Positive and negative effects of adenovirus type 5 helper functions on adeno-associated virus type 5 (AAV5) protein accumulation govern AAV5 virus production

Abstract

Full replication of adeno-associated virus type 5 (AAV5) is sustained by adenovirus type 5 (Ad5) helper functions E1a, E1b, E2a, E4Orf6, and virus-associated (VA) RNA; however, their combined net enhancement of AAV5 replication was comprised of both positive and negative individual effects. Although Ad5 E4Orf6 was required for AAV5 genomic DNA replication, it also functioned together with E1b to degrade de novo-expressed, preassembled AAV5 capsid proteins and Rep52 in a proteosome-dependent manner. VA RNA enhanced accumulation of AAV5 protein, overcoming the degradative effects of E4Orf6, and was thus required to restore adequate amounts of AAV5 proteins necessary to achieve efficient virus production.

FIG. 1.

(A) Replication of AAV 5 viral DNA in 293 cells in the presence of various combinations of Ad5 gene products. 293 cells were infected with AAV5 at an MOI of 10 and transfected with equal molar quantities of CMV E2a, CMV E4Orf6, and a VAI RNA-expressing clone in the combinations indicated. pBS-SK was used to compensate for the absence of any individual plasmid. Monomer length (mRF), dimer length (dRF), and single-strand progeny (ssDNA) DNA replication forms, either cell associated (C) or released into the media (M), are indicated. Markers (m; lane 1) are a mixture of untreated and denatured 4.6-kb Acc65I fragments from pAV5, which corresponds to the full-length AAV5 genome. (B) Quantification of cell-associated AAV5 generated in 293 cells in the presence of various combinations of Ad5 gene products: Slot blot analysis to detect cell-associated virus production, performed as described in the Materials and Methods, during the same experiment in which replication was assayed in panel A, is shown. The various combinations of Ad5 helper functions are shown on the left, and dilutions of samples are shown to the right. (C and D) Analysis of AAV5 Rep protein (C) and capsid protein (D) production in 293 cells in the presence of various combinations of Ad5 gene products: Immunoblot analysis to determine AAV5 protein expression during the same experiment in which viral replication (A) and virus production (B) was determined as described above, using either antibody against the viral Rep proteins (C) or the viral capsid proteins (D), is shown. The locations of the AAV5 proteins, as well as endogenously expressed 14-3-3 and actin proteins serving as loading controls, are shown.

FIG. 2.

(A) Accumulation of AAV5 capsid proteins in 293 cells in the presence of various combinations of Ad5 gene products. Immunoblot analysis of protein accumulation in 293 cells following transfection of the P41Cap construct together with various combinations of Ad5 gene products, as indicated, is shown. The complete set of samples was run on five separate gels and after transfer probed with antibodies to either AAV5 capsid proteins and actin (panel 1), E1a and tubulin (panel 2), E1b and actin (panel 3), E2a and actin (panel 4), or E4Orf6 and tubulin (panel 5). (B) Steady-state levels of total and cytoplasmic AAV5 capsid gene RNA generated in 293 cells in the presence of various combinations of Ad5 gene products, from the same experiment shown in panel A. Shown are the results from quantitative RNase protection assays, using the AAV5 RP probe as described in the Materials and Methods, of either total (T) or cytoplasmic (C) RNA generated in 293 cells following transfection of the P41Cap construct together with various combinations of Ad5 gene products, as shown from the same experiment in panel A. Bands protected by P41-generated unspliced and spliced RNAs are indicated on the left. RNase protections using a probe to cellular β-actin served as a loading control. A diagram of the P41Cap construct used for the experiments shown in panels A and B is shown at the bottom, with genetic landmarks and the location of the RP RNase protection probe indicated.

FIG. 3.

