Adeno-associated virus (AAV) is a member of the parvovirus family. It is a single-stranded DNA virus with a genome of ~4.7 kb that is dependent upon coinfection with helper viruses, such as adenovirus or herpesvirus, for efficient reproduction (5
). This small genome encodes four nonstructural proteins (Rep78, -68, -54, and -48) and three viral capsid proteins, Vp1, Vp2, and Vp3 (29
). The Rep proteins are multifunctional and play roles in almost every aspect of the life cycle of AAV, including replication, transcription, integration, and packaging of the genome into the preformed empty capsid (10
). Vp1, Vp2, and Vp3 are incorporated into capsids at a predicted ratio of 1:1:8 and have overlapping sequences, differing only at their N termini. Vp2 is 137 amino acids shorter than Vp1 and is the product of an alternative start codon, while Vp3 is 65 residues shorter than Vp2.
The structure of AAV type 2 (AAV2) has been determined at 3-Å resolution by X-ray crystallography. In addition, the capsid structures of other parvoviruses have been resolved by X-ray crystallography or cryoelectron microscopy (cryo-EM) and image reconstruction (1
). The parvovirus virion is composed of 60 subunits of the overlapping Vp (~530 C-terminal amino acids) region arranged with T
= 1 icosahedral symmetry (25
). Common features are a depression at the twofold axis of symmetry, three protrusions at or surrounding the threefold axes, and 12 narrow pores at the fivefold axes. The overlapping subunit structure is comprised of an eight-stranded β-barrel motif containing two antiparallel β-sheets (48
). Long loop insertions between the strands comprise about 60% of the structure. These interstrand loops contain small regions of β-structure and a conserved helix that forms the wall of the depressions at the twofold axes. Two small stretches of strand structure form a β-ribbon in each subunit, which cluster at the icosahedral fivefold axis of the capsid to create the cylindrical pores that connect the inside of the capsid to the outside. The 12 narrow pores at the fivefold axes are one of the most striking structural features of AAV2 capsids (Fig. ). The pores protrude from the surrounding capsid surface, surrounded by a depression that is lined by a loop between the βH and βI strands of the β-barrel. It is thought that the β-ribbons are dynamic structures, since the pore increases in diameter from the inside to the outside of the capsid. The β-ribbons are made up of residues 322 to 338 of the capsid sequence, including a highly conserved Leu336, and the residue type is found in all known parvoviruses. This residue surrounds the inside base of the channel, with side chains constricting the channel to ~8.7 Å in diameter (Fig. ).
FIG. 1. AAV2 crystal structure. (A) Surface topology of the whole capsid, viewed down the icosahedral fivefold symmetry axis, that contains a pore (left) bounded by five symmetry-related Vp subunits shown in salmon, blue, green, purple, and cyan ribbons in the (more ...)
The unique N termini of Vp1 and Vp2, proposed to be localized on the inside of the capsids, have not been visualized in any of the parvovirus crystal structures, although cryo-EM and image reconstruction studies of AAV2 interpreted “globules” observed under the twofold axis as structural candidates for these regions (24
). In the crystal structure of mature MVM virions, weak density within the fivefold pores could be modeled as a glycine-rich sequence within the N terminus of Vp1 to Vp3 of minute virus of mice (MVM) (3
). The crystal structure of canine parvovirus (CPV) virions supports the hypothesis that the weak density inside the fivefold pore corresponds to N-terminal peptide sequences (49
). There is biochemical and mutational evidence that the N-terminus of Vp1 and Vp2 in some parvoviruses can be externalized onto the capsid surface via the pores at the fivefold axes while the capsid remains intact. In parvoviruses, such as MVM and CPV, that undergo maturation cleavage of Vp2 to produce Vp3 following genomic-DNA packaging, it is thought that this exposure allows the processing (13
). It is also important to note that the unique N-terminal region of Vp1 of CPV became exposed in vivo within endosomal/lysosomal vesicles (35
). In vitro exposure of Vp1 N termini has been achieved by treatment of capsids with heat or urea (8
). However, in empty CPV, MVM, and AAV2 capsids, the N-terminal regions do not become accessible to antibodies while the capsid remains intact (13
), suggesting that the viral genome aids in the transition required for surface exposure.
