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. 2006 Jun;80(11):5199-210.
doi: 10.1128/JVI.02723-05.

Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly

Joshua C Grieger  1 , Stephen Snowdy, Richard J Samulski
Affiliations

Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly

Joshua C Grieger et al. J Virol. 2006 Jun.

Abstract

Adeno-associated virus (AAV) is gaining momentum as a gene therapy vector for human applications. However, there remain impediments to the development of this virus as a vector. One of these is the incomplete understanding of the biology of the virus, including nuclear targeting of the incoming virion during initial infection, as well as assembly of progeny virions from structural components in the nucleus. Toward this end, we have identified four basic regions (BR) on the AAV2 capsid that represent possible nuclear localization sequence (NLS) motifs. Mutagenesis of BR1 ((120)QAKKRVL(126)) and BR2 ((140)PGKKRPV(146)) had minor effects on viral infectivity ( approximately 4- and approximately 10-fold, respectively), whereas BR3 ((166)PARKRLN(172)) and BR4 ((307)RPKRLN(312)) were found to be essential for infectivity and virion assembly, respectively. Mutagenesis of BR3, which is located in Vp1 and Vp2 capsid proteins, does not interfere with viral production or trafficking of intact AAV capsids to the nuclear periphery but does inhibit transfer of encapsidated DNA into the nucleus. Substitution of the canine parvovirus NLS rescued the BR3 mutant to wild-type (wt) levels, supporting the role of an AAV NLS motif. In addition, rAAV2 containing a mutant form of BR3 in Vp1 and a wt BR3 in Vp2 was found to be infectious, suggesting that the function of BR3 is redundant between Vp1 and Vp2 and that Vp2 may play a role in infectivity. Mutagenesis of BR4 was found to inhibit virion assembly in the nucleus of transfected cells. This affect was not completely due to the inefficient nuclear import of capsid subunits based on Western blot analysis. In fact, aberrant capsid foci were observed in the cytoplasm of transfected cells, compared to the wild type, suggesting a defect in early viral assembly or trafficking. Using three-dimensional structural analysis, the lysine- and arginine-to-asparagine change disrupts hydrogen bonding between these basic residues and adjacent beta strand glutamine residues that may prevent assembly of intact virions. Taken together, these data support that the BR4 domain is essential for virion assembly. Each BR was also found to be conserved in serotypes 1 to 11, suggesting that these regions are significant and function similarly in each serotype. This study establishes the importance of two BR motifs on the AAV2 capsid that are essential for infectivity and virion assembly.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic of the identified BRs in the AAV capsid gene. A description of the four mutated BRs is portrayed in the schematic. (B) Western blot of AAV2 produced from each of the BR mutant helper plasmids. At 24 h posttransfection, the lysates were centrifuged through an iodixanol step gradient, followed by heparin column purification. Approximately equal titers of purified virus were added to each lane and detected by the B1 antibody. (C) Western blot of the B1 and A20 immunoprecipitations from nuclear (lanes 2 to 5) and cytoplasmic (lanes 6 and 7) fractions of pACG2-wt (lanes 1, 2, 4, and 6) and pACG2-BR4Δ (lanes 3, 5, and 7) transfected 293 cells 24 h posttransfection. Two exposures are presented (the upper exposure is four times longer) to clearly identify capsid proteins after B1 immunoprecipitation in the nucleus.
FIG. 1.
FIG. 1.