Accumulation of AAV5 Rep52 in 293 cells in the presence of various combinations of Ad5 gene products. Immunoblot analysis showing protein accumulation in 293 cells following transfection of the P19/P41-Rep52/Cap construct together with the various combinations of Ad5 gene products as indicated. After transfer, blots were probed together with antibody directed against the AAV Rep proteins and with antibody to cellular 14-3-3 proteins, and these proteins are indicated to the left. A diagram of the P19/P41-Rep52/Cap construct is shown at the bottom with genetic landmarks indicated. The band marked with an asterisk is a stable product generated from the AAV5 Rep open reading frame, which was previously observed (17).

FIG. 4.

(A) Inhibition of E4Orf6 activity by proteasome inhibitor MG132. 293 cells were transfected with P41Cap (lanes 1 and 2) and P41Cap with E4Orf6 (lanes 3 and 4). At 40 h posttransfection, 10 μM MG132 (lanes 2 and 4) or DMSO (lanes 1 and 3) was added to each of the samples as noted. The cell lysates were collected at 48 h posttransfection and then probed with antibody to AAV5 capsids. (B). Inhibition of E4Orf6 activity by proteasome inhibitor lactacystin. 293 cells were transfected with P41Cap (lanes 1 and 2) and P41 with E4Orf6 (lanes 3 and 4). At 40 h posttransfection, 5 μM lactacystin (lanes 2 and 4) or DMSO (lanes 1 and 3) was added to each of the samples as noted. The cell lysates were collected at 48 h posttransfection and then probed with antibody to AAV5 capsids. (C) E4Orf6 degrades AAV5 capsid proteins in the presence of cycloheximide. 293 cells were transfected with P41Cap and either pBS-SK or with CMV E4Orf6. At 30 h posttransfection, cycloheximide (100 μg/ml) or DMSO was added to each of the samples as noted above. Lysates were then collected at 30 h, 33 h, 36 h, or 39 h posttransfection. The samples then underwent 10% SDS-polyacrylamide gel electrophoresis and were immunoblotted with antibody to AAV5 capsid proteins.

FIG. 5.

Accumulation of AAV5 capsid proteins in HeLa cells in the presence of various combinations of Ad5 gene products. The results of immunoblot analysis showing protein accumulation in HeLa cells following transfection of the CMV P41Cap construct together with the various combinations of Ad5 gene products are shown. The complete set of samples was run on four separate gels and after transfer probed with antibodies to either AAV5 capsid proteins and actin (panel 1), E1a and tubulin (panel 2), E1b and actin (panel 3), or E4Orf6 and tubulin (panel 4). The locations of individual proteins are indicated.

FIG. 6.

Overexpression of AAV5 Rep52 and capsid proteins allows full replication of AAV5 in the absence of VA RNA. (A) 293 cells were infected with AAV5 at an MOI of 10 and transfected with equal molar quantities of CMV E2a, CMV E4Orf6, a VAI RNA-expressing clone, AAV5 Rep52-expressing CMV P19 Rep, and AAV5 capsid protein expressing CMV P41Cap, in the combinations indicated. pBS-SK was used to compensate for the absence of any individual plasmid. Monomer length (mRF), dimer length (dRF), and single-strand progeny (ssDNA) DNA replication forms are indicated. Markers (m; lane 9) are a mixture of untreated and denatured 4.6-kb Acc65I fragments from pAV5, which corresponds to the full-length AAV5 genome. (B) Accumulation of AAV5 capsid proteins in 293 cells in the presence of various combinations of Ad5 gene products, AAV5 Rep 52, and AAV5 capsid proteins. Shown are the results of immunoblot analysis demonstrating protein accumulation from the same experiment in which AAV5 replication was determined in the presence of various combinations of Ad5 and AAV5 gene products, as shown in panel A. The complete set of samples was run on two separate gels and after transfer probed with antibodies to either the viral Rep proteins and cellular 14-3-3 or the viral capsid proteins and cellular actin. The locations of the AAV5 proteins, as well as endogenously expressed 14-3-3 and actin proteins serving as loading controls, are shown. The band marked with an asterisk is a stable product generated from the AAV5 Rep open reading frame as previously observed (17).