The early steps of AAV2 infection begin with attachment to heparan sulfate proteoglycan and to a variety of cell surface receptors, such as FGFR, αVβ5 integrin, α5β1 integrin, and hepatocyte growth factor receptor (c-Met) (4
), followed by clathrin dependent endocytosis. Based on a number of studies, it has been proposed that AAV requires endosomal acidification to escape from the late endosome and traffic to the nucleus (16
). These studies also suggest that prior to escaping the endosome, the virion must undergo conformational changes leading to the exposure of the unique N terminus of Vp1 and the N terminus of Vp2 required for endososmal escape and nuclear entry (6
). The unique N terminus of Vp1 contains a conserved phospholipase A2 (PLA2) domain (8
) that is essential for infectivity and is thought to be required for endosomal escape. A suggestion that the N terminus of Vp1 are located within the virion and become surface exposed naturally within the cell via an induced conformational change is consistent with the observation that while intact capsids do not have PLA2 activity, heat or acidic-pH treatment of virions elicits this function (37
). Basic amino acid clusters, which are thought to control the nuclear import of incoming virions, are also contained within the unique Vp1 N terminus (27
) and the N-terminal amino acid stretch (166
) contained within an overlapping region of Vp1 and Vp2 (19
). With increasing knowledge of capsid motifs involved in AAV infection, it is currently unknown if the virions that have escaped the endosome successfully infect the host cell, because it has been previously shown that nuclear entry of intact AAV virions is very inefficient, suggesting that uncoating may occur before or during nuclear entry (30
In addition, the pore at the fivefold axis of the parvovirus capsid has been postulated as the site for genome import into the preformed capsid, genome export for uncoating, and Vp1/Vp2 N-terminus exposure during trafficking (6
). The purpose of this study was to further explore the role of the fivefold pores in the life cycle of AAV2. We decided to take a unique approach to determine the significance of the pore in the life cycle of AAV2 by generating Vp1 protein fusion constructs that would place the unique N terminus of Vp1 on the outside of the virus, similar to the autonomous parvovirus B19. A previous study of AAV2 capsids generated with the 8-kDa chemokine domain of fractalkine (FKN) fused to the end of the Vp2 protein had shown that the fusion protein was displayed on the capsid surface (6
). We rationalized that a construct made from a Vp2AFKN fusion to which the unique N-terminus of Vp1 was fused should also display the latter protein on the capsid surface. Our approach was to test the ability of such a fusion construct, carrying an active PLA2 domain with and without a nuclear localization signal (NLS) exposed on the capsid surface upon assembly, to enhance/restore the infectivity of Vp2/Vp3 and Vp3-only virions. In addition, we tested the abilities of these fusion constructs to rescue viruses with mutations at the conserved Leu336 residues positioned at the base of the fivefold axis, for which prevention of Vp1 N-terminus exposure through the fivefold pore had been reported to be detrimental to infection (6
). The Vp1 fusion proteins incorporated into the AAV capsid were surface exposed and functional based on native dot blot Western blotting and a PLA2 assay. Infectivity was enhanced/regained for Vp2/Vp3 and Vp3-only virions when Vp1FKN and Vp1NLSFKN were incorporated into the virion. However, the Vp1 fusion proteins did not rescue/enhance infectivity for the Leu336 mutants. In addition, the Leu336 mutant virions appeared to have altered permeability to transmission EM-negative stain compared to virions assembled from wild-type (wt) viral capsid proteins. Taken together, our data support the conclusions that (i) it is possible to generate infectious AAV virions that surface display their Vp1 N termini upon assembly, as has been reported for B19, and (ii) mutations of the conserved Leu336 cannot be rescued by surface-exposed Vp1 N termini, suggesting that Leu336 mutants may lead to a global conformational change in virions or inhibit an essential structural change that prevents successful infection.