ABSTRACT
The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant (76)HD/AN. The sPLA2 defect in (76)HD/AN, which severely impairs AAV's infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued (76)HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.
Subject(s)
Capsid Proteins/metabolism , Dependovirus/enzymology , Dependovirus/pathogenicity , Endosomes/virology , Phospholipases A2/metabolism , Virion/metabolism , Capsid Proteins/genetics , Dependovirus/classification , Dependovirus/genetics , Endosomes/physiology , HEK293 Cells , HeLa Cells , Humans , Mutation , Phospholipases A2/chemistry , Phospholipases A2/genetics , SerotypingABSTRACT
The actin-nucleating ARP2-ARP3 complex controls cell shape in plants in many different cell types. Its activity is controlled by a multimeric complex containing BRK1 (also known as HSPC300), NAP1, SRA1, ABI and SCAR/WAVE. In this study, we focus on the function of the five putative SCAR homologues in Arabidopsis and we provide biochemical evidence that AtSCAR2 can activate the ARP2-ARP3 complex in vitro. Among the single mutants, mutations in only AtSCAR2 result in a subtle or weak phenotype similar to ARP2, ARP3 and other ;distorted' mutants. Double-mutant analysis revealed a redundancy with AtSCAR4. Systematic application of the yeast two-hybrid system and Bimolecular Fluorescence Complementation (BiFC) revealed a complex protein-interaction network between the ARP2-ARP3 complex and its genetically defined regulators. In addition to protein interactions known in other systems, we identified several new interactions, suggesting that SPIKE1 may be an integral component of the SCAR/WAVE complex and that SCAR proteins in plants might act as direct effectors of ROP GTPases.
