With the availability of the AAV2 crystal structure (59
), many aspects of the AAV life cycle, including host cell recognition (3
), intracellular trafficking (5
), and uncoating (43
), are now possible to correlate with structure. The first of such studies have centered around the threefold loops and the determination that they are key topological features in host cell recognition (4
). Similar structure-function studies have extended from the threefold loops to the fivefold axis and the location of the virion pore and its potential role in viral genome packaging, capsid assembly, and VP1 unique N-terminal exposure (8
). Interestingly, the HI loop surrounds the fivefold pore and has an essential structural role in viral assembly by overlapping the neighboring VP3 subunit forming amino acid interactions with the underlying EF loop (Fig. ). Recently, Mavis Agbandje-McKenna has observed the HI loop flipping up 90° upon AAV2-heparan sulfate proteoglycan binding, suggesting a dynamic role for this structure in the viral infectious pathway (unpublished). To better understand the role of this capsid structure, we chose to characterize in detail the HI loop as it may contribute to specific stages in the virus life cycle such as viral genome packaging, assembly, and subsequent stages during the infectious pathway. The results of this study demonstrate that the AAV2 HI loop is essential for proper capsid assembly, packaging of the viral DNA, and viral infectivity when the conserved phenylalanine at amino acid position 661 is altered.
We carried out a comprehensive amino acid deletion-and-substitution study to uncover the role of the HI loop in the AAV life cycle. From these efforts, we determined that removal of the HI loop (AAV2 HI−/−
) leads to capsids that cannot assemble (Fig. ). We assayed viral assembly primarily with the A20 monoclonal antibody (52
), which detects tertiary structure of properly assembled AAV capsids. Viruses that were unable to form virions were further studied with gradients and Western blot analyses that confirmed the ability to synthesize VP subunits (data not shown). Although we relied primarily on A20 recognition to confirm the ability to form proper virion structures, additional studies such as iodixanol gradient purification of AAV2 HI−/−
cell lysate, followed by EM analysis, further determined that this mutant only appears to generate monomer subunits (data not shown). In contrast, replacement of the loop with glycines (AAV2 polyglycine) generated A20-recognizable assembled capsids; however, these capsids were deficient in the ability to package the viral DNA. Even though this mutant provided sufficient structure to assemble intact AAV particles, the glycine substitutions specifically ablate amino acid side chain interactions with the EF loop of the underlying subunit, suggesting that the HI loop structure and the backbone interactions of the HI loop with the underlying subunit are sufficient to facilitate capsid formation. However, the specific amino acid interactions are required for efficient packaging of the viral DNA. Although we cannot draw from our studies the exact mechanism of the viral genome packaging deficiencies of the glycine substitution mutant, it is interesting to speculate that this phenotype can possibly be attributed to gross conformational changes in the structure of the fivefold pore since the fivefold pore has been implicated as the site of Rep protein binding, a necessary step for efficient DNA encapsidation (7
). As a result, it will be of interest to determine the ability of HI loop mutants to bind Rep protein in pull-down experiments as previously described (7
In addition to the deletion-and-substitution studies described above, we decided to swap the AAV2 HI loop with those from representative serotypes. Domain swapping, specifically between virus serotypes, allows for determination of the role of that specific region of the capsid in the virus life cycle. For example, in adenovirus, the fiber necessary for host cell recognition (54
), was swapped between adenovirus subgroups, resulting in alteration of intracellular trafficking of the adenovirus vectors (29
). Alternatively, in the case of AAV, domains were swapped between AAV1 and -2 in order to characterize determinants necessary for AAV1 muscle tropism (19
). In a similar study regarding AAV, domains from AAV2 were swapped with those from AAV8, demonstrating that loop IV of AAV8 at the threefold axis of symmetry is responsible for dictating liver tropism (37
). Interestingly, in our studies, swapping the AAV2 HI loop with those of AAV1 and AAV8 did not affect titer, transduction, heparin binding, or gross conformation. However, this can be expected due to the relatively higher sequence homology between AAV2 and these serotypes. More importantly, these results suggest that the HI loop most likely does not contain determinants of tissue tropism or receptor binding. This was further confirmed via mouse intramuscular injections with AAV2 RGD 662. Briefly, bioluminescence imaging revealed similar in vivo transduction profiles for AAV2 RGD 662 and WT AAV2 1 week postadministration (data not shown).
