Involvement Of The Actin Cytoskeleton In Dengue Virus Infection

Dengue virus (DENV) has recently drawn renewed interest as the most geographically widespread of the arthropodborne viruses. Annually there are millions of infections and at times tens of thousands of deaths. The infecting DENV is able to produce an acute febrile syndrome characterized by clinically significant vascular permeability, dengue hemorrhagic fever (DHF). An estimated 500 000 cases of DHF require hospitalisation each year, and up to 50% may die if not properly diagnosed and treated. However, there is no vaccine against DENV because its pathogenic mechanisms are still not fully understood.DENV belongs to the family of Flaviviridae, which are enveloped, single-stranded RNA viruses. The viral genome RNA represents the only messenger RNA in infected cells, and encodes three structural proteins (C, capsid protein; M, membrane protein; E, envelope protein) and seven non-structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). DENV envelope surface projections are composed of dimers of the E glycoprotein, which binds to receptors and is the most immunologically important structural protein. During endocytosis and maturation process, DENV E proteins undergo pH-induced conformational changes, and result in the structural alteration from E dimmers to E trimers in endosome, or from prM-E heterotrimers to E homodimers in trans-Golgi network. It remained obscure whether or how viral E proteins interact with host cellular components during the life cycle.The actin, one major component of the cytoskeleton, has been implicated in many cellular functions including the maintenance of cell shape, cell motility, cell adhesion, cell division, intracellular transport and organelle localization. It has been reported that many microbes can exploit the host actin cytoskeleton to facilitate important aspects of their life cycle. Previous studies showed that two strategies are generally used by viruses for intracellular transport: viral component either hijack the cytoplasmic membrane traffic or they interact directly with the cytoskeletal transport machinery. The possibility of Rho GTPase involved in DENV2 infection was investigated.In this study, we investigated the involvement of microfilaments in DENV serotype 2 (DENV2) infection. We found that DENV2 infection and transient expression of E protein could induce rearrangement of microfilaments. Cofocal immunofluerescence analysis showed the close association of actin filaments with the viral E proteins. In accordance to that, actin was co-immunoprecipitated with monoclonal antibody against E protein in infected cells. Perturbing the integrity of the microfilament network with pharmacological agents interfered with DENV2 infectivity, production, and release. Moreover, Rac,a member of Rho GTPase family,was activated into GTP-Rac and recruited in the perinuclear region, where DENV2 proteins were sccumulated. These results indicated that actin filaments may play an essential role in DENV2 infection and viral E protein might be a key element in the interaction of DENV2 and actin filament. Results and ConclusionDENV2 infection induced reorganization of the actin cytoskeletonFirst, we examined whether DENV2 interaction with human umbilical vein endothelial cells (HUVECs) could induce the reorganization of the actin cytoskeleton. In mock infected HUVECs, strong actin stress fibers seemed unaffected and crossed the entire cytoplasm during the observation. Upon the infection of DENV2, the actin network displayed several different patterns that appeared sequentially during one hour post infection. Within 30 min p.i., actin fibers started to disassemble and formed dot-like short filaments. Some stress fibers had drastically disrupted and displayed a dispersed pattern throughout the cytoplasm. At 1 h p.i., interestingly, actin repolymerized to fibers in the cortical area and outlined the cell borders. With infection progressed, significant alterations of actin cytoskeleton were also observed at 24 h p.i. and 48 h p.i. In most of infected cells, stress fibers in perinuclear region disappeared and actin filaments showed a diffused dot-like pattern. Similar DENV2-mediated actin reorganizations were observed in endothelial cell line ECV304.Previous studies had reported that several bacterial/viral structural proteins could interact with actin filaments and induce the reorganization. Therefore, we were interested in whether DENV2 E protein involved in reorganizations of actin filaments. In ECV304 cells transfected with E protein genes, actin filaments disassembled and formed a dispersed pattern while E proteins accumulated in sufficient amounts in the cytoplasm. The alteration was similar to that observed in DENV2-infected cells. The results strongly suggested that E protein might be not the only, but an important viral component in the signal pathway triggering actin rearrangement.The DENV2 E protein interacted with the actin cytoskeletonTo confirm the interaction between actin cytoskeleton and DENV2 E protein, actin rearrangements and distribution of the viral E proteins were analyzed with confocal immunofluorescent assay. At 24 h p.i., actin filaments were observed in diffused pattern and it colocalized with DENV2 E protein in the perinuclear region of infected HUVECs. Meanwhile, reorganized actin filaments also colocalized with transiently expressed E proteins