Size does matter: overcoming the adeno-associated virus packaging limit

Recombinant adeno-associated virus (rAAV) vectors mediate long-term gene transfer without any known toxicity. The primary limitation of rAAV has been the small size of the virion (20 nm), which only permits the packaging of 4.7 kilobases (kb) of exogenous DNA, including the promoter, the polyadenylation signal and any other enhancer elements that might be desired. Two recent reports (D Duan et al: Nat Med 2000, 6:595-598; Z Yan et al: Proc Natl Acad Sci USA 2000, 97:6716-6721) have exploited a unique feature of rAAV genomes, their ability to link together in doublets or strings, to bypass this size limitation. This technology could improve the chances for successful gene therapy of diseases like cystic fibrosis or Duchenne muscular dystrophy that lead to significant pulmonary morbidity

Structure and Dynamics of Adeno-Associated Virus Serotype 1 VP1-Unique N-Terminal Domain and Its Role in Capsid Trafficking

The importance of the phospholipase A2 domain located within the unique N terminus of the capsid viral protein VP1 (VP1u) in parvovirus infection has been reported. This study used computational methods to characterize the VP1 sequence for adeno-associated virus (AAV) serotypes 1 to 12 and circular dichroism and electron microscopy to monitor conformational changes in the AAV1 capsid induced by temperature and the pHs encountered during trafficking through the endocytic pathway. Circular dichroism was also used to monitor conformational changes in AAV6 capsids assembled from VP2 and VP3 or VP1, VP2, and VP3 at pH 7.5. VP1u was predicted (computationally) and confirmed (in solution) to be structurally ordered. This VP domain was observed to undergo a reversible pH-induced unfolding/refolding process, a loss/gain of α-helical structure, which did not disrupt the capsid integrity and is likely facilitated by its difference in isoelectric point compared to the other VP sequences assembling the capsid. This study is the first to physically document conformational changes in the VP1u region that likely facilitate its externalization from the capsid interior during infection and establishes the order of events in the escape of the AAV capsid from the endosome en route to the nucleus

DNA damage alters DNA polymerase δ to a form that exhibits increased discrimination against modified template bases and mismatched primers

Human DNA polymerase δ (Pol δ4), a key enzyme in chromosomal replication, is a heterotetramer composed of the p125, p50, p68 and p12 subunits. Genotoxic agents such as UV and alkylating chemicals trigger a DNA damage response in which Pol δ4 is converted to a trimer (Pol δ3) by degradation of p12. We show that Pol δ3 has altered enzymatic properties: it is less able to perform translesion synthesis on templates containing base lesions (O6-MeG, 8-oxoG, an abasic site or a thymine-thymine dimer); a greater proofreading activity; an increased exonuclease/polymerase activity ratio; a decreased tendency for the insertion of wrong nucleotides, and for the extension of mismatched primers. Overall, our findings indicate that Pol δ3 exhibits an enhanced ability for the detection of errors in both primers and templates over its parent enzyme. These alterations in Pol δ3 show that p12 plays a major role in Pol δ4 catalytic functions, and provides significant insights into the rationale for the conversion of Pol δ4 to Pol δ3 in the cellular response to DNA damage

Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies

Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

The APOBEC3 proteins form a multigene family of cytidine deaminases with inhibitory activity against viruses and retrotransposons. In contrast to APOBEC3G (A3G), APOBEC3A (A3A) has no effect on lentiviruses but dramatically inhibits replication of the parvovirus adeno-associated virus (AAV). To study the contribution of deaminase activity to the antiviral activity of A3A, we performed a comprehensive mutational analysis of A3A. By mutation of non-conserved residues, we found that regions outside of the catalytic active site contribute to both deaminase and antiviral activities. Using A3A point mutants and A3A/A3G chimeras, we show that deaminase activity is not required for inhibition of recombinant AAV production. We also found that deaminase-deficient A3A mutants block replication of both wild-type AAV and the autonomous parvovirus minute virus of mice (MVM). In addition, we identify specific residues of A3A that confer activity against AAV when substituted into A3G. In summary, our results demonstrate that deaminase activity is not necessary for the antiviral activity of A3A against parvoviruses

Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome

The Effect of DNA-Dependent Protein Kinase on Adeno-Associated Virus Replication

BACKGROUND: DNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins. CONCLUSION/SIGNIFICANCE: Collectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs