Adeno-associated virus 9 (AAV9) viral proteins VP1, VP2, and membrane-associated accessory protein (MAAP) differentially influence in vivo transgene expression

ABSTRACT

Adeno-associated virus (AAV) is a Dependoparvovirus with a ssDNA ~4.7 kb genome in a ~25 nm icosahedral capsid structure. AAV genomes encode nine known functional proteins from two open reading frames between two inverted terminal repeats (ITRs). In recombinant AAV vectors for gene therapy use, the AAV genome is replaced with a transgene of interest flanked by ITRs and subsequently packaged within an AAV capsid made up of three viral structural proteins (VP1, VP2, and VP3) in an approximate 1:1:10 ratio, respectively. The AAV capsid, particularly VP3, has traditionally been ascribed to capsid-cellular receptor binding. However, AAV9 VP1/VP2 exhibits a capsid-promoter interaction that can alter neuronal cellular tropism in the rat and non-human primate central nervous system. This capsid-promoter interaction is altered by AAV9EU (AAV9 with six glutamates inserted at aa139) which exhibits a significant reduction in nuclear transgene DNA, a decrease in neuronal transduction, and a reduction in vivo relative transgene mRNA levels. AAV9EU has six amino acid insertions in VP1, VP2, and MAAP (membrane-associated accessory protein), but no combination of VP with MAAP recapitulated the AAV9EU in vivo phenotype. Surprisingly, AAV9 produced in the absence of MAAP9 exhibits an increase in relative transgene levels. While co-infusing two AAV9 vectors, differing only in transgene and MAAP9 presence during production, exhibit a significantly increased in vivo transgene fluorescence intensity by fivefold of both transgenes. Together, an MAAP9-related activity acts both in cis and in trans to increase AAV9 transgene mRNA levels and AAV9 transgene protein levels in vivo.

IMPORTANCE

Recombinant adeno-associated viruses (AAVs) are used extensively in clinical gene therapy for treating a range of tissues and pathologies in humans. In particular, AAV9 occupies a prominent position in central nervous system (CNS) gene therapy given its central role in ongoing clinical trials and an FDA-approved therapeutic. Despite its widespread use, recent studies have identified unique roles for the AAV capsid in in vivo transgene expression; for example, interior-facing capsid residues of AAV VP1 and VP2 modulate cellular transgene expression in vivo. The following experiments identified that the AAV9 MAAP protein exerts a significant influence on in vivo transgene expression. This finding could further explain how AAV can remain latent after infection in vivo. Together, these studies provide novel functional insights that highlight the importance of further understanding basic AAV biology.

INTRODUCTION

Adeno-associated virus (AAV) is a ~4.7 kb single-stranded DNA Dependoparvovirus. AAV2 was first discovered (1) and since then, several serotypes of AAV have been described and created (2). Recombinant AAV is the workhorse in the gene therapy field and has five FDA approvals. In particular, AAV9 occupies a prominent position due to its usefulness in the central nervous system (CNS) evidenced by multiple ongoing clinical trials and an FDA-approved therapy (3). Even with this prominence, elements of recombinant and wild-type AAV biology remain poorly characterized. The current understanding of AAV biology comes from in vitro studies of AAV2 performed 50 years ago. The AAV genome contains two genes, rep and cap, residing between two inverted terminal repeats (ITRs) that act as packaging beacons (1). The rep gene encodes four proteins required for replication of AAV (1). The cap gene encodes three structural capsid proteins (VP1, VP2, and VP3) present in an approximate ratio of 1:1:10 (VP1:VP2:VP3) in a fully assembled capsid (4, 5). VP3 is the predominant subunit on the exterior of the capsid and influences cellular/tissue tropism due to receptor binding accessibility. Also within the cap gene, novel proteins AAP (assembly-activating protein) and MAAP (membrane-associated accessory protein) were recently discovered. AAP is an accessory protein (6) that helps assemble some AAV capsids (7, 8). MAAP is a membrane-associated ~13 kDa protein (9) that enables AAV to associate with cellular secreted lipid membraned vesicles (exosomes/extracellular vesicles) during vector production and consequentially vector trafficking into the production cell media (10–12). MAAP is translated from an alternate reading frame (+2 with VPs + 1) on the VP2/VP3/AAP transcript (13). While AAV2 elements such as ITRs and rep are used in other AAV vector serotypes, differences among AAV serotypes beyond cellular/tissue tropism have not been dominant research questions.

