Investigation Of Interactions Between Rotavirus And Host Cells

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 RVFirstly, 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 diarrheaFirstly,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.