Abstract
Vectors from adeno-associated virus (AAV) appear very promising for use in some gene therapy applications, due to their relative safety and sustained gene expression. Different serotypes of AAV have been isolated, each having different properties. For example, it has been shown that AAV-8 uncoats more readily than AAV-2 in the nuclei of hepatocytes in mouse liver, possibly explaining the more robust transduction efficiencies of AAV-8 (Thomas et al., Journal of Virology, 2004, 78 (6) p. 3110-3224). Our goal is to better understand the mechanisms behind AAV-8 transduction by identifying cellular proteins that interact with the capsid, and that are necessary for optimal transduction. Towards this end, we used a yeast two-hybrid screen to identify cellular proteins that interact with different domains of the AAV-8 capsid. Using a portion of the AAV capsid protein VP3 as bait against a mouse liver cDNA library, we recovered around 700 clones. Analysis of the sequence of these clones revealed that over 100 genes had multiple hits, indicating that the interactions might be genuine. These included genes encoding many different types of proteins that may be involved in viral binding, entry and trafficking. In particular, some clones contained portions of cathepsins B and L, lysosomal proteases shown to be important in the uncoating of reovirus and the Ebola virus. These clones did interact with the corresponding portion of the AAV-2 capsid, but not AAV-5, when using the same two-hybrid system. Infection of murine fibroblast NIH-3T3 cells by AAV-2 and AAV-8 was severely inhibited by treating the cells with the cathepsin B inhibitor Ca-074Me. In addition, capsid proteins from assembled AAV-2 and AAV-8, but not AAV-5, were cleaved specifically when incubated with purified cathepsin B protein, which was consistent with our two-hybrid data. Cathe sin B seems to be involved in the uncoating of both AAV-2 and 8. This data confirms that some of the roteins isolated in the screen are indeed involved in AAV transduction, and further analysis of the other genes recovered in our screen may hel reveal other cellular roteins im ortant for AAV8-mediated gene transfer.
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