Rab 5 is required for the cellular entry of dengue and West Nile viruses - PubMed (original) (raw)
Comparative Study
Rab 5 is required for the cellular entry of dengue and West Nile viruses
Manoj N Krishnan et al. J Virol. 2007 May.
Abstract
The mechanisms of cellular entry of dengue and West Nile viruses are not well characterized. We show that both these viruses enter HeLa cells by clathrin-dependent endocytosis and require vacuolar acidic pH. Inhibition of the GTPase Rab 5 or 7, which regulates transport to early or late endosomes, respectively, demonstrated that Rab 5 was essential for survival of both dengue and West Nile virus. These data broaden our understanding of the pathways required for productive dengue and West Nile virus infection and may facilitate new strategies for combating disease.
Figures
FIG. 1.
Effect of NH4Cl (A) and chlorpromazine (D) and depletion of VATPase and Eps15 (B, C, E, and F) on DNV or WNV infectivity of HeLa cells. (A and D) HeLa cells preadsorbed with the viruses for 1 h at 4°C (MOI of 10) were transferred to 37°C and incubated for the desired time periods, culture medium containing 20 mM NH4Cl or 10 μM chlorpromazine was added, and the cells were grown for 16 h or 10 h for DNV or WNV, respectively. After the incubation, cells were fixed and immunofluorescence was performed. (B and C) HeLa cells transfected with siRNA against VATPase and Eps15 for 4 days were infected with DNV and WNV (MOI of 10) for 16 h or 10 h, respectively, and analyzed by immunofluorescence (B) or Q-RT-PCR to quantify viral E-gene copies (C). (E and F) HeLa cells transfected with pEGFP-C vector expressing a dominant-negative mutant of Eps15 (E95/295) for 30 h were infected with DNV or WNV (MOI of 10) for 16 h or 10 h, respectively, and analyzed by immunofluorescence (E) or Q-RT-PCR (F) to quantify viral E-gene copies. E-gene copies were normalized using beta actin gene copies. Images were captured using a magnification ×40 objective by fluorescence microscopy. Quantification was done by counting the total number of infected cells in each captured image (corresponds to an average of 100 cells per image) and expressed as percent infected cells of the total cells. Results are expressed as means ± standard deviations from triplicates of a representative experiment.
FIG. 2.
Effect of overexpression of green fluorescent protein (GFP)-tagged dominant-negative mutant of Eps15 (D, E, and F or P, Q, and R), Rab 5 (G, H, and I or S, T, and U), or Rab 7 (J, K, and L or V, W, and X) on DNV or WNV infection, respectively. HeLa cells transfected with EGFP-C vector expressing a dominant-negative mutant gene of Eps15 (E95/295) or pQCXIP vector having a dominant-negative Rab 5 or 7 mutant (Rab 5 S34N or Rab 7 T22N) for 30 h was infected with DNV or WNV (MOI of 10) for 16 or 10 h, respectively, and analyzed by immunofluorescence. Control cells were transfected with GFP vector (A, B, and C or M, N, and O). Images were captured by fluorescence microscopy using an ×40 objective lens. Green, either the mutant gene or GFP alone (control); red, virus; blue, nucleus.
FIG. 3.
Virus-mediated, pH-induced cell-cell fusion by DNV and WNV. Cells (labeled with the fluorescent probe CellTracker Green CMFDA; Molecular Probes) infected with virus for 20 h (MOI of 10) or preadsorbed with virus on surface by incubation with virus at an MOI of 200 for 1 h at 4°C (both labeled with the fluorescent probe CellTracker Green CMFDA) were mixed at a 1:1 ratio at 1 × 106 cells/ml in buffers of various pHs for up to 5 min at 37°C. Soon after the incubation, the low-pH buffer was replaced with serum-free medium. The cell mixture was then plated onto 384-well plates at a density of 9,000 cells per well, spun down at 220 × g for 2 min, and imaged under a fluorescence microscope using a ×10 objective lens. Results were prepared by visually counting three random fields of images from representative experiments and expressed as percentages of the total cells involved in cell-cell fusion. Results are expressed as means ± standard deviations from a representative experiment.
FIG. 4.
Effect of functional repression of Rab 5 and 7 on DNV or WNV infection, using RNAi (A and B) or dominant-negative mutant overexpressions (C and D). HeLa cells treated with the corresponding siRNA for 4 days were infected with DNV for 16 h or WNV for 10 h (MOI of 10) and processed for immunofluorescence (A) or Q-RT-PCR to quantify viral E-gene copies (B). Images were captured by fluorescence microscopy (×40 objective). Panels C and D shows the results of the effect of Rab 5 and Rab 7 dominant-negative mutant overexpression on DNV and WNV infections. HeLa cells transfected with mutant genes for 30 h were infected with DNV or WNV (MOI of 10) for 16 h or 10 h, respectively, and analyzed by immunofluorescence (C) or Q-RT-PCR to quantify viral E-gene copies (D). Control cells were transfected with GFP vector. E-gene copies were normalized with beta actin gene copies. Results in panels A and C were prepared by visually counting three random fields of images from representative experiments and expressed as percent infected cells of the total cells. Results are expressed as mean ± standard deviations from a representative experiment.
References
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