathway.
At last, it is well known that neonatal BALB/c mice are far more susceptible to RVdiarrhea than older (>2weeks) ones. To find out whether CYPA plays an important role in thesusceptibility to RV diarrhea in BALB/c mice, we compared the expression of CYPA in theintestine of BALB/c mice of different ages. We found that in neonatal (5days old) BALB/cmice, CYPA was only expressed in the bottom of intestinal villus, mainly in the enterocytesof intestine crypts, and hardly detected in the upper section of villus of small intestine wherethe enterocytes were the main targets of RV infection. In contrast, in older (>15days old)BALB/c mice CYPA was abundantly expressed in the all the enterocytes of intestinal villus of small intestine. These results suggested that lack of CYPA expression in epithelial cells ofsmall intestine might contribute to host susceptibility to RV diarrhea.
Section II: Discovery of new mechanism of RV diarrhea
Firstly，to find out the effects of RV infection in enterocytes in a digestive environmentsimilar to that in intestinal tract, we infected Caco-2cells with human rotavirus (Wa strain)and100ug/ml trypsin was added into the culture medium. We found that RV infection in adigestive environment resulted in a quick shedding of host cells. FACS showed that there wasno significant apoptosis or necrosis in the detached cells. Trypan blue staining showed that theviability of the detached cells remained well. We further tested the trypsin activity insidedifferent segments of small intestine. We found that the trypsin activity in the middle anddownstream of jejunum as well as upstream of ileum of BABL/c pups was highest.Accordingly, by intravital staining with hexamethylpararosaniline it was found that there wasmost severe shedding of enterocyts in these segments of small intestine mentioned above.
Secondly, to find out the reason why RV infection in a digestive environment elicited asignificant shedding of enterocytes, we analysed the total cell response in Caco-2cells atdifferent times of RV infection by a quantitive proteomic method Stable Isotope Labeling withAmino acids in Cell culture (SILAC). It was found that RV infection significant impaired theexpression of host proteins involved in cell adhesions including both cell-cell adhesion andcell-extracellular matrix adhesion. The impairment of these adhesion molecules such asE-cadherin, plakoglobin, desmoplakin, laminin B2, fibronectin1, plectin1, cingulin as well asZO-1were further validated by western blot and qRT-PCR. Immunofluorescence assay furtherconfirmed the disruption of cell-cell junctions including tight junction, adhesion junction andmacula adherens in RV infection. Further, by hexamethyl-pararosaniline staining, it wasdiscovered that RV infection significantly disrupted the barrier between enterocytes that wasformed by cell junctions, which leaded to a severe leak of trypsin from the culture medium inthe lumen to the bottom of cell where it disrupted cell-extracellular adhesion and ultimatelyresulted in detachment of enterocytes.
At last, to find out the mechanism by which RV infection caused a desruption of celladhesions, we analysed the effects of some kinases and phosphotase that might be involved inRV infection. We found that inhibition of tyrosine protein kinase by genistain significantlyrestored the cell shedding in RV infection and inhibition of tyrosine phosphatase by sodium orthovanadate enhanced cell shedding both in RV-infected and mock-infectedenterocytes. Inhibition of protein phosphatase2A by okadaic acid also significantly enhancedcell shedding in both RV-infected and mock-infected enterocytes. Additionally, western blotshowed that the expression of PP2A subunits like PPP2R1A and PPP2R1B were reduced inRV. Increasing cytosol Ca2+by ionomycin and thapsigargin greatly enhanced the shedding ofcells in RV infection, however, inhibition of cytosol Ca2+by BAMPT-AM and U73122didnot restore the cell shedding in RV infection, indicating that the increase of cytosol Ca2+inRV infection could contribute to the shedding of host cells but was not necessary.
In conclusion, in this study we firstly identified CYPA as a novel host restrictive factorthat confers protection against rotavirus infection and might contribute to host susceptibilityto RV diarrhea. CYPA could also inhibit RV infection by conducting host IFN-β productionthat was independent on its PPIase activity but dependent on the activation of JNK signalingpathway. Then we discovered that RV infection caused severe shedding of enterocytes in adigestive environment, which was attributed to a disruption of cell adhesions caused byactivation of tyrosine protein kinase and a repression of protein phosphatase2A in RVinfection.
