Adeno-associated virus type 2-mediated gene transfer: altered endocytic processing enhances transduction efficiency in murine fibroblasts

Abstract

Adeno-associated virus type 2 (AAV) is a single-stranded-DNA-containing, nonpathogenic human parvovirus that is currently in use as a vector for human gene therapy. However, the transduction efficiency of AAV vectors in different cell and tissue types varies widely. In addition to the lack of expression of the viral receptor and coreceptors and the rate-limiting viral second-strand DNA synthesis, which have been identified as obstacles to AAV-mediated transduction, we have recently demonstrated that impaired intracellular trafficking of AAV inhibits high-efficiency transduction of the murine fibroblast cell line, NIH 3T3 (J. Hansen, K. Qing, H. J. Kwon, C. Mah, and A. Srivastava, J. Virol. 74:992-996, 2000). In this report, we document that escape of AAV from the endocytic pathway in NIH 3T3 cells is not limited but processing within endosomes is impaired compared with that observed in the highly permissive human cell line 293. While virions were found in both early and late endosomes or lysosomes of infected 293 cells, they were localized predominantly to the early endosomes in NIH 3T3 cells. Moreover, treatment of cells with bafilomycin A1 (Baf), an inhibitor of the vacuolar H(+)-ATPase and therefore of endosomal-lysosomal acidification, decreased the transduction of 293 cells with a concomitant decrease in nuclear trafficking of AAV but had no effect on NIH 3T3 cells. However, after exposure of NIH 3T3 cells to hydroxyurea (HU), a compound known to increase AAV-mediated transduction in general, virions were detected in late endosomes and lysosomes, and these cells became sensitive to Baf-mediated inhibition of transduction. Thus, HU treatment overcomes defective endocytic processing of AAV in murine fibroblasts. These studies provide insights into the underlying mechanisms of intracellular trafficking of AAV in different cell types, which has implications in the optimal use of AAV as vectors in human gene therapy.

FIG. 1

(A) Slot blot analysis of viral DNA in membranes and cytoplasm of cells. Equivalent numbers of 293 or NIH 3T3 cells were infected for 1 h with the recombinant vCMVp-lacZ (3,000 particles/cell) and homogenized, and the membranes were separated on a sucrose cushion by ultracentrifugation as described in Materials and Methods. Fractions were collected from the bottom of the tube, and viral DNA was quantified by slot blot analysis using the ³²P-labeled lacZ DNA probe as described previously (25). The bracket identifies the fractions containing the sucrose cushion. As a control, purified AAV virions were also loaded onto the sucrose cushion. (B) Analysis of endocytic markers. Cells were pulsed for 10 min with biotinylated holotransferrin prior to separation of homogenate on the sucrose cushion. Each fraction was then analyzed by Western blotting for the presence of biotinylated transferrin (Tfn), an early endosome marker, and by an enzymatic assay for acid β-galactosidase (Acid β-gal) activity, a lysosomal marker. Results are reported as percentages of the maximum signal intensity based on densitometric scanning of the autoradiogram or percentages of maximum enzymatic activity. (C) Subcellular distribution of AAV. Autoradiograms from three separate experiments similar to that in panel A were densitometrically scanned, and the signal intensities in each fraction were quantified. The combined values from fractions 1 to 4 and 5 to 8 were averaged and represent the amounts of AAV DNA in the cytoplasm (Cyt) and membranes (Mb), respectively.

FIG. 2

(A) Slot blot analysis of viral DNA in endocytic organelles from cells. Equivalent numbers of 293 or NIH 3T3 cells were infected for 1 h with vCMVp-lacZ (3,000 particles/cell) and homogenized, and the membranes were separated on a Percoll density gradient as described in Materials and Methods. Fractions collected from the bottom of the tube were then analyzed for the presence of viral DNA by slot blot hybridization as described in the legend to Fig. 1. (B) Detection of endosomal and lysosomal markers. For each fraction, markers for early endosomes (Tfn) and lysosomes (Acid β-gal) were assayed as described in the legend to Fig. 1, and the results are shown as percentages of the maximum signal. (C) Distribution of AAV within endocytic vesicles. Autoradiograms similar to that in panel A from two separate experiments were densitometrically scanned, and the viral DNA in each faction was quantified, averaged, and plotted as a percentage of the total signal.

