Identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein

Abstract

Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2(144-184)). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase-AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2(144-184) has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly.

Importance: Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolar-localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.

FIG 1

Amino acid sequences of the wild type (wt) and mutants (mt) of AAP2 near the C terminus and their roles as an NLS and/or NoLS. The sequence of amino acid positions from 144 to 184 in the wild-type AAP2 is shown at the top, followed by the sequences of a total of 30 AAP2 mutants with arginine/lysine-to-alanine substitutions (mt1 to mt27) or deletions (mt28 to mt30). The 5 BR clusters are indicated by lines above the wild-type sequence. The red and light-blue letters show basic amino acids and alanine mutations, respectively. The blue underlines indicate deletions. The dots in the sequences indicate residues that are the same as those of the wild type. The presence of a fully functional NLS (N) and/or NoLS (No), determined by immunofluorescence microscopy (Fig. 3), is indicated on the right. A plus in the N column indicates that the protein is observed exclusively in the nucleus regardless of its subnuclear localization. A plus in the No column indicates that the protein is strongly associated with the nucleolus. The mutants that showed a nucleolar-exclusion pattern are indicated by Ex.

FIG 2

Intracellular localization of GFP or bacterial β-galactosidase fused with the AAP2^144–184 region at the C or N terminus, respectively. HeLa or HEK 293 cells were transiently transfected with plasmid pCMV-GFP-AAP2^144–184 (A), pCMV-GFP (B), pAAV-CMV-lacZ-AAP2^144–184 (C), or pAAV-CMV-lacZ (D), using PEI. pCMV-GFP-AAP2^144–184 and pAAV-CMV-lacZ-AAP2^144–184 are plasmids expressing the GFP-AAP2^144–184 fusion protein and the β-galactosidase–AAP2^144–184 fusion protein, respectively, under the control of the CMV promoter. For HeLa cells, the signals were detected by immunofluorescence microscopy using corresponding antibodies, except for GFP, for which direct fluorescence was imaged. For HEK 293 cells, X-Gal staining was performed. Scale bars, 20 μm.

FIG 3

Intracellular localization of various AAP2 mutants with arginine/lysine-to-alanine substitutions or deletions within the BR clusters, AAP2BR1 to AAP2BR5. HeLa cells were transiently transfected with a plasmid expressing the wild-type or a mutant AAP2 with an N-terminal FLAG tag. The cells were fixed 48 h posttransfection, immunostained with anti-FLAG antibody (green) and anti-nucleostemin antibody (red), and counterstained with DAPI (blue), before being imaged on a Zeiss LSM 710 confocal microscope with a 100× objective. Representative cell images are shown for the wild type and each mutant. (A) Wild-type AAP. (B) AAP2mt10, showing a staining pattern representing all the AAP2BR1 mutants, AAP2mt1 to -mt10 (the data for AAP2mt1 to -mt9 are not shown). (C to V) AAP2mt11 to -mt30. Formation of multiple nuclear bodies containing both AAP2 and nucleostemin was evident in many cells expressing the wild-type AAP2 (A) and the AAP2 mutants showing an intracellular localization pattern similar to that of the wild type (B, C, F, G, T, and U). The identity and the role of these speckled structures have yet to be determined. The 5-digit numbers in the bottom right corner of each FLAG-AAP2 panel indicate mutations introduced in each AAV2BR in each mutant (1, wild type; 0, alanine mutation; -, deletion). For example, 01110 (mt12) indicates BR1⁻/BR2⁺/BR3⁺/BR4⁺/BR5⁻. Scale bar, 20 μm.

FIG 4

Immunofluorescence microscopic analysis of a FLAG-tagged AAP2 fused with GFP in HeLa cells with or without proteinase treatment. (A to C) HeLa cells were transiently transfected with pCMV-FLAG-cmAAP2-GFP expressing the wild-type full-length AAP2 fused with a FLAG tag and GFP. Intracellular localization of the fusion protein was analyzed with or without trypsin treatment by anti-FLAG antibody immunostaining (A), direct detection of GFP fluorescence (B), and anti-GFP antibody immunostaining (C) under a fluorescence microscope. (D) Merged images. Scale bar, 20 μm.

