Figure 4. AAV2 Infection Requires Arf1 and Cdc42.

(A) AAV2-cherry transduction of HeLa and 293T cells expressing EGFP-tagged Arf1 dominant-negative (DN) and constitutively active (CA) mutants. Flow cytometry analysis was performed as described in Figure 1A. Insets represent the selected EGFP+/mCherry+ populations, and numbers indicate the percentage of mCherry+ cells among the EGFP+ population.

(B) AAV2-cherry transduction of HeLa and 293T cells expressing EGFP-tagged Cdc42 DN and CA mutants; analysis as described above.

(C) AAV2-luc transduction of 293T cells expressing dominant-negative Arf1 or Cdc42. Values are normalized to GFP vector-transfected controls.

(D) AAV2 binding and endocytosis in 293T cells transfected with dominant-negative Arf1 or Cdc42. Transfection efficiency was approximately 80% (not shown).

(E) Co-localization of Cdc42 with AAV2 or Ad5 in HeLa cells. Cells were transfected with EGFP-Cdc42WT, infected with AAV2-luc or Ad5-luc and stained for AAV2 or Ad5 capsids. Insets show higher magnification of boxed areas. Values indicate the percentage of virus (red) overlapping with Cdc42 (green). Bar, 20 μm.

(F) CtxB internalization (30 min) in HeLa cells treated with 100 μM EIPA, 80μM dynasore, or both. Surface-bound CtxB was removed by acid-stripping for intracellular fluorescence quantification.

(G) Confocal micrographs of HeLa cells treated as above. Acid wash was omitted to show surface accumulation of CtxB in cells treated with dynasore and EIPA. Bar, 10 μM.

(H) Synergistic effect of EIPA and dynasore on AAV2-luc transduction (left panel) and internalization (right panel). Values are normalized to DMSO controls.

Error bars indicate SD, *p < 0.05, **p < 0.005.