Mutational Analysis of Narrow Pores at the Fivefold Symmetry Axes of Adeno-Associated Virus Type 2 Capsids Reveals a Dual Role in Genome Packaging and Activation of Phospholipase A2 Activity

Abstract

Adeno-associated virus type 2 (AAV2) capsids show 12 pores at the fivefold axes of symmetry. We mutated amino acids which constitute these pores to investigate possible functions of these structures within the AAV2 life cycle. Mutants with alterations in conserved residues were impaired mainly in genome packaging or infectivity, whereas few mutants were affected in capsid assembly. The packaging phenotype was characterized by increased capsid-per-genome ratios. Analysis of capsid-associated DNA versus encapsidated DNA revealed that this observation was due to reduced and not partial DNA encapsidation. Most mutants with impaired infectivity showed a decreased capability to expose their VP1 N termini. As a consequence, the activation of phospholipase A2 (PLA2) activity, which is essential for efficient infection, was affected on intact capsids. In a few mutants, the exposure of VP1 N termini and the development of PLA2 activity were associated with enhanced capsid instability, which is obviously also deleterious for virus infection. Therefore, PLA2 activity seems to be required on intact capsids for efficient infection. In conclusion, these results suggest that the pores at the fivefold axes function not only as portals for AAV2 single-stranded DNA packaging but also as channels for presentation of the PLA2 domain on AAV2 virions during infection.The viral capsid can be considered as a multiprotein complex which not only protects the viral genome against extreme environmental influences outside the cell but also combines a number of functional elements which are needed for host cell recognition, intracellular trafficking, and disassembly in order to successfully deliver genetic information to the host cell (for reviews, see references 23, 60, and 75). These functional elements either are concomitantly transported with the virion as separate proteins or are part of the structural proteins themselves. Identifying such functional elements on viral capsids is one of the most attractive goals of viral structural research. Over the past few years, several such functional domains have been identified in parvoviruses (for a review, see reference 70).Adeno-associated virus (AAV) type 2 (AAV2) is a member of the parvovirus family. AAV2 requires coinfection with helper viruses, such as adenovirus or herpesvirus, for efficient reproduction (4, 9, 58) and has a nonenveloped icosahedral capsid. It encloses a 4.7-kb single-stranded DNA (ssDNA) genome containing two large open reading frames, rep and cap (61). The Rep proteins, encoded by the rep gene, are required for DNA replication, DNA encapsidation, and control of gene expression (36,45). The viral capsid is composed of three structural viral proteins (VPs), designated VP1, VP2, and VP3, which are encoded by the cap open reading frame and expressed from the p40 promoter. Capsids are formed with the VPs in an approximate molar ratio of 1:1:10 due to alternative splicing and an unconventional start codon for VP2. This ratio is also maintained in assembled capsids (6, 34). The ...

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“…There are also reports that mutation of AAV2 residues in the 5-fold channel, structurally adjacent to the HI loop, both in the ␤-ribbons that extend outward (with VR-II at its apex) and the residues on the interior narrow region of the channel, results in an inability of the capsid to package its genome (105,106). It has been suggested that this is principally due to altered Rep-VP complexes that form at the 5-fold channel, proposed to be the portal for DNA packaging.…”

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“…Capsid assembly probably occurs in the nucleolus and is then relocalized into the nucleoplasm, with some influence of Rep proteins. The colocalization of the empty assembled capsid, the Rep proteins and the ssDNA AAV genome in the nucleoplasm allows the encapsidation process to occur (Im and Muzyczka, 1989;Wistuba et al, 1997;King et al, 2001;Bleker et al, 2005). Finally, the novel viral particles exit the cell, probably by an unknown exocytosis mechanism.…”

“…These three capsid proteins differ among them at the N terminus. VP1, VP2 and VP3 assemble in a molar ratio of 1:1:10, respectively, forming a near-spherical protein shell of 60 subunits that leads to perfect icosahedral symmetry (Bleker et al, 2005). A conserved phospholipase A 2 (PLA2) motif, located within the VP1 N-terminal region (Zádori et al, 2001), was reported to have a biological significance in AAV2 infection (Girod et al, 2002).…”

“…A conserved phospholipase A 2 (PLA2) motif, located within the VP1 N-terminal region (Zádori et al, 2001), was reported to have a biological significance in AAV2 infection (Girod et al, 2002). It seems to play a crucial role in the trafficking of the AAV genome from the endosome to the nucleus and in the initiation of viral gene expression (Girod et al, 2002;Bleker et al, 2005).…”

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“…Recombinant AAV vectors were quantified by replicative Dot Blot titration 50 giving infectious titers expressed as replicating units (r.u.). Instead of HeLa cells, HeLaRC cells expressing Rep and Cap proteins 35 were used.…”