RV感染是导致全球范围内5岁以下婴幼儿急性脱水腹泻的主要原因。RV腹泻发生的确切的分子机制到目前为止仍然没有定论。最初RV腹泻被认为是由于小肠绒毛顶部肠上皮细胞被RV感染后生理功能受到破坏引起的。一些体外实验研究表明RV感染可以引起肠上皮细胞的凋亡和坏死。在对RV腹泻动物模型的研究中进一步发现RV感染可以导致小肠粘膜发生病理改变及功能异常，其程度的轻重取决于RV病毒株的毒力。然而，这种机制难以解释为什么在RV腹泻中常常发现肠道粘膜只有轻微的组织病理学改变且没有肠上皮细胞的破坏的现象。其它一些研究发现，RV感染后肠上皮细胞刷状缘相关的一些酶如蔗糖酶异麦芽糖酶（SI）、乳糖酶-根皮苷水解酶，二肽基肽酶IV（DPPIV）、Na+-D-葡萄糖转运体1（SGLT1）以及Na+L-丙氨酸转运体的表达及活性的出现损害，由此推测RV感染导致肠上皮细胞对葡萄糖和氨基酸的消化吸收能力减弱可能是RV感染引起腹泻发生的一种可能的机制。但是这些研究都未能给出一个被广泛接受的RV感染引起腹泻发生的具体的分子机制。
近年来，蛋白质组技术被广泛应用于病毒感染的研究之中，这对揭示病毒感染的病理生理机制，深化我们对病毒-宿主相互作用的认识发挥了巨大作用。在本研究中，我们通过蛋白质组技术来发现新的RV宿主限制性因子以及探究RV腹泻的新机制。本研究分为两部分，第一部分采用二维凝胶电泳(2-DE)等技术发现新的RV宿主限制因子，观察宿主细胞的天然免疫机制是如何应对RV感染的；第二部分则运用一种先进的定量蛋白质组技术SILAC来研究RV感染肠上皮细胞引发腹泻的分子机制，观察RV是如何破坏宿主的天然免疫屏障从而致病的。本研究将会加深对RV感染及RV与宿主细胞相互作用分子机制的认识，为RV腹泻的预防和治疗提供实验依据。
第一部分：新的RV宿主限制性因子发现
首先，我们确定了RV感染的合适时间和MOI以获得最好的感染效果和细胞活力，这对随后的二维凝胶电泳分析十分重要。为进行二维凝胶电泳样本的制备，我们将MA104培养在直径10cm的组织培养皿中，当细胞完全汇合时，用用纯化的人轮状病毒Wa株感染（moi=3），同时用等量的DMEM作为对照感染。细胞感染12后用冷0.9%NaCl冲洗感染的细胞，蛋白裂解液提取细胞总蛋白。采用13cm ReadyStrip IPG胶条(非线性,pI3-10)在IPGphor电泳系统上进行蛋白质的等电聚焦。等电聚焦结束后，将胶条置于预先制好的12.5%聚丙烯酰胺凝胶上垂直电泳以进行多肽分离。电泳结束后，用胶体蓝对凝胶进行染色，并采用GS800扫描仪对染色后的凝胶进行扫描，用PDQuest6.1软件进行图像分析。我们选择RV感染后表达显著上调50%以上的蛋白点，通过MALDI-TOF-MS进行蛋白序列鉴定。结果显示在人RV Wa株感染的MA104细胞中共发现24个蛋白点的表达显著上调50%以上，质谱成功鉴定了其中16个蛋白点。在成功鉴定的蛋白点中，11个为宿主蛋白（10种），5个点为病毒蛋白（4种）。我们随机选取了一些宿主蛋白如ACP1、ALDOA、CYPA和KRT20对它们的表达采用Western blot和qRT-PCR等方法进一步验证。
其次，为了明确CYPA在RV感染中的作用，我们先进行了免疫荧光实验。结果发现在RV感染MA104细胞后，CYPA与RV非结构蛋白NSP5出现显著共定位，提示CYPA被招募到RV病毒粒质。继而我们通过免疫共沉淀实验发现， RV结构蛋白VP2与CYPA能够相互共沉淀，表明这两种蛋白质可能存在直接或间接的相互作用。在此基础上为了进一步明确CYPA是否对RV感染有抑制或促进作用，我们在宿主细胞内过表达野生型CYPA及其脯氨酰顺反异构酶功能缺失突变体CYPA/R55A，或干扰宿主细胞内源性CYPA的表达，观察这些处理对RV感染的影响。结果显示，过表达野生型CYPA显著降低RV复制，而过表达CYPA/R55A会显著促进RV复制。降低内源性CYPA表达同样显著促进RV的复制。这些结果显示CYPA可以通过其脯氨酰顺反异构酶活性抑制RV复制。同时，过表达CYPA及其酶功能缺失突变体CYPA/R55A均能降低宿主对RV的易感性。进一步研究发现CYPA还可以通过促进宿主细胞IFN-I反应来抑制RV感染，这种功能不依赖于其脯氨酰顺反异构酶活性但是可能依赖于宿主细胞JNK信号路径的激活。
