Background/Aims: Human parvovirus B19 (B19V) may cause inflammatory cardiomyopathy (iCMP) which is accompanied by endothelial dysfunction. The B19V capsid protein

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Down-Regulation of Na+/K+ ATPase Activity by Human Parvovirus B19 Capsid Protein VP1

The complex infection process of parvoviruses is not well understood so far. An important role has been attributed to a phospholipase A2 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.

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

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.

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

Parvovirus B19 is the causative agent of erythema infectiosum. In addition, parvovirus B19 infection may be associated with other disease manifestations, namely, thrombocytopenia or granulocytopenia, spontaneous abortion or hydrops fetalis in pregnant women, acute and chronic arthritis, and systemic lupus erythematosus. Based on sequence homology data, a phospholipase A2 motif has been identified in the VP1 unique region of parvovirus B19. (Y. Li et al., J. Gen. Virol. 82:2821-2825, 2001; Z. Zadori et al., Dev. Cell 1:291-302, 2001). We have established a new in vitro assay based on electrospray ionization tandem mass spectroscopy to show that phospholipase A2 activity is present in the VP1 unique region produced in Escherichia coli and in virus-like particles consisting of combinations of VP1 and VP2 proteins expressed by recombinant baculovirus. The enzyme activity of the VP1 unique region showed typical Ca(2+) dependency and could be inhibited by manoalide and 4-bromophenacylbromide, which bind covalently to lysine and histidine residues, respectively, as part of the active center of the enzyme. By using subfragments, we demonstrated an association between the phospholipase A2-like activity and the carboxy-terminal domain of the VP1 unique region.

The VP1 unique region of parvovirus B19 and its constituent phospholipase A2-like activity.

Release of canine parvovirus from endocytic vesicles.

Bombyx mori densovirus (BmDNV-1), on the basis of the previously reported genome sequence, constitutes by itself a separate genus (Iteravirus) within the Densovirinae subfamily of parvoviruses. Inconsistencies in the genome organization, however, necessitated its reassessment. The genome sequence of new clones was determined and resulted in a completely different genome organization. The corrected sequence also contained conserved sequence motifs found in other parvoviruses. Some amino acids in the highly conserved domain in the unique region of VP1 were shared by critical amino acids in the catalytic site and Ca2+-binding loop of secreted phospholipase A2, such as from snake and bee venoms. Expression of this domain and determination of enzyme activity demonstrated that capsids have a phospholipase A2 activity thus far unknown to occur in viruses. This viral phospholipase A2, which is required shortly after entry into the cell, showed a substrate preference for phosphatidylethanolamine and phosphatidylcholine over phosphatidylinositol.

G. Fédière

Papers

Genome organization of the densovirus from Bombyx mori (BmDNV-1) and enzyme activity of its capsid

Genome Organization of Casphalia extranea Densovirus, a New Iteravirus