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

Abstract

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.

FIG. 1.

Sequence homology of the VP1 unique region with different phospholipases A2 (PLA2 IB, from bovine pancreas; PLA2 IIC, from a rat; PLA2, from bee venom) (1) and schematic representation of the location of functional activities in the sequence of the VP1 protein. The VP1 protein consists of the VP1 unique region (aa 1 to 227) and the VP2 protein (aa 227 to 780). The hypothesized phospholipase A2 motif ranges from aa 130 to 195 in the VP1 unique region and the VP1N-B fragment. A potential globoside binding site is in the region from aa 577 to 677. The amino acid sequence of the VP1 unique region from positions 130 to 195 shows the important catalytic residues that are observed (shaded gray), the residues important for binding of Ca²⁺ (arrows), the residues forming the catalytic network (circles), and the phospholipid binding site (squares).

FIG. 2.

Phospholipase A2 assay. Comparison of the phospholipase A2 activities of the VP1 unique region (B19-VP1N) and shortened subfragments of the protein domain with that of a control bee venom phospholipase A2. The VP1 unique region (B19-VP1N), subfragments VP1N-A and VP1N-B (1 μM), and the bee venom phospholipase A2 (0.017 μM) were incubated at 37°C for 10 min with or without 10 mM calcium chloride. The basic reaction mixture was comprised of 100 μl of phospholipid liposomes containing 25 or 50 nM PC in 10 mM Tris buffer (pH 8.5). The reaction was stopped by the addition of methanol-chloroform (2:1 [vol/vol]). Lipids were extracted according to the method of Bligh and Dyer (3). The dried lipid extracts were dissolved in methanol-chloroform (3:1 [vol/vol]) containing 10 mM ammonium formate and characterized by electrospray ionization tandem mass spectrometry. We used a Quattro LC triple-quadrupole mass spectrometer (Micromass, Manchester, United Kingdom) with the following settings: capillary, 3.5 kV; cone, 41 V; collision energy, 24 V; and collision gas pressure, 1.3 10⁻³ torr. Samples were injected at a constant flow rate of 75 μl/min with the Waters (Milford, Mass.) Alliance model 2790 filtration system and analyzed by a parent scan of m/z 184 specific for phosphocholine-containing lipids (6). The intensity of the ratio of hydrolyzed product (LPC) to substrate (PC) was used to determine the degree of phospholipase A2 activity. Standard deviations are indicated by bars.

FIG. 3.

Kinetics showing the reactivities of the VP1 unique region (2 μM) (B19-VP1N) and bee venom phospholipase A2 (0.034 μM) (PLA2 bee venom). The proteins were incubated at 25°C for 32 min in the presence of 10 mM calcium chloride. The reaction was stopped after 0, 0.5, 1, 2, 4, 8, 16, and 32 min, and the LPC/PC ratio was determined.

FIG. 4.

Inhibition of the phospholipase A2 activity with manoalide (MLD) and 4-bromophenacylbromide (BPB). The VP1 unique region (1 μM) (VP1N) and the bee venom phospholipase A2 (0.017 μM) (PLA2) were preincubated with 100 μM MLD and 1 mM BPB for 1, 2, 3, 4, and 5 h. The negative sample (neg) was incubated for 5 h without inhibitor. After incubation, the LPC/PC ratio was determined and calculated.

FIG. 5.

Comparison of enzyme activities of the VP1 unique region (1 μM protein) (B19-VP1N), recombinant VP1-VP2 capsids (1 μM protein), and VP2 capsids (1 μM protein).