Fig. 6

Silencing of CDK4 or CDK1 or Cyclin B1 in MEG-01 cells reduced DENV2 loads and enhanced CDK1 and Cyclin B1 interactions is enhanced upon DENV2 infection. QRT-PCR and immunoblotting analyses showing silencing of CDK4 (A, D) or CDK1 (B, E) or Cyclin B1 (C, F) in MEG-01 cells transfected with either scrambled siRNA or siRNA for respective genes (CDK4, CDK1, and Cyclin B1) for 24 h and followed by DENV2 infection (MOI of 5, at 24 h p.i.). QRT-PCR and immunoblotting analyses is shown for DENV2 capsid gene or protein loads determined upon silencing of CDK4 (D, J), or CDK1 (H, K) or Cyclin B1 (I, L) in MEG-01 cells transfected with either scrambled siRNA or siRNA for respective genes (CDK4, CDK1, and Cyclin B1) for 24 h and followed by DENV2 infection (MOI of 5, at 24 h p.i.). DENV2-loads and mRNA expression levels of CDK4, CDK1 and Cyclin B1 genes were normalized to human beta-actin mRNA levels. Y-axis represents levels of transcripts and X-axis indicates uninfected or DENV2-infected groups. P value determined by student’s t-test is shown. Scrambled siRNA-treated samples serve as negative control. (M) Immunoprecipitation assay performed with MEG-01 cell lysates from uninfected or DENV2-infected sample (MOI of 5, at 72 h p.i.) using Cyclin B1 antibody and detection with CDK1 antibody is shown for interaction between Cyclin B1 and CDK1 upon DENV2 infection. Total protein profile images serve as loading control (in panels D, E, F, J, K, L and M). (N) Schematic representation shown as model to summarize the current findings. DENV2 enters megakaryocyte cells and upregulates CDK4 protein, which eventually may enter the nucleus and facilitate cell progression from G1 to S phase. DENV2 induces the expression of CDK1 and Cyclin B1 and their interactions that may further assist in the progression of megakaryocytes through the G2 phase of the cell cycle into mitosis. Once the megakaryocytes enter the metaphase, DENV2 may reduce the expression of certain molecules, which are inhibitory to the progression of anaphase A. As a result, DENV2 modulates the cell cycle process in megakaryocytes by pushing from one phase to the next and eventually creating a favorable environment for DENV2 to selectively replicate inside megakaryocytes that undergoes polyploidy and endomitosis.