bIcosahedral viral capsids are obligated to perform a thermodynamic balancing act. Capsids must be stable enough to protect the genome until a suitable host cell is encountered yet be poised to bind receptor, initiate cell entry, navigate the cellular milieu, and release their genome in the appropriate replication compartment. In this study, serotypes of adeno-associated virus (AAV), AAV1, AAV2, AAV5, and AAV8, were compared with respect to the physical properties of their capsids that influence thermodynamic stability. Thermal stability measurements using differential scanning fluorimetry, differential scanning calorimetry, and electron microscopy showed that capsid melting temperatures differed by more than 20°C between the least and most stable serotypes, AAV2 and AAV5, respectively. Limited proteolysis and peptide mass mapping of intact particles were used to investigate capsid protein dynamics. Active hot spots mapped to the region surrounding the 3-fold axis of symmetry for all serotypes. Cleavages also mapped to the unique region of VP1 which contains a phospholipase domain, indicating transient exposure on the surface of the capsid. Data on the biophysical properties of the different AAV serotypes are important for understanding cellular trafficking and is critical to their production, storage, and use for gene therapy. The distinct differences reported here provide direction for future studies on entry and vector production.

We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs) from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i) low isoelectric point of a capsid and (ii) relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP) column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP) grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.A deno-associated virus (AAV) is a nonenveloped singlestranded DNA virus that has a relatively simple capsid consisting of 3 proteins that share a C-terminal amino acid sequence (2). The three capsid proteins (VP1, VP2, and VP3) assemble in an approximate ratio of 1:1:10 to form an icosahedral particle ϳ246 nm in diameter. Recently the X-ray crystal structure of AAV serotype 8 (AAV8) identified a surface region that undergoes a conformational shift when the virus is incubated at acidic pH (14). This region of four amino acids (the pH quartet) is formed from three different symmetry-related capsids at the intersection of the 2-, 3-, and 5-fold axes. Two of these amino acids, E566 and Y707, interact through hydrogen bonds at pH 7.4 but progressively migrate from each other as the pH is lowered to 6.0 or 5.5, which are the approximate pHs of early and late endosomes. Similar amino acid rearrangements have been seen in the crystal structures of AAV1 (unpublished observation). In AAV2 (Fig. 1), E563 is structurally equivalent to E566 in AAV8, and it is flanked by two other acidic residues (E562 and E564) that are conserved in all AAV serotypes that have been sequenced to date (Table 1). In addition, a tyrosine (Y704), arginine (R389), and histidine (H526) are also highly conserved, as well as another acidic cluster (528DDEE531) near this region ( Fig. 1 and Table 1). Acidification of...

The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ϳ5.0-Å resolution (medium) and also at 3.8-and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ϳ10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the ␤A strand region under the icosahedral 2-fold axis rather than antiparallel to the ␤B strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCEThe mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ϳ1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.T he adeno-associated viruses (AAVs) have emerged as attractive gene therapy vectors because they can package foreign genes and achieve stable, long-term gene expression in a broad range of tissues, with no known pathogenicity (1-3). AAVs are small, nonenveloped, icosahedral viruses (ϳ260 Å in diameter) that package ϳ4.7 kb of single-stran...

Twenty-eight bacteriophages infecting the local host Bacillus pumilus BL-8 were isolated, purified, and characterized. Nine genomes were sequenced, of which six were annotated and are the first of this host submitted to the public record. The 28 phages were divided into two groups by sequence and morphological similarity, yielding 27 cluster BpA phages and 1 cluster BpB phage, which is a BL-8 prophage. Most of the BpA phages have a host range restricted to distantly related strains, B. pumilus and B. simplex, reflecting the complexities of Bacillus taxonomy. Despite isolation over wide geographic and temporal space, the six cluster BpA phages share most of their 23 functionally annotated protein features and show a high degree of sequence similarity, which is unique among phages of the Bacillus genera. This is the first report of B. pumilus phages since 1981.

Viruses are a diverse class of nanoparticles. However, they have evolved a few common mechanisms that enable successful infection of their host cells. The first stage of this process involves entry into the cell. For enveloped viruses this process has been well characterized. For nonenveloped viruses, the focus of this review, the entry mechanisms are less well understood. For these viruses, a typical pathway involves receptor attachment followed by internalization into cellular vesicles and subsequent viral escape to the cytosol and transport to the site of genome replication. Significantly, these viruses have evolved numerous mechanisms to fulfill this seemingly simple infection scheme. We focus on the latest observations for several families of nonenveloped viruses and highlight specific members for eukaryotic families: Adenoviridae, Papillomaviridae, Parvoviridae, Picornaviridae, Polyomaviridae and Reoviridae; and prokaryotic families: Microviridae, Myoviridae, Podoviridae and Siphoviridae.

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