Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins

Abstract

The proteins encoded by the adeno-associated virus type 2 (AAV-2) rep and cap genes obtained during a productive infection of HeLa cells with AAV-2 and adenovirus type 2 were fractionated according to solubility, cellular localization, and sedimentation properties. The majority of Rep and Cap proteins accumulated in the nucleus, where they distributed into a soluble and an insoluble fraction. Analysis of the soluble nuclear fraction of capsid proteins by sucrose density gradients showed that they formed at least three steady-state pools: a monomer pool sedimenting at about 6S, a pool of oligomeric intermediates sedimenting between 10 and 15S, and a broad pool of assembly products with a peak between 60 and 110S, the known sedimentation positions of empty and full capsids. While the soluble nuclear monomer and oligomer pool contained predominantly only two capsid proteins, the 30 to 180S assembly products contained VP1, VP2, and VP3 in a stoichiometry similar to that of purified virions. They probably represent different intermediates in capsid assembly, DNA encapsidation, and capsid maturation. In contrast, the cytoplasmic fraction of capsid proteins showed a pattern of oligomers continuously increasing in size without a defined peak, suggesting that assembly of 60S particles occurs in the nucleus. Soluble nuclear Rep proteins were distributed over the whole sedimentation range, probably as a result of association with AAV DNA. Subfractions of the Rep proteins with defined sedimentation values were obtained in the soluble nuclear and cytoplasmic fractions. We were able to coimmunoprecipitate capsid proteins sedimenting between 60 and 110S with antibodies against Rep proteins, suggesting that they exist in common complexes possibly involved in AAV DNA packaging. Antibodies against the capsid proteins, however, precipitated Rep78 and Rep68 predominantly with a peak around 30S representing a second complex containing Rep and Cap proteins.

High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant

“…The data presented here strongly suggest an involvement of Rep78 and Rep68 in the AAV assembly process. This is in line with the results of Wistuba et al, showing that Rep78 and Rep68 could be coprecipitated with different monoclonal antibodies against capsid proteins from fractionated extracts of HeLa cells productively infected with AAV and Ad2 (32).…”

“…The cells were subsequently infected with Ad2 and induced with dexamethasone (10 Ϫ6 M) to express Rep78. After 32 h, cells were fixed with 2% paraformaldehyde and immunostained for AAV capsid proteins by using monoclonal antibody B1 (32).…”

Enhancement of Adeno-Associated Virus Infection by Mobilizing Capsids into and Out of the Nucleolus

“…However, for several reasons it is unlikely that the virus is subject to this system. During replication, AAV monomers and subunits can be readily detected in cytoplasm and nuclei by antibody B1 (64,65), but in our studies and others, researches have failed to detect significant B1 staining during infection (references 38 and 57 and data not shown). Thus, the supply of free monomers and subunits would likely not be great enough to support reassembly during infection.…”

The packaging capacity of adeno‐associated virus (AAV) and the potential for wild‐type‐plus AAV gene therapy vectors

“…Furthermore, interaction, or lack of interaction, of the inserted foreign DNA with the AAV encoded Rep78 protein may also alter the efficiency of packaging. The AAV encoded Rep78 protein been shown to interact both with the AAV capsid proteins [23]and AAV TR DNA [24], as well as multiple sequences within human chromosomal DNA [19, 25]. Thus, the specific foreign DNA which is inserted into the AAV vector will likely affect packaging, due to differing interactions with the packaging machinery.…”