FIG. 3

(A) Effect of Baf on AAV-mediated transduction of 293 and NIH 3T3 cells. All cells were pretreated for 2 h with 750 μM tyrphostin 1 and incubated for 1 h with or without 20 nM Baf. The cells were then either mock infected or infected with vCMVp-lacZ (5,000 particles/cell) for 2 h, and transgene expression was measured 48 h later as described in Materials and Methods. Results are expressed in RLU per microgram of total protein. (B) Southern blot analysis of the subcellular distribution of viral DNA in Baf-treated cells. 293 cells were either mock treated or treated for 1 h with 20 nM Baf and subsequently infected with 5,000 particles of vCMVp-lacZ per cell for 2 h. The cells were trypsinized and washed, after which nuclear (Nuc) and PNS fractions were isolated as described previously (18). Low-M_r DNA was isolated from each fraction, and analyzed by Southern blotting using a ³²P-labeled lacZ probe. ssDNA denotes the viral single-stranded DNA genomes.

FIG. 4

(A) Infectivity of AAV isolated from the cytoplasm of cells. Virions were isolated from the cytoplasm of vCMVp-luc-infected 293 (293 virions) or NIH 3T3 (NIH 3T3 virions) cells as described in Materials and Methods. After the physical particle titer of the isolated virions was determined by slot blot analysis, 293 cells were either mock infected or infected with equivalent numbers of AAV particles and the luciferase activity was measured 48 h later. Values are expressed in RLU per microgram of total protein. These results are representative of data from two independent experiments. (B) Effect of pH on the transducing ability of AAV. vCMVp-luc was incubated for 30 min at 37°C in buffers of pH 3.0 or 7.0, diluted 100-fold in IMDM, and then used to infect 293 cells (5,000 particles/cell). Luciferase activity was measured 48 h postinfection as described for panel A.

FIG. 5

(A) Time course of transduction of HU-treated NIH 3T3 cells. Cells were either mock treated (AAV), pretreated for various times with 10 mM HU or for 2 h with 750 μM Tyrphostin 1 (Tyr), and subsequently infected with vCMVp-lacZ (5,000 particles/cell) for 2 h. β-Galactosidase activity was measured 48 postinfection and is expressed as RLU per microgram of protein. (B) Detection of AAV in density-fractionated membranes from HU-treated NIH 3T3 cells. Cells were either mock treated or pretreated with 10 mM HU for 18 h and infected with vCMVp-lacZ for 1 h, and endocytic organelles were fractionated on a Percoll density gradient as described in the legend to Fig. 2. Viral DNA in each fraction was detected by slot blot analysis as described in the legend to Fig. 1. (C) Localization of AAV in subcellular fractions. Autoradiograms similar to that in panel B from three separate experiments were densitometrically scanned, and the signal in each fraction was quantified, averaged, and plotted as a percentage of the total signal. (D) Effect of Baf on AAV-mediated transduction of HU-treated NIH 3T3 cells. The cells were either mock treated (AAV) or treated with 10 mM HU for 18 h. Following a 1-h incubation in medium with or without 20 nM Baf, the cells were mock infected or infected with vCMVp-lacZ (5,000 particles/cell) for 2 h, and β-galactosidase activity was measured 48 h postinfection. Data are expressed as RLU per microgram of protein.

FIG. 6

Hypothetical model comparing intracellular trafficking of AAV in 293 (A) and NIH 3T3 (B) cells. AAV binds and enters the early endosomes of both cell types efficiently (a). In 293 cells, most of the virions progress down the endocytic pathway (b), enter a dense endocytic organelle with a low pH, undergo a putative capsid modification (?), and subsequently enter the nucleus by an unknown mechanism (c). This process can be blocked by inhibitors of endosomal acidification, in which case virions enter the nucleus by a less efficient pathway (d). In contrast to the viral trafficking observed in 293 cells, the virions in NIH 3T3 cells fail to pass through dense, acidic endosomes and therefore do not traffic efficiently to the nucleus. Instead, AAV escapes from early endosomes and inefficiently enters the nucleus by an alternate route (b).