最后，由于新生BALB/c小鼠对RV非常易感, RV感染容易产生腹泻症状。随着日龄的增大(>2周) BALB/c小鼠对RV易感性显著降低。为了明确小肠上皮细胞CYPA的表达是否与BALB/c小鼠对RV易感性的变化相关，我们比较了不同日龄段BALB/c小鼠小肠上皮细胞中CYPA的表达，发现在新生小鼠(5日龄) CYPA仅在小肠绒毛底部，主要是在肠隐窝处上皮细胞中有少量表达，而在RV容易感染到的小肠绒毛上部的上皮细胞中几乎不见CYPA的表达。相反，在稍大的BALB/c小鼠(15日龄)的小肠绒毛，不管是绒毛顶端还是基底部，其上皮细胞中CYPA均有丰富的表达。这些结果表明，新生BALB/c小鼠小肠绒毛上段上皮细胞中CYPA表达缺失可能是导致其对RV易感的重要原因。
第二部分：RV感染肠上皮细胞引发腹泻新机制的发现
首先，我们用人轮状病毒(Wa株)感染人肠上皮细胞Caco-2，同时在培养基中加入100μg/ml胰蛋白酶，以此模拟肠道消化性的环境，观察肠腔内消化性环境对RV感染肠上皮细胞后果的可能影响。实验发现，在消化性环境中RV的感染会导致肠上皮细胞快速脱落。台盼蓝染色表明脱落的细胞活性良好，流式细胞术结果显示这些脱落的细胞在脱落时没有出现明显的凋亡或坏死。在非消化性环境下肠上皮细胞只会在RV感染的末期才出现明显的脱落，此时脱落细胞几乎全部凋亡及坏死。结果提示，体内RV感染中，被感染的肠上皮细胞可能还没有来的及凋亡或坏死或其它明显功能损害就已经从基底膜脱落，彻底失去功能。我们进一步检测了小肠内不同段肠腔内的胰蛋白酶的活性，结果显示在BABL/c小鼠幼仔的空肠中、下段和回肠的上段胰蛋白酶活性最高。此外，通过结晶紫对BABL/c肠上皮细胞进行活体染色发现，RV感染引起的肠上皮细胞脱落在上述肠段中最为明显。我们的结果提示RV感染导致肠上皮细胞在肠道消化性环境中快速脱落可能才是RV腹泻产生的根本原因。
其次，为了寻找消化性环境中RV感染引肠上皮细胞快速脱落的直接原因，我们采用了一种定量蛋白质组学方法-细胞培养稳定同位素氨基酸标记（SILAC）来分析RV感染后Caco-2细胞蛋白表达谱的变化。结果显示，RV感染显著削弱了与细胞间的粘附相关蛋白以及细胞与细胞外基质的粘附相关的蛋白质的表达，如E-caherin, Plakoglobin,Desmoplakin,层粘连蛋白B2,纤维连接蛋白1, Plectin1, Cingulin以及ZO-1等。Western blot及qRT-PCR进一步证实了这些蛋白质在RV感染后显著下降。免疫荧光实验进一步验证了RV感染引起细胞间连接包括紧密连接，粘附连接以及桥粒连接的严重损坏。进一步通过结晶紫染色发现，RV感染显著破坏肠上皮细胞屏障功能，增大细胞间隙，导致胰蛋白酶向细胞底部严重渗漏，从而破坏细胞-细胞外基质粘附，最终引起上皮细胞脱落。
最后，为了明确RV感染导致细胞粘附功能破坏从而引起肠上皮细胞脱落的的相关信号机制，我们对RV感染中可能涉及到的激酶和磷酸酶与细胞脱落之间的关系进行了研究。我们发现抑制酪氨酸蛋白激酶可以显著抑制消化性环境中RV感染引起的肠上皮细胞的脱落，抑制蛋白酪氨酸磷酸酶则显著促进RV感染及感染的肠上皮细胞的脱落。抑制蛋白酶磷酸2A（PP2A）同样显著促进RV感染引起肠上皮细胞的脱落。此外，免疫印迹法显示RV感染引起PP2A两个主要结构亚单位PPP2R1A和PPP2R1B表达减少。RV感染可以引起细胞胞浆内Ca2+浓度升高，我们结果显示，通过离子霉素和毒胡萝卜素增大胞浆Ca2+浓度加速RV感染引起的细胞脱落。然而，使用BAMPT-AM和U73122抑制胞浆内Ca2+增加却不能阻止消化性环境中RV感染引起的肠上皮细胞的快速脱落。这表明RV感染引起胞浆Ca2+的增加只是导致宿主细胞的脱落促进因素而非决定因素。
综上所述，本研究发现CYPA是一种新的抗RV感染的宿主限制性因子。CYPA通过本身的脯氨酰顺反异构酶活性抑制RV的复制，其在肠上皮细胞中的表达可能与宿主对RV的易感性密切相关。CYPA同时还可以通过介导宿主细胞IFN-I反应来抑制RV感染。CYPA的这种功能不依赖于其脯氨酰顺反异构酶活性，而可能依赖于宿主细胞中JNK信号通路的激活。另一方面，我们发现在消化性环境中RV感染会导致肠上皮细胞快速脱落，这种现象可能才是RV腹泻产生的真正原因。而造成细胞脱落的原因与RV感染引起肠上皮细胞粘附功能及屏障功能的破坏，胰蛋白酶渗透到细胞基底部破坏细胞与细胞外基质的粘附密切相关。在这个过程中，RV感染引起的酪氨酸蛋白激酶的激活以及PP2A的抑制起到了关键作用，而RV感染引起的Ca2+的内流是加重而非决定因素。