(A) Schematic of the identified BRs in the AAV capsid gene. A description of the four mutated BRs is portrayed in the schematic. (B) Western blot of AAV2 produced from each of the BR mutant helper plasmids. At 24 h posttransfection, the lysates were centrifuged through an iodixanol step gradient, followed by heparin column purification. Approximately equal titers of purified virus were added to each lane and detected by the B1 antibody. (C) Western blot of the B1 and A20 immunoprecipitations from nuclear (lanes 2 to 5) and cytoplasmic (lanes 6 and 7) fractions of pACG2-wt (lanes 1, 2, 4, and 6) and pACG2-BR4Δ (lanes 3, 5, and 7) transfected 293 cells 24 h posttransfection. Two exposures are presented (the upper exposure is four times longer) to clearly identify capsid proteins after B1 immunoprecipitation in the nucleus.
FIG. 1.
FIG. 1.
(A) Schematic of the identified BRs in the AAV capsid gene. A description of the four mutated BRs is portrayed in the schematic. (B) Western blot of AAV2 produced from each of the BR mutant helper plasmids. At 24 h posttransfection, the lysates were centrifuged through an iodixanol step gradient, followed by heparin column purification. Approximately equal titers of purified virus were added to each lane and detected by the B1 antibody. (C) Western blot of the B1 and A20 immunoprecipitations from nuclear (lanes 2 to 5) and cytoplasmic (lanes 6 and 7) fractions of pACG2-wt (lanes 1, 2, 4, and 6) and pACG2-BR4Δ (lanes 3, 5, and 7) transfected 293 cells 24 h posttransfection. Two exposures are presented (the upper exposure is four times longer) to clearly identify capsid proteins after B1 immunoprecipitation in the nucleus.
FIG. 2.
FIG. 2.
Fluorescence microscopy images of AAV2, AAV2-BR3Δ-GFP, and coinfection with AAV2-luciferase for a complementation study. HeLa cells were infected with 3,000 viral genomes/cell of AAV2-GFP (A) or AAV2-BR3Δ-GFP (B) and coinfected with AAV2-BR3Δ-GFP and AAV2-Luc (C). Positive cells express GFP. Images were obtained with a Leitz DMIL fluorescence microscope.
FIG. 3.
FIG. 3.
Confocal microscopy images of the subcellular localization of AAV2 and AAV2-BR3Δ capsid proteins (A) or fluorescent in situ hybridization against the GFP gene after infection of HeLa cells (B). HeLa cells were grown in chambered glass slides and infected with either AAV2-GFP (i) or AAV2-BR3Δ-GFP (ii). Cells were processed 24 h later for immunohistochemistry with the MAb B1 and fluorescence in situ hybridization for the GFP genomes. The samples were then processed by using TSA. (B) Two cells were selected from a population of cells to illustrate the low concentration (left panels) and high concentration (right panels) of GFP genomes in the nucleus. The red staining represents capsid protein (A) and GFP genomes (B).
FIG. 4.
FIG. 4.
Fluorescence microscopy images of AAV2-infected HeLa cells. Substitution of the canine parvovirus NLS for the BR3Δ mutant sequence rescues transduction. HeLa cells were infected with 3,000 viral genomes/cell of AAV2-GFP (A), AAV2-BR3Δ-GFP (B), and AAV2-CPVNLS-GFP (C). Positive cells express GFP. Images were obtained with a Leitz DMIL fluorescence microscope.
FIG. 5.
FIG. 5.
(A) Fluorescence microscopy images of HeLa cells infected with assorted AAV2 virions in the presence of adenovirus at an MOI of 5. HeLa cells were infected with AAV2 (i), Vp3 only (ii), Vp2/Vp3 (iii), Vp1/Vp2/Vp3 (iv), Vp1/Vp3 (v), Vp1BR3Δ/Vp3 (vi), and Vp1BR3Δ/Vp2/Vp3 (vii) GFP viruses at 3,000 viral genomes/cell to determine whether the redundant BR3 sequence on Vp2 is functional. AAV2 was produced from a single helper plasmid supplying all three capsid proteins. The Vp2/Vp3 virus was produced from the Vp2/Vp3 expressing plasmid. The Vp1 only, Vp1BR3Δ only, and Vp3 only expressing plasmids were produced for the present study by mutating the start codons of Vp2/Vp3 and Vp1/Vp2, respectively. These plasmids were then utilized together and in combination with the Vp2/Vp3 plasmid to produce the Vp1/Vp2/Vp3, Vp1BR3Δ/Vp2/Vp3, Vp1/Vp3, and Vp1BR3Δ/Vp3 viruses. (B) A native dot blot assay of wtAAV2 and Vp2/Vp3 virions. Each virus was heated to 60°C for 5 min and then reacted with A20 (intact capsids), B1 (unassembled capsids), A1 (Vp1 N termini), or A69 (Vp1 and Vp2 N termini) under nondenaturing conditions.
FIG. 5.
FIG. 5.
