Human hepatocyte growth factor receptor is a cellular coreceptor for adeno-associated virus serotype 3

Abstract

Adeno-associated viruses (AAVs) use a variety of cellular receptors/coreceptors to gain entry into cells. A number of AAV serotypes are now available, and the cognate receptors/coreceptors for only a handful of those have been identified thus far. Of the 10 commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells in general. However, in our more recent studies, we observed that AAV3 vectors transduced human liver cancer cells remarkably well, which led to the hypothesis that AAV3 uses hepatocyte growth factor receptor (HGFR) as a cellular coreceptor for viral entry. AAV3 infection of human liver cancer cell lines was strongly inhibited by hepatocyte growth factor, HGFR-specific small interfering RNA, and anti-HGFR antibody, which corroborated this hypothesis. However, AAV3 vectors failed to transduce murine hepatocytes, both in vitro and in vivo, suggesting that AAV3 specifically uses human HGFR, but not murine HGFR, as a cellular coreceptor for transduction. AAV3 may prove to be a useful vector for targeting human liver cancers for the potential gene therapy.

FIG. 1.

(A) AAV3 vector-mediated transgene expression in Huh7 cells in the presence of various concentrations of HGF. Cells were transduced with vector at 1 × 10⁴ VG/cell, and EGFP-positive cells were enumerated 72 hr posttransduction. (B) Effect of pre- and postincubation of Huh7 cells with HGF (5 μg/ml) on the transduction efficiency of AAV2 and AAV3 serotype vectors. (C) AAV3 vector-mediated transgene expression in Hep293TT cells in the presence of various concentrations of HGF. Cells were transduced with vector at 2 × 10³ VG/cell, and EGFP-positive cells were enumerated 72 hr posttransduction. (D) Effect of pre- and postincubation of Hep293TT cells with HGF (10 μg/ml) on the transduction efficiency of and AAV3 serotype vectors.

FIG. 2.

(A) Effect of transfection with 10 pmol of either negative control siRNA or HGFR-specific siRNA on AAV3 vector-mediated transduction of Huh7 cells. (B) AAV3 vector-mediated transgene expression in Huh7 cells in the absence or presence (100 μg/ml) of anti-HGFR antibody. Transgene expression was determined 72 hr posttransduction.

FIG. 3.

(A) Transduction efficiency of scAAV2, scAAV3, and scAAV8 serotype vectors in murine hepatocytes in vivo. Male C57BL/6J mice were either mock-injected, or injected with 1 × 10¹⁰ VG each of scAAV2-CBAp-EGFP, scAAV3-CBAp-EGFP, or scAAV8-CBAp-EGFP vectors via the tail vein. Eight weeks postinjection, liver tissues were harvested and sections of each of the lobes were examined for EGFP expression, using a fluorescence microscope. Original magnification, × 100. (B) Crystal structure of the HGF β chain (gray) in complex with the Sema (extracellular) domain of hHGFR (green). In hHGFR, the C_α positions of amino acids that differ from mHGFR are depicted as green spheres; the amino acids that contact HGF in the crystal structure are depicted as red spheres; and amino acids that differ between hHGFR and mHGFR, and also make contact with HGF, are depicted as blue spheres. A close-up of the HGF and hHGFR contact region is also shown. The RCSB Protein Data Bank accession code for the coordinates used for this image is 1SHY. The program used to generate this image was PyMol.