A Conformational Change in the Adeno-Associated Virus Type 2 Capsid Leads to the Exposure of Hidden VP1 N Termini

Abstract

The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A 2 domain which is located within the unique N terminus of the capsid protein VP1. Based on the structural difference between adeno-associated virus type 2 wild-type capsids and capsids lacking VP1 or VP2, we show via electron cryomicroscopy that the N termini of VP1 and VP2 are involved in forming globules inside the capsids of empty and full particles. Upon limited heat shock, VP1 and possibly VP2 become exposed on the outsides of full but not empty capsids, which is correlated with the disappearance of the globules in the inner surfaces of the capsids. Using molecular modeling, we discuss the constraints on the release of the globularly organized VP1-unique N termini through the channels at the fivefold symmetry axes outside of the capsid.For infection, nonenveloped viruses are involved in a number of interactions with macromolecular structures of the host cell that enable virus uptake, disassembly, and delivery of the genome. Such interactions not only implicate recognition of key elements required for cell entry and intracellular passage but also enable activities of viral proteins to overcome the barriers of the host cell. This is exemplified by the binding of viral capsids to cellular receptors, the active release from endocytic vesicles, the use of cellular filament systems for trafficking, and different kinds of delivery of the viral genomes to the cell nucleus (30, 60). These processes are often supported by signaling events which reflect the reaction of the cell to the attachment and uptake of the virus (16). The information for these interactions is maintained within the structure of the virus shell.The infection process of adeno-associated virus type 2 (AAV-2) is initiated by attachment to the cell surface via binding to heparan-sulfate proteoglycan (53). Upon interaction with a secondary receptor, either fibroblast growth factor receptor 1 or ␣V␤5 integrin (41, 52), virions enter the cell by means of clathrin-coated pits (5) into the endosomal pathway. While a large number of viral particles seem to accumulate in perinuclear vesicular compartments (4), they have to escape into the cytoplasm for successful infection (21, 63). Rac signaling and interaction with cellular filament systems have an impact on trafficking toward the nucleus (44). There is increasing evidence that virions can enter the cell nucleus (5, 45, 63), possibly by an as yet unknown pathway which is independent of passage through the nuclear pores (22, 63). According to this scenario, the final uncoating reaction has to take place in the nucleus.The AAV-2 capsid harbors a 4.7-kb linear single-stranded genome which contains two open reading frames coding for four nonstructural proteins and three capsid proteins (48). They are flanked by two identical inverted terminal repeats. The nonstructural proteins have functions in the control of gene expression, in DNA replication, and in genome encapsidation (...

Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6

“…3A, VRII), consistent with dynamics which might be required for genome packaging or the PLA2 externalization (34,35). The HI loop lines the floor of the depression around the icosahedral 5-fold axes and is implicated in capsid assembly as well as capsid dynamics associated with receptor attachment (16,38).…”

“…2C and 3A), between the ␤DE and ␤HI strands, respectively, play essential structural and functional roles in the life cycle of the AAVs and other parvoviruses. The DE loops in five, symmetry-related monomers interact and form the channel at the 5-fold axis through which genomic ssDNA is postulated to be packaged (35). This is also where a phospholipase A2 (PLA2) domain, located within the VP1 unique N termini, is proposed to be externalized during cellular trafficking (reviewed in reference 10) (35).…”

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

“…10,16,33 Endosomal escape is mediated by lipolytic pore formation via phospholipase A2 activity upon endosomal acidification. [34][35][36] The latter also leads to the exposure of additional domains required for nuclear delivery of vector/viral genomes. 37 In view of the reliance of AAV2 on such specific intracellular conditions for successful infection, it is not surprising that ligands that direct rAAV targeting vectors to alternative pathways can lead to less efficient intracellular processing and lower transduction efficiency.…”

Structural Insights into Adeno-Associated Virus Serotype 5

“…Structures determined for AAVs show capsids assembled from the common VP3 region (20)(21)(22)(23)(24)(25)(26)(27)(28)(29). There is currently no experimentally determined structure for VP1u or the overlapping VP1/ VP2 N terminus.…”

“…There is currently no experimentally determined structure for VP1u or the overlapping VP1/ VP2 N terminus. Low-resolution density globules located inside the capsid under the icosahedral 2-fold axes have been interpreted as the VP1u (25,28). The VP3 topology contains a core eightstranded (␤B to ␤I) ␤-barrel motif and large loop insertions between the ␤-strands which form the surface of the capsid.…”