(A) Fluorescence microscopy images of HeLa cells infected with assorted AAV2 virions in the presence of adenovirus at an MOI of 5. HeLa cells were infected with AAV2 (i), Vp3 only (ii), Vp2/Vp3 (iii), Vp1/Vp2/Vp3 (iv), Vp1/Vp3 (v), Vp1BR3Δ/Vp3 (vi), and Vp1BR3Δ/Vp2/Vp3 (vii) GFP viruses at 3,000 viral genomes/cell to determine whether the redundant BR3 sequence on Vp2 is functional. AAV2 was produced from a single helper plasmid supplying all three capsid proteins. The Vp2/Vp3 virus was produced from the Vp2/Vp3 expressing plasmid. The Vp1 only, Vp1BR3Δ only, and Vp3 only expressing plasmids were produced for the present study by mutating the start codons of Vp2/Vp3 and Vp1/Vp2, respectively. These plasmids were then utilized together and in combination with the Vp2/Vp3 plasmid to produce the Vp1/Vp2/Vp3, Vp1BR3Δ/Vp2/Vp3, Vp1/Vp3, and Vp1BR3Δ/Vp3 viruses. (B) A native dot blot assay of wtAAV2 and Vp2/Vp3 virions. Each virus was heated to 60°C for 5 min and then reacted with A20 (intact capsids), B1 (unassembled capsids), A1 (Vp1 N termini), or A69 (Vp1 and Vp2 N termini) under nondenaturing conditions.
FIG. 6.
FIG. 6.
Immunofluorescence and confocal microscopy images of HeLa cells infected with wtAAV2 and transfected with pACG2 and pACG2-BR4Δ. Panels A through F were analyzed by indirect immunofluorescence using a Leitz DMIL fluorescence microscope. Panel G is an overlay image that was analyzed by confocal microscopy using the Leica SP2 aobs confocal microscope. HeLa cells were infected with wtAAV2 and adenovirus (A and B), transfected with pACG2 and adenovirus helper plasmid XX680 (C and D), and transfected with pACG2-BR4Δ (E and F) and adenovirus helper plasmid. Each sample was probed with primary antibodies B1 (anti-capsid subunits) and fibrillarin (nucleolar protein). Capsid proteins are identified by green fluorescence (Alexa-Fluor 488 secondary) (A, C, and E), fibrillarin is identified by red fluorescence (Alexa-Fluor 568) (G), and nuclei are identified by blue fluorescence (DAPI) (B, D, F, and G). A series of horizontal sections (each 0.3 μm) were obtained for panel G. The white arrow points to intranuclear assembly centers identified by the B1 antibody. GFP exposure times were identical for panels C and E, while a shorter exposure time was used for panel B due to increased abundance of capsid protein in the cells.
FIG. 7.
FIG. 7.
(A) Schematic representation of the infectious entry pathway of AAV2 (step 1). The first step in AAV2 infection is binding to its primary receptor and a secondary receptor (step 2). AAV2 enters the cell by endocytosis via clathrin-coated pits and is brought into the cytoplasm in an early endosome (step 3). The early endosome then matures into a late endosome as the pH begins to drop to around 5. A pH-dependent conformational change occurs that is thought to expose the N terminus of Vp1, and possibly BR3, providing the phospholipase activity for endosome escape and region essential for genome import into the nucleus (step 4). At this point in the pathway, AAV either fails to escape the late endosome, where it later becomes degraded by the lysosome or escapes into the cytoplasm perinuclearly, where it becomes ubiquitinated (step 5). The ubiquitinated virions can then be recognized by cytoplasmic proteasomes on their way to the nucleus, where they are degraded, but those that avoid interaction with the proteasomes continue on their path to the nucleus (step 5). (B) Schematic representation of the pathway involved in AAV2 capsid protein synthesis and assembly of wt capsid proteins and those containing the BR4 mutation. AAV DNA is found in the nucleus of transfected cells, and mRNA is produced and translated into the capsid proteins, which shuttle into the nucleus for assembly. The red capsid proteins represent wt capsid sequence, while the blue proteins represent the capsid proteins with the BR4 mutations (depicted in yellow). The wt capsid subunits are capable of intersubunit interactions, leading to the intermediate pentamer/trimer formation, leading to capsid assembly. We propose that the misfolded/destabilized BR4 capsid subunits are incapable of intersubunit interactions and cannot form intact virions.
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