Rotavirus (RV), a member of the family Reoviridae, is a non-enveloped icosahedral viruswhose capsid is formed by three concentric layers of structural proteins and contains agenome made up of11segments of double-stranded RNA which encode12viral proteinsincluding6structural proteins (VP1-4,6,7) and6non-structural proteins (NSP1-6). RV hasstrict cell tropism and only efficiently infects epithelial cells of gastrointestinal tract andkidney. Infants of human and animals are generally susceptible to RV infection; however, asthey grow older their susceptibility is greatly reduced. The reason for this change could bedue to the expression of some host restrictive factors of RV in enterocytes during growth. Infact, some host proteins such as APOBEC3G, Lv1, Ref1and EWI-2wint have been reported toplay a repressive role in viral infections. The expressions of such host restrictive factors aresupposed to contribute to host resistance to viral infections. However, as few such host factorsthat could protect host against RV infection were identified, the exact reason remains unclear.As many other viral infections, RV infection also elicits a global shutoff of host proteinsynthesis. However, several studies revealed that some host proteins such as α2β1and β2integrins, Grp78and Grp94were up-regulated and required for successful virus reproduction.Therefore, it is possible that RV is able to promote the expression of certain host proteins forefficient viral replication. On the other hand, it is wondering that whether host cells could alsoelicit the expression of some self-proteins to protect themselves against RV infection.
