| Dengue virus (DENV) is an RNA virus of the family Flaviviridae; genus Flavivirusand there are four serotypes referred to as DENV1-4. The dengue virus genome containsabout11,000nucleotide bases, which code for the three different types of structure proteinmolecules (C, prM and E) and seven other types of nonstructure protein molecules (NS1,NS2a, NS2b, NS3, NS4a, NS4b, NS5). Dengue virus is mainly transmitted by mosquitoesand widely spread in tropical and subtropical regions.Infection with DENV can lead to aspectrum of clinical diseases ranging from self-limited dengue fever(DF) to the most severeforms, dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS). According to WorldHealth Organization, there are about50-100million infections and500,000DHF/DSSpatients annually. To date, neither vaccines nor specific antiviral drugs are available toprevent or treat DENV infection and the mortality rate of DHF/DSS can be as high as50%if untreated. Therefore, DENV infection has become a severe problem of the public health.As obligate intracellular paramicrobe,viruses use and manipulate the cell’s machineryfor most steps in there life cycles. The formation of the virus factories in many viruses hasbeen described,which consist of perinuclear area or cytoplasmic foci.It involves a numberof complex interactions and signalling events between organelles,cytoplasmic membranesand cytoskeletal components,supplying a relative separate space to enhance replicationefficiency and protection from host defenses. The virus factories of flaviviridae virusesoften involve endoplasmic reticulum(ER),golgi apparatus and mitochondrial,but thecomponents of DENV-induced virus factories in host cells is unknown.The formation ofvirus factories in several viruses has been associated with vimentin intermediate filaments.Our previous work has shown that vimentin retracted from the cellperiphery,surrounded the nucleus in DENV2-infected cells and colocalize with viral NS1/Eproteins.Disruption of vimentin inhibited DENV2replication and production.However, themechanism of this process remains unknown. Rearrangement of vimentin often involvesphosphorylation of head N-terminal domain by cellular kinases. Rho-associated coiledcoil-containing kinase (ROCK) is a key molecule in regulation of this process. ROCK is aserine/threonine kinase that induced the complete collapse of vimentin network to form avimentin-containing area near the perinuclear region. In the present study, we investigatewhether ROCK plays critical role in DENV2infection-induced vimentin rearrangement.RESULTS1.Association of vimentin and cellular organelles with the formation of DENV2virusfactoriesIn our experiments, vimentin and cellular organelles of ECV304cells were markedwith specific antibodies or fluorescent probes. Immunofluorescence staining showed thatDENV2infection maily induced vimentin filaments rearrange into two types:(1)vimentinfilaments retracted from the cell periphery to one side of nucleus, forming a vimentin cagesurrounding the viral proteins,(2) vimentin filaments retracted and arranged to surround thenucleus,both of these vimentin structure colocalized with viral NS1/E proteins. DENV2induced endoplasmic reticulum(ER) retracting and accumulating in perinuclear region,colocalizing with viral NS1/NS3proteins. Golgi apparatus remained unchange duringDENV2infection while colocalized with viral E protein at48h p.i.. DENV2induced thetubular network of mitochondria shift to fragmentation,but didn’t colocalized with viralproteins.These data suggested that the formation of DENV2virus factories in ECV304cellsmay involve vimentin and ER while not golgi apparatus or mitochondria.2. DENV2-induced rearrangement of vimentin filaments was associated withphosphorylation at ser71of vimentinRearrangement of vimentin has been involved phosphorylation of N-terminal domainsby the activition of ROCK.In our experiments,the possibility that DENV2causedphosphorylation of vimentin by ROCK was analyzed by immunoflurescence staining withan antibody specific for phospho-ser71vimentin. The results showed that phosphorylationat Ser71of vimentin was similar to the strctures of vimentin,surrounding the virus factories and colocalizing with DENV2viral proteins. These data suggusted that DENV2-inducedrearrangement of vimentin filaments was associated with phosphorylation at ser71ofvimentin.DENV2infection may activate ROCK that lead to phosphorylation at ser71ofvimentin.3.DENV2induced rearrangement of vimentin filaments in ECV304cells, andinactivation of ROCK with Y-27632inhibited the rearrangementImmunoflurescence staining showed that DENV2infection induced rearrangement ofvimentin filaments in ECV304cells.In mock infection group,the vimentin filamentsemanated from the nucleus and extended into the cytoplasm, remaining almost unchange.Ininfection group, vimentin filaments retracted from the cell periphery early at30min p.i., andaccumulated to one side of nucleus, forming a vimentin cage surrounding the viral proteinsat1h p.i.. Then vimentin filaments retracted and arranged to surround the nucleus at8h p.i..These two vimentin structures can be both observed at24h p.i..These data suggested thatthe rearrangement of vimentin was closely associated with DENV2infection.ECV304cells were pretreated with ROCK inhibitor Y-27632or0.1%DMSO for1h at37℃.Then cells were infected with DENV2maintaining Y-27632or0.1%DMSO for1h at37℃.The DENV2-induced rearrangement of vimentin filaments was analyzed byimmunofluorescence staining.Results showed that Y-27632prevented rearrangement ofvimentin in DENV2infected cells.These data suggested that ROCK activation was closelyassociated with DENV2-induced vimentin filaments rearrangement.4. DENV2infection leads to phosphorylation at Ser71of vimentin, and inactivationROCK with Y-27632inhibited the phosphorylation at Ser71of vimentinTo further study the role of phosphorylation of ser71-vimentin played during DENV2infection, the expression of vimentin and phosphorylated vimentin were analyzed atdifferent time points by Western blot.The results showed that vimentin expression showedno significant difference between infected group and control group.Compared to controlgroup, vimentin-Ser71phosphorylation level was increased to176.3%at30min duringDENV2entry, and it was increased to333.2%at8h p.i., then was decreased to203.5%.These data suggusted that DENV2infection lead to phosphorylation of serine71ofvimentin.ECV304were pretreated with ROCK inhibitor Y-27632or0.1%DMSO for1h at37 ℃.Then cells were infected with DENV2for1h at37℃with Y-27632or0.1%DMSO.Thevimentin expression and vimentin-Ser71phosphorylation level were analyzed at differenttime points by Western blot.Results showed that compared to control group, vimentin-ser71phosphorylation of Y-27632treated group were decreased51.42%,42.42%,54.01%,46.76%at30min,1h,8h,24h p.i.,respectively. These data suggested that ROCK activationwas closely associated with DENV2-induced vimentin-ser71phosphorylation.5.DENV2infection leads to activation of ROCKIn our experiments,ECV304cells were infected with heat inactivated DENV2orDENV2,collected the sample at different time points p.i..Using96-well ROCK ActivityAssay Kit to detect intracellular ROCK activity.The results showed that ROCK wasactivated during DENV2infection.Compared to mock group,ROCK activity increased243.75%,177.19%,216.18%at15min,30min and8h p.i.,respectively. However,ROCKactivity showed no significant difference between infected and uninfected cells at othertime points.These data suggested that DENV2infection lead to activation of ROCK.In conclusion, this research tentatively analyzed the composition of DENV2virusfactories and the role of ROCK in DENV2-induced vimentin filaments rearrangement. Ourresults suggested that the formation of DENV2virus factories involved vimentin and ER,and ROCK implicated in DENV2-induced vimentin reorganization. |
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