Caveolin-1 in lipid rafts interacts with dengue virus NS3 during polyprotein processing and replication in HMEC-1 cells

Abstract

Lipid rafts are ordered microdomains within cellular membranes that are rich in cholesterol and sphingolipids. Caveolin (Cav-1) and flotillin (Flt-1) are markers of lipid rafts, which serve as an organizing center for biological phenomena and cellular signaling. Lipid rafts involvement in dengue virus (DENV) processing, replication, and assembly remains poorly characterized. Here, we investigated the role of lipid rafts after DENV endocytosis in human microvascular endothelial cells (HMEC-1). The non-structural viral proteins NS3 and NS2B, but not NS5, were associated with detergent-resistant membranes. In sucrose gradients, both NS3 and NS2B proteins appeared in Cav-1 and Flt-1 rich fractions. Additionally, double immunofluorescence staining of DENV-infected HMEC-1 cells showed that NS3 and NS2B, but not NS5, colocalized with Cav-1 and Flt-1. Furthermore, in HMEC-1cells transfected with NS3 protease, shown a strong overlap between NS3 and Cav-1, similar to that in DENV-infected cells. In contrast, double-stranded viral RNA (dsRNA) overlapped weakly with Cav-1 and Flt-1. Given these results, we investigated whether Cav-1 directly interacted with NS3. Cav-1 and NS3 co-immunoprecipitated, indicating that they resided within the same complex. Furthermore, when cellular cholesterol was depleted by methyl-beta cyclodextrin treatment after DENV entrance, lipid rafts were disrupted, NS3 protein level was reduced, besides Cav-1 and NS3 were displaced to fractions 9 and 10 in sucrose gradient analysis, and we observed a dramatically reduction of DENV particles release. These data demonstrate the essential role of caveolar cholesterol-rich lipid raft microdomains in DENV polyprotein processing and replication during the late stages of the DENV life cycle.

Figure 1. Localization of lipid raft resident proteins and viral DENV proteins.

(A) HMEC-1cells were stained with specific antibodies against Cav-1 and Flt-1, and immunofluorescence were analyzed by confocal laser scanning microscopy. (B) HMEC-1 cells were mock infected or infected with DENV-2 and then double-stained with specific antibodies (developed in our laboratory) targeting NS2B (green) and NS3 (red) or NS3 (red) and NS5 (green).

Figure 2. NS3 and NS2B are present in detergent-resistant membrane fractions.

HMEC-1cells were infected with DENV-2 at 10 MOI for 48 h and lysed using 1% Brij in TNEV buffer. Lysates were analyzed by sucrose gradient ultracentrifugation (A). The floating bands from sucrose gradient ultracentrifugation were collected and analyzed by dot blot with an anti-Flt-1 antibody (a lipid resident protein). (B) The samples from each fraction were then analyzed by western blotting using specific antibodies against NS3, NS2B, Flt-1, Cav-1 and TfR as a marker of non-raft fractions.

Figure 3. Colocalizationanalysis of DENV NS2B, NS3, and NS5 with caveolin and flotillin in infected cells.

HMEC-1 cells were mock infected or infected with DENV-2, fixed 36 h after infection with 4% paraformaldehyde, permeabilized, and immunolabeled. Cells were double stained with mouse monoclonal antibodies against NS2B (A), NS3 (B), or NS5 (C); the lipid raft markers Cav-1 (upper panel) and Flt-1 (middle panel); and the non-raft marker TfR (bottom panel). DAPI was used for nuclear DNA staining. Slides were analyzed by confocal laser scanning microscopy. (D) The degree of overlap between the green and red signals for each viral protein was statistically analyzed and expressed as a Pearson's coefficient by the microscopy software.

Figure 4. DENV-2 NS3 with Cav-1 colocalize at early stages after infection.

(A) HMEC-1cells were infected with DENV-2 and double stained at different times after infection (12, 24 and 48hpi) or were mock infected (not shown). In all cases, cells were double stained with mouse monoclonal antibodies against NS3 (green) and the lipid raft marker Cav-1 (red). Nuclear DNA was stained with DAPI. (B) Slides were analyzed by confocal laser scanning microscopy (Leica SP5 Objective HCXPLAPO63 63Oil). The graph shows the colocalization index. (C) HMEC-1 cells were transfected with (a) NS2BNS3pro-GFP, (b) NS3pro-GFP, and,(c) NS2B-GFP. After 24 h, they were stained with the lipid raft marker Cav-1 (red), and colocalization was analyzed.

Figure 5. dsRNA weakly colocalizes with lipid raft markers in DENV-infected HMEC-1.

(A) HMEC-1 cells, mock infected or infected with DENV-2, were fixed 48 h after infection, permeabilized, and analyzed by immunofluorescence. Cells were double stained with mouse monoclonal antibodies against dsRNA (red always) and NS2B (green) (a); dsRNA and NS3 (green) (b); dsRNA and NS5 (green) (c);(B) dsRNA and Cav-1 (green) (a); or dsRNA and flotillin (green) (b). Colocalization of dsRNA was observed at different times after infection. (C)HMEC-1cells were infected with DENV-2 and double stained at different times after infection (18 h and 24 h) dsRNA, red, Cav-1, green; and mock infected, not shown. Nuclear DNA was counterstained with DAPI, and the merged image is shown at higher magnification.

Figure 6. Association of NS3 with caveolin.

(A) HMEC-1cells were infected with DENV-2 at 10 MOI for 48 h. The cells were lysed, and lysates were divided into two aliquots. One aliquot was used for western blotting with anti-NS2B, anti-NS3, anti-NS5, and anti-Cav-1 antibodies. (B) The other aliquot was used for immunoprecipitation with anti-Cav-1 and developed with anti-NS3 antibody.(C) a experiment performed as described above the lysed was subjected to sucrose gradient ultracentrifugation The floating bands corresponding to lipid rafts (3, 4, and 5) were mixed and used in immunoprecipitation assays with an antibody targeting α-Cav-1. Immunoprecipitated samples were analyzed by western blotting with rat-anti NS2B, rat monoclonal anti-NS5, and mouse monoclonal anti-NS3 and anti-Cav-1 antibodies.

Figure 7. Depletion of cholesterol by MβCD affects DENV-2 infection in HMEC-1.

HMEC-1cells were infected at 10 MOI. At 120 min after adsorption, the inoculated virus was washed off, and the cells were cultured in fresh medium. At 2 or 6 h post infection, the cells were mock treated (A) or treated with 10 mM MβCD (B) for 2 h. The drug was then washed off, and the cells were cultured in fresh serum-free medium for 48 h. Detergent-resistant membrane fractions obtained from sucrose gradient ultracentrifugation were collected, and Cav-1 and NS3 were analyzed. The recovered fractions were numbered from 1 (top fraction) to 11 (bottom fraction). (C) The culture supernatants were harvested to determine the viral titration by plaque-forming assays. (D) Disaggregation of lipid rafts after10 mM MβCD treatment.