FIG 5

Microscopic assessment of the abilities of the AAP2BRs to target GFP to the nucleus and the nucleolus. The method used was the same as that for Fig. 2. (A to F) GFP-AAP2^144–184mt13 to -mt26 are GFPs fused with the 41-amino-acid-long AAP^144–184 region containing their corresponding AAP2BR mutations. (G and H) GFP-AAP2^144-150 and GFP-AAP2^144-156 are GFPs fused with AAP2BR1 only (7 amino acids) and AAP2BR1-AAP2BR2 only (13 amino acids), respectively. Scale bar, 20 μm.

FIG 6

AAV2 VP3 capsid production titers and wild-type and mutant AAP2 protein expression levels in HEK 293 cells. HEK 293 cells seeded on 6-cm dishes were transfected with pAAV2-RepVP3, pCMV-FLAG-cmAAP2 expressing either the full-length wild-type AAP2 or a full-length AAP2 mutant with an AAP2BR mutation(s), and pHelper, using PEI (12). The cells were harvested at 48 h posttransfection and made into a 100-μl crude cell lysate in HEPES-buffered saline after three cycles of freezing and thawing. The AAV2 particle concentration in each crude lysate was quantified by an A20 antibody-based ELISA. (A) Viral titers relative to the titer obtained with the wild-type AAP2 (black bars). A relative titer of 1.0 corresponds to 7.2 × 10¹² particles/ml. The thick horizontal black line between the graph and the x axis labels indicates the wild type and AAP2 mutants that showed exclusive nuclear localization with enhanced nucleolar association. The thick horizontal gray line indicates the mutants showing nucleolar exclusion. The data were collected from biologically triplicate experiments. *, P < 0.05 (two-tailed Welch's t test) compared to the wild-type values. In a separate experiment, HEK 293 cells seeded on 6-cm dishes were transfected with pCMV-FLAG-cmAAP2 (the wild type or mutant) using PEI. The cells were harvested 48 h posttransfection, and mutant AAP2 protein expression levels relative to the level of the wild type were determined by a quantitative Western blot analysis using biologically duplicate samples (gray bars). The error bars represent standard errors of the mean (SEM) (black bars) or the difference between each value and the mean value (gray bars). (B) Correlations between AAP2 protein levels of the wild type and mutants indicated by the thick horizontal black line in panel A and AAV2 VP3 capsid production titers are shown in a scatter plot. All values are relative to the levels of the wild type.

FIG 7

Western blot analysis of the wild-type and mutant AAP2 proteins expressed in HEK 293 cells. Cells seeded on 6-cm dishes were transfected with pCMV-FLAG-cmAAP2 (wild type or mutant) using PEI. The same quantity of crude lysates prepared at 48 h posttransfection were separated on an 8% SDS-PAGE gel and blotted onto a membrane, which was then probed with both anti-FLAG and anti-α-tubulin antibodies. The AAP2 mutant numbers are shown above the lanes.

FIG 8

Intracellular localization of AAV2 VP3 in the presence or absence of wild-type AAP2 or in the presence of the nuclear-targeted, nucleolar-excluded AAP2 mutant, AAP2mt26. HeLa cells were transfected with the following plasmids: pCMV-AAV2VP3 only (A), pCMV-AAV2VP3 and pCMV-FLAG-cmAAP2 (wild type) (B), and pCMV-AAV2VP3 and pCMV-FLAG-cmAAP2mt26 (C). The cells were fixed at 48 h posttransfection and immunostained with anti-FLAG, anti-AAV VP1/VP2/VP3, and anti-nucleostemin antibodies. Scale bar, 20 μm.