Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jan;74(2):992-6.
doi: 10.1128/jvi.74.2.992-996.2000.

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts

J Hansen  1 , K Qing, H J Kwon, C Mah, A Srivastava
Affiliations

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts

J Hansen et al. J Virol. 2000 Jan.

Abstract

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
(A) Southern blot analysis of viral DNA entry into cells. Equivalent numbers of Raji, 293, and NIH 3T3 cells were either mock infected or infected with the recombinant vCMVp-lacZ vector (104 particles/cell), trypsinized, and washed extensively, and low-Mr DNA samples were isolated and analyzed with the 32P-labeled lacZ DNA probe as described previously (19, 24). ssDNA, viral single-stranded DNA genomes. (B) EMSA for detection of the phosphorylation status of the ssD-BP in NIH 3T3 cells. Equivalent numbers of cells were either mock treated or treated with the indicated amounts of tyrphostin 1 at 37°C for 2 h. Whole-cell extracts were prepared and used in EMSAs with the 32P-labeled single-stranded AAV DNA probe, as described previously (16, 25). Phosphorylated and dephosphorylated forms of the ssD-BP are denoted by the arrow and the arrowhead, respectively. (C) AAV-mediated transgene expression. Equivalent numbers of HeLa (left) and NIH 3T3 (right) cells were infected with 5 × 103 particles/cell of vCMVp-lacZ and then treated with the indicated amounts of tyrphostin 1 for 2 h under identical conditions. Forty-eight hours postinfection, β-galactosidase activity was determined as described in the text.
FIG. 2
FIG. 2
Confocal microscopy for localization of AAV particles in 293 and NIH 3T3 cells. At 15 min (panels 1 and 3) and 2 h (panels 2 and 4) postinfection with 104 particles/cell of Cy3-labeled AAV, 293 (panels 1 and 2) and NIH 3T3 (panels 3 and 4) cells were visualized as described in the text. Cy3-labeled AAV (red) and cellular nucleic acids (green) are shown in images representative of the center of the cells. Magnification, ×630.
FIG. 3
FIG. 3
(A) Southern blot analysis of subcellular distribution of the viral DNA in 293 and NIH 3T3 cells. Approximately 2 × 106 cells of each type were either mock infected or infected with the recombinant vCMVp-lacZ vector (104 particles/cell), trypsinized, and washed extensively. Low-Mr DNA samples were isolated at various indicated time points from purified nuclear (Nuc.) and cytoplasmic (Cyt.) fractions and analyzed with the 32P-labeled lacZ DNA probe as described in the legend to Fig. 1. (B) Southern blot analyses of AAV entry and trafficking into the nucleus of permissive human cell lines. Equivalent numbers of HeLa, KB, and 293 cells were infected with the recombinant vCMVp-lacZ vector, trypsinized, and washed extensively, and low-Mr DNA samples were isolated 2 h postinfection from purified nuclear and cytoplasmic fractions. The rest of the analysis on Southern blots was performed as described above.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alexander I E, Russell D W, Spence A M, Miller A D. Effects of gamma irradiation on the transduction of dividing and nondividing cells in brain and muscle of rats by recombinant adeno-associated virus vectors. Hum Gene Ther. 1996;7:841–850. - PubMed
    1. Bartlett J S, Samulski R J, McCown T J. Selective and rapid uptake of adeno-associated virus type 2 in brain. Hum Gene Ther. 1998;9:1181–1186. - PubMed
    1. Berns K I, Bohenzky R A. Adeno-associated viruses: an update. Adv Virus Res. 1987;32:243–307. - PubMed
    1. Berns K I, Giraud C. Biology of adeno-associated virus. Curr Top Microbiol Immunol. 1996;218:1–23. - PubMed
    1. Carter B J, Flotte T R. Development of adeno-associated virus vectors for gene therapy of cystic fibrosis. Curr Top Microbiol Immunol. 1996;218:119–144. - PubMed

Publication types

LinkOut - more resources