| The adeno-associated virus type-2 (AAV2) has received considerable attention as a gene therapy vector. All current AAV2 vectors share two elements, the terminal repeats (TRs) and the capsid. The TRs are believed to be essential cis-DNA elements that participate in the majority of viral intracellular activities including priming DNA replication, transcriptional regulation, directed integration, proviral rescue, and genome packaging. The TRs fold back on themselves in solution forming a stable T-shaped secondary structure that can be endonucleolytically nicked in a strand- and site-specific manner by the Rep protein. Here we produce recombinant viruses with mutations in the TR known to enhance and inhibit nicking and we examine the effects during an infection in vivo. We demonstrate that Rep binding and nicking are required for efficient replication from a plasmid template. Importantly, we generated a recombinant virus that is wild type in every respect except that it is unable to integrate into human chromosome 19. In addition, we show that in vivo the p40 Cap proteins are expressed at high levels independent of detectable Rep expression.; The capsid is an icosahedral protein shell (composed of 60 structurally equivalent subunits) that protects the AAV genome from degradation and that physically docks to the cell to initiate a virus infection. Here we identify the specific amino acids on the surface of the capsid that facilitate to binding to the cell surface receptor heparin sulfate proteoglycan (HSPG). Our data indicate that a spatially clustered array of four arginines and one lysine, including residues 484, 487, 532, 585, and 588, are primarily responsible for HSPG binding, and therefore for infectivity. Substituting a six amino acid stretch containing residues 585 and 588 into the HSPG binding-deficient serotype AAV5 conferred heparin binding in vitro, suggesting that these are the minimal necessary and sufficient requirements for HSPG binding. |