Rotavirus (RV) is the primary cause of acute dehydrating diarrhea worldwide in infantsand young children under5years of age. However, the precise mechanism of the occurrenceof rotavirus diarrhea is still inconclusive until now. Previously, rotavirus diarrhea was thoughtbe due to enterocyte destruction from the top of intestinal villus. Some reports revealed thatrotavirus infection induced apoptosis and necrosis of enterocyts in vitro and extensive studiesin animal models have reported the presence of histopathologic changes and functionalabnormalities in intestinal infected mucosa that varied from mild to severe depending on the rotavirus strain virulence. However, this mechanism cannot explain the facts in which mildhistopathologic changes without enterocyte destruction are usually found in rotavirus diarrhea.Some recent reports provided some other insights into the mechanism of the occurrence ofrotavirus diarrhea. They found a default in the expression and activity of the brush bordermembrane (BBM)-associated enzymes such as sucrase-isomaltase(SI) Lactase-phlorizinhydrolase, dipeptidyl peptidase IV (DPP IV) as well as Na+-D-glucose symport activity(SGLT1), Na+-L-alanine symporter and Na+-L-alanine symporter after rotavirus infection.These reports presumed that the diminished absorptive capacity of glucose and amino acids ofenterocytes might contribute to the occurrence of rotavirus-induced diarrhea. However, thesestudies still failed to exhibit a convinced molecular mechanism of the occurrence of rotavirusdiarrhea.
In recent years proteomic techniques have been widely used in studying viral infectionsand these have given a better understanding of the underlying virus-host interactions thatmight contribute to pathogenesis following viral infection. In this study, we focused ourinterest on identifying novel host restrictive factors to RV infection and new mechanism ofRV diarrhea by proteomic tools. There were two sections of our study. In section I, we were todiscover novel host restrictive factors to RV using2-dimensional gel electrophoresis (2-DE)coupled with mass spectrometry (MS); in section II, we were to explore new mechanism ofRV diarrhea where a more advance proteomic technique SILAC was used. Our study wouldmake a great advance of the knowledge on RV infection and was beneficial to the preventionand therapy of RV diarrhea.
Section I: Discovery of novel host restrictive factors to RV
Firstly, we determined the appropriate moi and time of infection in order to obtain thebest infectivity and cell viability, which was important for later2DE analysis. For2-DEsample preparation, MA104monolayers cultured in tissue culture dishes (100mm×20mm)were infected with purified RV (Wa strain) at a moi of3, or with DMEM as mock infection.At12h p.i., the infected cells were washed with cold0.9%NaCl, lysed in lysis buffer.2-DEanalysis was carried out using13cm ReadyStrip IPG strips (nonlinear, pI3-10). Then theIPG strips were focused by the IPGphor electrophoresis system. After separation in the firstdimension, the strips were then laid atop precast12.5%gels for separation of polypeptides byvertical electrophoresis. The gels were stained using colloidal blue, scanned with a GS800 scanner and analyzed with PDQuest6.1software; Expression intensity ratios larger than1.5(ratioHRV/mock≥1.5) were set as a threshold indicating significant changes (p≤0.05). A totalof24spots were found significantly up-regulated in HRV-infected MA104cells, amongwhich11protein spots representing10host protein and5protein spots representing4viralproteins were successfully identified by MALDI-TOF-MS analysis. Some of host proteinsidentified such as ACP1, ALDOA, CYPA and KRT20were further validated by western blotand qRT-PCR.
Secondly, to clarify the possible roles of CYPA in RV infection, we carried outImmunofluorescence and Co-immunoprecipitation assays. We found that CYPA was recruitedto the viroplasms upon RV infection in MA104cells. RV structural protein VP2and CYPAcould be co-immunoprecipitated with each other, indicating an interaction between these twoproteins. To make sure whether CYPA played an important role in RV infection, weoverexpressed wild type of CYPA and its PPIase negative mutant CYPA/R55A and knockeddown the endogenous CYPA. We found that Overexpressing wild type of CYPA significantlyreduced RV reproduction in MA104cells, while overexpressing CYPA/R55A greatlyenhanced RV reproduction. Knockdown of endogenous CYPA also remarkably facilitated thereproduction of RV. Therefore, it was indicated that CYPA inhibited RV reproduction throughthe PPIase activity. Notably, the fact that the PPIase activity of CYPA was necessary ininhibiting RV reproduction but was unnecessary in reducing host susceptibility to RVsuggested that there were different ways for CYPA to inhibit RV infection. We further foundthat CYPA could inhibit RV infection by promoting host IFN-β response that was independenton its Pease activity but dependent on JNK signaling pathway.