Building on the findings in vitro AAV biology data, in vivo studies continue to shed light on AAV biology within a cell. Two host factors were identified that aid in AAV cellular transduction, AAVR (14) and GPR108 (15). However, the bulk of current AAV research still focuses on manipulating the capsid protein sequence, mainly VP3, to generate novel tissue/cell specificity for gene therapy. While VP3 is thought to be a major determinant in AAV cellular tropism, multiple point mutations in AAV2 VP3, in the twofold region of the capsid, were shown to exhibit nuclear DNA uncoating but did not exhibit transgene transcription suggesting a role for VP3 (capsid) beyond cellular receptor binding (16, 17).

VP1/VP2 unique regions are internalized within the capsid (18–20) but become externalized to facilitate AAV transduction. VP1 includes a phospholipase A2 (PLA2) domain required for endosomal escape during AAV transduction (21, 22). While both VP1 and VP2 contain basic regions that serve as nuclear localization signals during the life cycle of AAV (23, 24), specifically alterations in basic region 3 (BR3) contained in VP1 and VP2 were shown to reduce AAV transduction (24, 25). Further study indicated that BR3 in VP2 can compensate for a mutant BR3 in VP1, suggesting that VP2 has a role in transduction (24) and is not necessarily dispensable (26). Also, studies have shown that VP1 residues are important for cellular transduction (25). Recent in vitro studies found that serine residues in a conserved region of AAV2 VP1/2 that when mutated to alanines cause a decrease in mRNA levels, suggesting a role for VP1/2 in regulating transcription (27). We recently discovered that the VP1/VP2 region can interact with the transgene promoter (CBA, CAG, CBh, and JetI) leading to significant alternation in the reporter expression in both rat (28) and non-human primate (29) CNS. The following studies further elaborate on the AAV9 VP1/VP2 capsid-promoter interaction within the rat CNS by assessing the cellular tropism of recombinant AAV9 viruses with a six glutamate insertion (EU) in either VP1 or VP2 alone and combination with MAAP9 or MAAP9EU (MAAP9 with a RRKRRK insert) (Table 1). We further demonstrate that MAAP9 influences in vivo transgene expression in the rat CNS.

TABLE 1.

AAV capsids and their AAV9 and AAV9EU components that are used in this study^a

AAV capsid	AAV9 components	AAV9EU components
AAV9	VP1, VP2, VP3, MAAP, AAP	–
AAV9EU	VP3, AAP	VP1, VP2, MAAP
AAVE99	VP2, VP3, AAP	VP1, MAAP
AAV9E9	VP1, VP3, AAP	VP2, MAAP
AAVEE9 (AAV9EU produced with two rep/cap plasmids)	VP3, AAP	VP1, VP2, MAAP

^a The AAV capsid name is indicated with the serotype of each known cap gene encoded component. VP1, VP2, and VP3 are the AAV capsid structural proteins, AAP is the assembly-activating protein, and MAAP is the membrane-associated accessory protein. AAV vectors were produced with either one rep/cap plasmid or two as further diagrammed in Fig. S2.

