Adeno-Associated Virus Type 2 Protein Interactions: Formation of Pre-Encapsidation Complexes

Abstract

The nonstructural adeno-associated virus type 2 Rep proteins are known to control viral replication and thus provide the single-stranded DNA genomes required for packaging into preformed capsids. In addition, complexes between Rep proteins and capsids have previously been observed in the course of productive infections. Such complexes have been interpreted as genome-linked Rep molecules associated with the capsid upon successful DNA encapsidation. Here we demonstrate via coimmunoprecipitation, cosedimentation, and yeast two-hybrid analyses that the Rep-VP association also occurs in the absence of packageable genomes, suggesting that such complexes could be involved in the preparation of empty capsids for subsequent encapsidation steps. The Rep domain responsible for the observed Rep-VP interactions is situated within amino acids 322 to 482. In the presence of all Rep proteins, Rep52 and, to a lesser extent, Rep78 are most abundantly recovered with capsids, whereas Rep68 and Rep40 vary in association depending on their expression levels. Rep78 and Rep52 are bound to capsids to roughly the same extent as the minor capsid protein VP2. Complexes of Rep78 and Rep52 with capsids differ in their respective detergent stabilities, indicating that they result from different types of interactions. Rep-VP interaction studies suggest that Rep proteins become stably associated with the capsid during the assembly process. Rep-capsid complexes can reach even higher complexity through additional Rep-Rep interactions, which are particularly detergent labile. Coimmunoprecipitation and yeast two-hybrid data demonstrate the interaction of Rep78 with Rep68, of Rep68 with Rep52, and weak interactions of Rep40 with Rep52 and Rep78. We propose that the large complexes arising from these interactions represent intermediates in the DNA packaging pathway.

Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication

“…These findings indicate that the mutant Rep-D371Y proteins may be deficient for packaging of the viral ssDNA genome into pre-assembled virus capsids which results in rAAV2-D371Y stocks that are not compatible with efficient virus replication. This could be due to two reasons: first, the mutant Rep proteins may not be capable of binding to pre-assembled capsids, a process necessary to form the ssDNA-capsid complex [ 32 , 67 ], or second, the mutant Rep proteins are not capable of binding to ssDNA. To test the latter possibility, we performed gel filtration chromatography to assess the capability of Rep-D371Y to form DNA-protein complexes with either ssDNA or dsDNA.…”

“…Notably, we were not able to successfully produce recombinant AAV2 progeny virus stocks harboring the mutant rep-D371Y gene. Hence, we hypothesized that the mutant Rep-D371Y proteins may be deficient for packaging of the viral ssDNA genome into pre-assembled virus capsids, a process involving both, the binding of Rep to pre-assembled capsids and the formation of Rep-ssDNA complexes at the same time [ 32 , 67 ]. A variety of mutations in the Rep SF3 helicase have been tested in the past for their ability to form the Rep-capsid complex.…”

Adeno-associated virus interactions with B23/Nucleophosmin: Identification of sub-nucleolar virion regions

“…From our chemical cross-linking experiments, the relatively short length of the DSP crosslinker (12Ǻ) suggests that NPM exists in close proximity to Cap or Rep proteins. Since Rep proteins associate with the virion or viral DNA by noncovalent and covalent interactions respectively (Dubielzig et al, 1999;Prasad and Trempe, 1995), it is not possible to verify that NPM directly interacts with both viral proteins in the coinfected cell. That AAV Cap proteins are co-immunoprecipitated with anti-NPM from non-crosslinked extracts suggests there is a stronger affinity between these proteins than between NPM and Rep. Our inability to coimmunoprecipitate NPM with Cap-specific antibodies suggests that NPM interactions may block the structural epitopes required for immunoprecipitations.…”

Adeno-Associated Virus-Mediated MicroRNA Delivery and Therapeutics

“…The ITRs form two T-shaped structures at each end of the genome and contribute to AAV genome replication, packaging, integration and rescue from host genome. 35,36 The Rep gene is involved in viral genome replication and encapsidation during AAV replication 37,38 in the presence of a helper virus, such as adenovirus 39 and herpes simplex virus. 40 The cap ORF provides structural proteins for the viral capsid which determine the tissue tropism and immune biology in viral infection.…”