At last, it is well known that neonatal BALB/c mice are far more susceptible to RVdiarrhea than older (>2weeks) ones. To find out whether CYPA plays an important role in thesusceptibility to RV diarrhea in BALB/c mice, we compared the expression of CYPA in theintestine of BALB/c mice of different ages. We found that in neonatal (5days old) BALB/cmice, CYPA was only expressed in the bottom of intestinal villus, mainly in the enterocytesof intestine crypts, and hardly detected in the upper section of villus of small intestine wherethe enterocytes were the main targets of RV infection. In contrast, in older (>15days old)BALB/c mice CYPA was abundantly expressed in the all the enterocytes of intestinal villus of small intestine. These results suggested that lack of CYPA expression in epithelial cells ofsmall intestine might contribute to host susceptibility to RV diarrhea.
Section II: Discovery of new mechanism of RV diarrhea
Firstly，to find out the effects of RV infection in enterocytes in a digestive environmentsimilar to that in intestinal tract, we infected Caco-2cells with human rotavirus (Wa strain)and100ug/ml trypsin was added into the culture medium. We found that RV infection in adigestive environment resulted in a quick shedding of host cells. FACS showed that there wasno significant apoptosis or necrosis in the detached cells. Trypan blue staining showed that theviability of the detached cells remained well. We further tested the trypsin activity insidedifferent segments of small intestine. We found that the trypsin activity in the middle anddownstream of jejunum as well as upstream of ileum of BABL/c pups was highest.Accordingly, by intravital staining with hexamethylpararosaniline it was found that there wasmost severe shedding of enterocyts in these segments of small intestine mentioned above.
Secondly, to find out the reason why RV infection in a digestive environment elicited asignificant shedding of enterocytes, we analysed the total cell response in Caco-2cells atdifferent times of RV infection by a quantitive proteomic method Stable Isotope Labeling withAmino acids in Cell culture (SILAC). It was found that RV infection significant impaired theexpression of host proteins involved in cell adhesions including both cell-cell adhesion andcell-extracellular matrix adhesion. The impairment of these adhesion molecules such asE-cadherin, plakoglobin, desmoplakin, laminin B2, fibronectin1, plectin1, cingulin as well asZO-1were further validated by western blot and qRT-PCR. Immunofluorescence assay furtherconfirmed the disruption of cell-cell junctions including tight junction, adhesion junction andmacula adherens in RV infection. Further, by hexamethyl-pararosaniline staining, it wasdiscovered that RV infection significantly disrupted the barrier between enterocytes that wasformed by cell junctions, which leaded to a severe leak of trypsin from the culture medium inthe lumen to the bottom of cell where it disrupted cell-extracellular adhesion and ultimatelyresulted in detachment of enterocytes.
At last, to find out the mechanism by which RV infection caused a desruption of celladhesions, we analysed the effects of some kinases and phosphotase that might be involved inRV infection. We found that inhibition of tyrosine protein kinase by genistain significantlyrestored the cell shedding in RV infection and inhibition of tyrosine phosphatase by sodium orthovanadate enhanced cell shedding both in RV-infected and mock-infectedenterocytes. Inhibition of protein phosphatase2A by okadaic acid also significantly enhancedcell shedding in both RV-infected and mock-infected enterocytes. Additionally, western blotshowed that the expression of PP2A subunits like PPP2R1A and PPP2R1B were reduced inRV. Increasing cytosol Ca2+by ionomycin and thapsigargin greatly enhanced the shedding ofcells in RV infection, however, inhibition of cytosol Ca2+by BAMPT-AM and U73122didnot restore the cell shedding in RV infection, indicating that the increase of cytosol Ca2+inRV infection could contribute to the shedding of host cells but was not necessary.
In conclusion, in this study we firstly identified CYPA as a novel host restrictive factorthat confers protection against rotavirus infection and might contribute to host susceptibilityto RV diarrhea. CYPA could also inhibit RV infection by conducting host IFN-β productionthat was independent on its PPIase activity but dependent on the activation of JNK signalingpathway. Then we discovered that RV infection caused severe shedding of enterocytes in adigestive environment, which was attributed to a disruption of cell adhesions caused byactivation of tyrosine protein kinase and a repression of protein phosphatase2A in RVinfection.
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