in transfected ECV304 cells. The co-localizations indicated the possible interaction between E protein and actin microfilaments during DENV2 infection.To confirm the interaction, co-immunoprecipitation (co-ip) of actin with antibodies against DENV E protein was performed. As shown in Fig. 4, no actin was detected when co-ip was carried out with mock-infected cells. However, the actin (42KD) was precipitated with anti-E MAb in DENV2-infected C6/36 cells. Meanwhile, co-ip was also performed with DENV2- infected suckling mouse brain and the same result as above-mentioned was obtained. The results strongly suggested the close association of E proteins with the actin filaments.The effect of microfilament-disrupting drugs on DENV2 infectionNext, we were interested whether the interaction of the DENV2 with actin filaments is of any significance for viral life cycle. To assess the effect of jas and cyt D on early stages of the infection, we measured the plaque-forming on Vero cells pretreated with the drugs for 5, 3, or 1 h and then infected with DENV2. Compared with that in mock treated cells, the numbers of plaques formation in drug-treated cells were reduced in a time and dose dependent manner. The plaques of cells pretreated with 2μg/ml of cyt D or 0.1μmol/L of jas for 5 h were significantly reduced to 50% or 75%, respectively. This indicated that the efficiency of DENV2 entry might decrease with the disturbance of actin network.To determine whether DENV2 infection requires proper function of actin filaments, medium with drugs was added immediately post initiation of infection. At 9 h p.i., we quantitated virus titers of the supernatant and cell associated in drug-treated and mock-treated cells. The titers of extracellular and cell-associated DENV2 significantly decreased compared to results from mock-infected cells, indicating that disturbing the actin network showed inhibitory effect on DENV2 infection.To determine the stage of virus life cycle during which the requirement of actin were most evident, the drugs were added into the medium at different time points post infection. When the infected cells were incubated with jas from 3 to 9 h p.i., there was little change in intracellular viral titer whereas extracellular viral titer decreased. In case of the same period treatment with cyt D, decreased viral titers in both intracellular and extracellular space were observed, especially the extracellular viral titer showed a dramatically decreased tendency. When drugs were maintained in the medium from 6 to 9 h p.i., jas seems not affect on intracellular and extracellular viral titers. In contrast, cyt D showed obvious inhibitory effect on extracellular viral titer and slight inhibitory effect on cell-associated virus titers as compared with mock-treatment. These results suggested that extracellular viral titer appeared to be more sensitive to cyt D induced-disassembly of actin filaments than cell-associated virus.Interestingly, when the ratios of supernatant virus titer to cell-associated virus titer were further calculated, the possible role of actin filaments in the infection was identified. The significantly decreased ratios indicated that the drug has more inhibitory effects on extracellular viral titer than intracellular viral titer, and viral release might be partially blocked. In this assay, Jas treatment reduced the ratios in a time dependent manner, whereas cyt D treatments did not. When cells were treated with cyt D from 0 h p.i., the ratio was 81.0%. However, the ratio was reduced to 49.5% when treated from 3 h p.i., and remained 50% around when treated from 6 h p.i. The results suggested that DENV2 release might depend more on actin treadmilling than stabled actin.To further confirm effects of actin filaments on viral release, distributions of viral antigen were observed at 72 h p.i. In mock-treated cells, viral antigens were usually observed clustering on one side of the nucleus. However, in cyt D-treated or jas-treated cells, numerous viral antigens in pattern of large fluorescent spots accumulated throughout the cytoplasm, indicating that disturbing of actin network is able to inhibit the virus release. Rac was activated after DENV2 infectionFirst, we observed the distributions of Rac and DENV2 proteins. In the mock infected cells, Rac proteins were dispersed throughout the cytoplasma. However, in DENV2-infected cells, Rac proteins were recuited to the perinuclear region, and colocalized with DENV2 antigens.Next, the active forms of Rac, GTP-Rac, were detected by histochemisty assay with GST-CRIB. The result showed that there was no significant positive signal in mock-infected cells, while strong positive spots were observed on the one side of nuclear in DENV2-ifnected cells. This indicated that Rac proteins were activated and reorganized after DENV2 infection.In summary, this study demonstrated that actin network plays several important roles in whole process of DENV2 infection, and DENV2 E protein induces profound cytoskeletal rearrangements and co-localizes with actin filaments. Furthermore, the activated Rac proteins may also contributed to the actin reoganization after DENV2 infection. These observations might lead novel insights into the involvement of actin filaments in pathogenesis of DENV2 infection. However, the exact nature of the interactions and the identity of various signal transduction pathways involved remain to be defined.