The research objective of this project is to investigate the interactions of adeno-associated virus (AAV) with its host cell. The underlying hypothesis is that by understanding these interactions as they apply to the biology of the virus we can contribute to the use of AAV vectors for gene therapy. Staff members focus on two types of interactions: those involved in viral transduction of the target cell, and those between the Rep proteins of the wild type AAV and their cellular partners. Current projects study the tropism and transduction pathways of AAV serotypes, the lifecycle of these viral isolates, and identify and functionally characterize novel cellular proteins that interact with the AAV Rep proteins. One project the Unit has focused on is the characterization of two new AAV serotypes, AAV4 and AAV5. These new viral isolates are being studied both as natural mutations of other serotypes for understanding the biology of this genus of virus and because of their unique cell tropism, as novel vectors for gene transfer. AAVs are dependent viruses and require other viruses and cellular products for vital functions. This dependency coupled with the stability of the AAV2 particles has made AAV2 an attractive vector for gene therapy. AAV2 vectors can be maintained stably in the host cell, allowing long term expression of the transduced genes. However, AAV2 demonstrates a wide variability in transduction efficiency of different cell types, and poor transduction efficiency upon repeat administration of the vector. While some of these limitations may be overcome as our understanding of the basic biology of AAV increases, others may require employing different naturally occurring isolates of AAV serotypes. To date 8 primate isolates have been cloned and are referred to as AAV1-8. Our previous work with AAV4 and AAV5 has demonstrated distinct tropisms for these viruses compared to AAV2. While heparin sulfate proteoglycans are important in AAV2, and AAV3 binding and transduction, AAV4 and AAV5 interact with the cell surface by a distinct mechanism. In previous studies we have identified sialic acid as an important cell component in transduction with these serotypes. To aid in our investigation of virus tropism, we have begun to develop new tools to identify genes involved in AAV transduction. Through the use of microarray technology, we have linked AAV5 transduction with the expression of the platelet derived growth factor receptor alpha (PDGFRa) or beta (PDGFRb) expression. Our knowledge of the interactions necessary for transduction with these vectors has been helpful in defining their biology and likely will identify new applications for these vectors, as well as aid in the development of a new generation of vectors for gene transfer. We have also demonstrated a stable interaction between some of the non-structural proteins of AAV (Reps) and the cyclic AMP dependent protein kinase, PKA, and a novel homolog, PrKX. The AAVBU's investigations over the last three years have demonstrated this interaction constitutes a novel form of viral interference which has lead to our current concept of interferons. We have shown that one role of this interaction in the lifecycle of AAV is to control the replication of its helper virus during a co-infection with adenovirus (Ad). Our future studies will examine the biology of AAV2's interaction with other viruses and test whether this antiviral response is conserved in other isolates of AAV.
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