In the case of the AAV4 and AAV5 HI loops, significant phenotypic variations were seen, possibly due to lower sequence homology with AAV2. For instance, the AAV4 HI loop is composed of a higher number of hydrophobic residues than the loop from AAV2 based on the amino acid sequence and crystal structure (16
). The three-dimensional structure of the AAV2 VP3 monomer (Fig. ) shows that the side chains of residues 659 and 660 point away from the capsid and do not interact with the residues in the underlying subunit. On the other hand, the conserved phenylalanine at position 661 interacts with proline 373 in the EF loop of the underlying subunit, as mentioned in Results. The alanine-to-serine change at position 663 of the AAV2 HI4 mutant might contribute a hydrogen bond interaction due to a potentially accessible hydroxyl group that is not present in the AAV2 HI loop. The K665P change in AAV2 HI4 suggests a possible contribution to increased hydrophobic interactions with proline, valine, phenylalanine, and methionine residues found in the underlying subunit of AAV2. However, this assessment is based on a structure model of AAV2 HI4, and a more accurate analysis of the AAV4 HI loop amino acid contributions to AAV2 capsid stability is dependent upon knowing the crystal structure of the AAV2 HI4 mutant.
Collectively, the aforementioned amino acid changes in AAV2 HI4 could enhance HI loop-EF loop interactions and thereby could well account for increased capsid stability, as demonstrated through increased resistance to heat treatment in comparison to AAV2. In addition, such increases in capsid stability and possible gross conformational changes to the fivefold pore might account for lower packaging efficiency and titers seen with the AAV2 HI4 mutant. It is possible that the AAV capsid “breathes” or expands in volume during viral genome packaging, and if the capsid is too stable or held too tightly together, it may be more difficult for the Rep protein to package the viral genome. The idea of capsid expansion has been studied in bacteriophage, and it has been shown that during the DNA packaging process a conformational change occurs which causes an increase in capsid volume (21
Additionally, previous data suggest that the Rep protein is bound in higher quantities to the capsids of packaging-deficient mutants (7
), possibly due to “jamming” of the genome threading machinery. Such has also been noted in the case of AAV2 HI4, wherein the particle bound increased amounts of Rep protein in comparison with AAV2 (data not shown). For the AAV2 HI4 mutant, it is possible that there is increased stability of the particle based on the presence of another protein or proteins on the capsid surface.
In the case of AAV2 HI5, despite normal expression of capsid subunit proteins, no intact capsid assembly is observed. This was further confirmed via EM analysis on cesium chloride gradient fractions of the AAV2 HI5-transfected cell lysate. It was determined that the AAV2 HI5 mutant may form pentamers but does not form proper intact particles (data not shown). This phenotype is likely attributable to the fact that the AAV5 HI loop is one amino acid shorter, based on the crystal structure (48
), than the WT AAV2 HI loop. In corollary, insertion of the missing threonine at position 659 minimally rescues capsid assembly. Therefore, the length of the HI loop in relation to the underlying subunit appears to be crucial for proper capsid assembly, while the loop amino acid interactions with the underlying subunit dictate genome packaging efficiency. This was further corroborated by a new collection of HI mutants that increased the length of the HI loop by 1, 3, 5, and 7 glycine residues. The fact that the AAV2 HI loop glycine extensions formed intact virus particles but were unable to package the viral genome, based on DNA dot blot (data not shown) and Western dot blot (data not shown) analyses, further supports the importance of the length of the HI loop.
