Laboratorium für Molekulare Biologie, Genzentrum, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 München, Germany1
Deutsches Krebsforschungszentrum, Forschungsschwerpunkt Angewandte Tumorvirologie, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany2
Centre de Microbiologie et Biotechnologie, INRS Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Laval, Quebec, Canada H7V 1B73
Medizinische Klinik III, Klinikum Grosshadern, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, D-81377 München, Germany4
GSF National Research Center for Environment and Health, Klinische Kooperationsgruppe Gentherapie, Hämatologikum, Marchioninistrasse 15, D-81377 München, Germany5
Author for correspondence: Michael Hallek at Genzentrum, Ludwig-Maximilians-Universität München. Fax +49 89 7095 6039. e-mail mhallek{at}med3.med.uni-muenchen.de
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Abstract |
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Recently, a conserved phospholipase A2 (PLA2) motif, resembling the catalytic motif of secreted PLA2 (sPLA2), was identified by sequence alignment in VP1up of most parvoviruses, including AAV-2 (Zádori et al., 2001 ). Mutations of critical amino acid residues in the putative parvoviral PLA2 (pvPLA2) of porcine parvovirus (PPV) resulted in strongly reduced PLA2 activity and virus infectivity (Zádori et al., 2001
). Phospholipases are enzymes that hydrolyse phospholipids to generate free fatty acids and lysophospholipids (reviewed by Murakami et al., 1997
; Balsinde et al., 1999
). They are classified according to the bond cleaved in a phospholipid. Thus, PLA2 hydrolyses specifically the 2-acyl ester (sn-2) bond of phospholipid substrates to generate lysophospholipids and free fatty acids. PLA2s are found in prokaryotes, protists, animals and plants and vary greatly in both size and structure. They are divided into three main types based on their biological properties: sPLA2, cytosolic Ca2+-dependent PLA2 and intracellular Ca2+-independent PLA2, resulting in many diverse functions (Dennis, 1997
; Dessen, 2000
).
In order to characterize the function of VP1up in the AAV-2 life cycle, we initially constructed AAV-2 deletion and insertion mutants maintaining the approximate length of VP1. Later, a double point mutation in the proposed PLA2 catalytic centre was added to our analysis (Fig. 1AC). AAV-2 mutant constructs were generated using plasmid pUC-AV2 (Girod et al., 1999
) containing the full-length AAV-2 genome as the template. Mutant
XX, constructed for comparison (Hermonat et al., 1984
; Tratschin et al., 1984
), and mutant
BH were generated by removing the XhoIXhoI (186 bp) and the BsrBIHincII (87 bp) fragments of the cap gene, respectively. The remaining mutants were generated using the ExSite PCR-based Site-Directed Mutagenesis kit (Stratagene), as described previously (Girod et al., 1999
). Mutant B+L was constructed by the insertion of the L14-encoding sequence previously used for successful AAV-2 retargeting (Girod et al., 1999
) at the BsrBI restriction site of VP1up using primers K-
BH+L14 (5' CTCTGCGGGCTTTGGTGGTGGTGGGCCAGGTTT 3') and L-B+L14 (5' CAAGCCGGCACTTTTGCCCTCCGCGGTGATAATCCACAAGGACGGCAT AAGGACGACA-GCAGGGGTCTT 3'). Mutant
BH+L contains a concomitant deletion of the BsrBIHincII fragment and was constructed using primers K-
BH+L14 and L-
BH+L14 (5' CAAGCCGGCACTTTTGCCCTCCGCGG TGATAATCCACAAGGAAACGAGGCAGACGCCGCGGCCCTC 3'). Mutant 76HD/AN was generated by mutating two key residues, 76HD, of the catalytic centre in the putative pvPLA2 to 76AN using primers K-76HD/AN (5' GCGGCCCTCGAGGCCAACAAAGCCTACGACCGG 3') and L-76HD/AN (5' CCGGTCGTAGGCTTTGTTGGCCTCGAGGGCCGC 3').
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To test whether the reduction in infectivity was due to defects in the putative pvPLA2, the enzymatic activity of each mutant was analysed as described previously (Zádori et al., 2001 ). Briefly, VP1up from wt and mutant AAV-2 genomes were amplified using primers AAV2-PLA2-a (5' GCGGATCCATGGCTGCCGATGGTTATCTTC 3') and AAV2-PLA2-b (5' GCTCTAGACGTATTAGTTCCCAGACCAG 3') and the PCR products were cloned into the pBADTBX bacterial expression vector. VP1up proteins, expressed as thioredoxin fusion proteins, were incubated with phosphatidylcholine for 10 min to measure PLA2 activity in the mixed micelles assay (Manjunath et al., 1994
; Zádori et al., 2001
). The relative specific PLA2 activity (Fig. 1D
) was calculated after quantification of radiolabelled products separated by thin layer chromatography (Fig. 1E
). The enzymatic activities of mutants
XX,
BH,
BH+L and 76HD/AN were strongly reduced in comparison to wt but only slightly reduced in the case of mutant B+L. Sequence alignment of putative pvPLA2 and established sPLA2 domains, as presented by Zádori et al. (2001)
, was used to visualize the position of each mutation in relation to the predicted secondary structures within the putative pvPLA2 domain (Fig. 1D
). This illustrated that a mutation in the catalytic centre (76HD/AN) or any deletion affecting the calcium-binding loop (CaBL) (
XX,
BH and
BH+L) of the pvPLA2 domain strongly decreased enzyme activity. The slight reduction in enzymatic activity observed for mutant B+L suggested the region upstream of CaBL is not critical for pvPLA2 function.