Following the domain swaps, we decided to use peptide substitutions in order to mutate multiple residues of the HI loop. Many groups have successfully inserted peptides, specifically at the threefold loops, on the capsid surface as a means to retarget the virus for specific tissue types (14
). In this study, we used peptides not as insertions but as substitutions in a novel region of the capsid. Peptide substitution within the AAV2 HI loop showed that certain amino acid changes do not affect virus titer and transduction, as seen with the AAV2 RGD 658, AAV2 RGD 662, and AAV2 QPEHSST mutants. However, some peptide substitutions resulted in marked changes in phenotype that were dependent on the amino acid position substituted. For instance, replacement with peptide RGD at position 660 (AAV2 RGD 660), SGRGDS starting at position 658 (AAV2 SGRGDS), VNTANST starting at position 658 (AAV2 VNTANST), and SIGYPLP also starting at position 658 (AAV2 SIGYPLP) resulted in decreased titer and infectivity. In all of these mutants, the conserved F661 residue observed in all serotypes is replaced.
Interestingly, a number of these mutants, such as AAV2 VNTANST, SIGYPLP, RGD 660, and SGRGDS, also revealed differential banding patterns seen with B1 antibody staining of a Western blot (Fig. ). Specifically, there appears to be a decreased incorporation of VP1 capsid subunits in these mutant capsids. It is likely that such a phenotype, which would reduce the effectiveness of the PLA2 domain (located in the VP1 N-terminal domain) required for endosomal escape and nuclear entry of the viral capsid, could explain the decrease in transduction seen with these mutants (15
). The lower titers of the aforementioned mutants can possibly be attributed to improper capsid assembly (7
) and defective packaging (7
). In the AAV2 VNTANST mutant, there is an additional protein band seen between VP2 and VP3 with B1 staining that has yet to be identified. The observed protein product is most likely due to proteolytic processing of VP1, which could also account for the decreased amount of VP1 present in this capsid mutant. The 77-kDa protein band in AAV2 RGD 660 and AAV2 SGRGDS shown by A1 staining further corroborates these speculations (Fig. ).
As mentioned above, one trait shared by these defective peptide substitution mutants is that they span the conserved phenylalanine at amino acid position 661. F661 interacts with P373 in the EF loop in the underlying subunit of all serotypes through stacking interactions (Fig. ). This interaction appears critical for the stability (9
) of assembled capsid subunits since the HI loop is the only region at the fivefold axis of symmetry that extends from one subunit and overlaps the underlying subunit. Mutation of F661 results in a phenotype similar to that seen with peptide substitutions spanning this region. Based on data shown in Fig. , we hypothesize that the interaction between F661 and P373 is necessary to stabilize the capsid around the fivefold axis of symmetry, being of great importance due the fivefold pores’ contribution to viral genome packaging and infectivity (8
). Disruption of this interaction appears, in particular, to reduce the amount of VP1 incorporated into these mutant capsids (Fig. ).
Additionally, such mutagenesis could result in improper incorporation of VP1 subunits at the fivefold axis of symmetry, which would expose the critical PLA2 domain to cellular proteases during virus production. If unassembled VP1 monomers or loosely assembled particles exposing the VP1 unique N terminus are present, it is possible that they may be susceptible to cellular proteases. This may not occur as readily in the WT or other mutant viruses that are able to assemble the VP monomers efficiently in a stable configuration. Further studies will help elucidate these hypotheses.
In conjunction with this observation, a similar phenomenon may be occurring in the AAV baculovirus production system (41
). There appears to be inefficient incorporation of VP1 into the AAV2 capsid during production in insect cells, and this may be due to the susceptibility of VP1 to cellular protease in the nonmammalian cell environment (24
). The notion that VP1 is susceptible to cellular proteases is further substantiated by the fact that when mammalian cells were transfected with VP1 constructs, specifically, VP1NLSFKN and VP1FKN, a second-molecular-mass band was detected between VP1 and VP2 in the cell lysates (17
), similar to the result obtained in this study. Upon mutation of F661, this molecular mass species was not only generated but also incorporated into the intact capsid.
In addition, it is not surprising that a single amino acid on the AAV capsid, such as F661, could significantly impact the biology of the virus. A recent study in our laboratory has shown that single amino acid mutations, specifically, K531E in AAV6 and E531K in AAV1, suppress and enhance heparin binding, respectively (56
). Taken together, our data support the role of the HI loop as an important AAV capsid structural element necessary for proper incorporation of VP1 into an assembled infectious particle and a functional fivefold pore that allows efficient packaging of viral genomes.