In order to determine the role of the putative pvPLA2 during the AAV-2 life cycle, early steps of infection, namely binding and entry, were studied. Equal amounts (as judged by A20 ELISA) of purified wt AAV-2, XX (deletion of the CaBL) and 76HD/AN (mutation in the catalytic centre) mutant particles were fluorescently labelled with Cy3, as described by the manufacturer (Amersham), and incubated on HeLa cells (2000 particles per cell) for 30 min on ice. Cells were washed with PBS to remove unbound virus and fixed with 1% paraformaldehyde immediately (0 min) or after 2 or 4 h at 37 °C. Analysis of the distribution of fluorescently labelled particles by confocal microscopy showed that mutant and wt particles bound equally well to cells at 0 min (Fig. 2AC
). In addition, no significant difference in the perinuclear accumulation of the virus particles at 2 and 4 h (Fig. 2AC
; data not shown) was observed between the VP1 mutants and wt AAV-2. Similar results were obtained using A431 cells (data not shown). Taken together, these results indicated neither virus binding to cells nor virus entry and trafficking through the cytoplasm up to the point of accumulation in the nuclear periphery were affected in these mutants.
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AAV-2 is gaining increased importance as a vector for gene therapy. To continuously improve the vector design, i.e. modifying the immunoreactivity of AAV-2 capsids or targeting to specific cell types, a detailed understanding of functional domains of the AAV-2 capsid proteins is important. In this report, we focused on the unique region of VP1, which contains a PLA2 motif and a corresponding PLA2 activity that is required for the full infectivity of the virus. Analysis of individual steps in the life cycle of different VP1up mutants and wt AAV-2 leads to the following conclusions: (i) mutations in VP1up did not affect DNA replication or packaging but lead to a strong reduction in infectivity (Table 1); (ii) this reduction in virus infectivity correlated with a loss in pvPLA2 activity (Fig. 1
); (iii) binding to and entry into cells was unaffected in VP1up mutants (Fig. 2AC
); (iv) however, these mutations showed drastically reduced and delayed Rep expression (Fig. 2D
). Taken together, our results suggest the pvPLA2 activity is required for a step in the virus life cycle following perinuclear accumulation of virions but prior to the onset of early gene expression. Similar studies were performed with PPV where mutations in the catalytic centre of pvPLA2 had no effect on virus binding, entry or trafficking to the perinuclear late endosomal/lysosomal compartment, while mutants failed to initiate viral DNA replication in the nucleus (Zádori et al., 2001
). Both studies suggest that the pvPLA2 activity may be required for endosome exit and viral genome transfer into the nucleus but neither the exact step nor the exact function of the enzymatic activity (i.e. signalling to the nucleus for stimulating early gene expression) during the virus life cycle are known. Furthermore, full pvPLA2 activity might require an activation step. For PPV, isolated particles exhibit pvPLA2 activity only at very high concentrations and a level of enzymatic activity in virions similar to bacterially expressed VP1up is only obtained after alkali denaturation and renaturation of particles (Zádori et al., 2001
). This points to an activation of the pvPLA2 activity in the endosomal compartment where a partial disintegration of the AAV capsid is likely to occur.
In summary, analysis of AAV-2 capsid mutants in VP1up confirmed the presence of a viral PLA2 activity and underlined the importance of this enzymatic activity in the virus life cycle, namely the timely onset and correct amount of early gene expression. The acronym lip used by Hermonat et al. (1984) to describe mutants characterized by a low infectious particles phenotype may now also be used to describe the phospholipase activity contained in VP1up. Further characterization will be needed to determine the molecular events leading to the activation of pvPLA2 as well as enzymatic targets. However, as this study showed, retaining a functional pvPLA2 domain in cis in any future modification of the capsid, i.e. for vector targeting, is crucial for virus infectivity. Moreover, an increase in the viral PLA activity might improve virus infectivity and thereby the efficiency of AAV-2 as a vector for gene therapy. We are currently assessing this possibility by mutating pvPLA2 and replacing it with other PLA motifs.
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Acknowledgments |
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Footnotes |
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b Present address: Department of Pathology, Washington University School of Medicine, Box 8118, 660 South Euclid Avenue, St Louis, MO 63110, USA.
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References |
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Received 18 September 2001;
accepted 21 December 2001.