RESULTS

AAV9EU has reduced relative transgene mRNA levels and nuclear transgene genomes

Our previously published studies showed that AAV9EU [AAV9 with six glutamates inserted at aa139 (VP1 numbering)] exhibits reduced neuronal transduction compared to AAV9 with an overall oligodendrocyte transduction pattern in the rat CNS (28). To further define the mechanism underlying AAV9EU’s reduced neuronal transduction within the rat striatum (28), relative transgene mRNA levels of AAV9EU were quantified and compared to AAV9 within the same animal. Vectors were produced with a transgene encoding green fluorescent protein (GFP) driven by a strong constitutive promoter, chicken β-actin (CBA), and were produced to a high titer with appropriate capsid subunit ratios (Fig. S1A; Table S1). AAV9-CBA-GFP and AAV9EU-CBA-GFP were infused bilaterally into the rat striatum (1.0E9vg in equal volumes). The rats were sacrificed 2 weeks post-infusion, the striata harvested, RNA isolated, cDNA synthesized, and the relative transgene mRNA levels quantified. Relative GFP mRNA levels were determined by comparison of endogenous GAPDH mRNA levels in which both were measured by qPCR Taqman assays using the ΔΔct method. The fold change of GFP/GAPDH mRNA of AAV9EU was determined by comparing it to the AAV9 ΔΔct value from the same animal. AAV9EU exhibits a decrease (more than a 2,000-fold reduction) in relative GFP mRNA levels compared to AAV9 (Fig. 1A). It should be noted that assessing intra-animal comparisons to control for animal variability limited the statistical tests and reduced power for determining statistical significance. The mRNA data agree with the microscopy data in this manuscript and previously reported data showing a significant reduction in AAV9EU neuronal transduction compared to AAV9 (Fig. 2) (28).

Fig 1.

AAV9EU has reduced relative GFP mRNA levels and nuclear GFP copies compared to AAV9. AAV9-CBA-GFP and AAV9EU-CBA-GFP were infused bilaterally into the rat striatum in an equal amount (1.0E9vg) and volume. Animals were sacrificed 2 weeks post-infusion, striata resected, and nuclear DNA and RNA isolated. qPCR was used to determine relative GFP mRNA levels and nuclear transgene copies. (A) The fold change (ΔΔct) in relative GFP mRNA levels was compared to endogenous GAPDH assayed using qPCR Taqman expression assays performed on synthesized cDNA. Comparable amounts of RNA were used as input in cDNA synthesis and qPCR assays. Measurements were made in triplicate, then averaged for each animal and the fold change was determined within each animal. Values are graphed on box and whisker plots where the whiskers are the min and max with all data points indicated. There was no significant difference (ns) determined by the Wilcoxon test. (B) The number of nuclear GFP copies per ng of genomic DNA was measured by Taqman assays for GFP and GAPDH. Equal amounts of nuclear DNA were included in the assay with DNA concentration determined by GAPDH qPCR Taqman assay using a standard curve. Measurements were made in triplicate and then averaged for each animal. Values are graphed on box and whisker plots where the whiskers are the min and max with all data points indicated. Statistical significance (* indicates P < 0.05) was determined using the Mann-Whitney test.

Fig 2.

AAV9E9 and AAVE99 exhibit a similar dominant neuronal cellular tropism as AAV9 in the rat striatum. All vectors were infused in an equal amount (1.0E9vg) and volume into the rat striatum then 2 weeks later the animals were sacrificed, fixed, and the brain sliced (40 µm). Native mCherry and immunohistochemistry for NeuN (green), a neuronal cell marker, was performed followed by confocal fluorescence microscopy. The white dashed boxes indicate the area enlarged in the far column. White arrows indicate some of the cells that exhibit mCherry and NeuN colocalization. Representative maximum projected z-stacks images are shown of AAV9-CBA-mCherry, AAV9EU-CBA-mCherry, AAV9E9-CBA-mCherry, and AAVE99-CBA-mCherry (top to bottom, respectively).

AAV9EU-induced reduction of relative GFP mRNA levels could be explained by a compromised ability to enter the nucleus. AAV9EU is an AAV9 variant with six glutamates (negatively charged) inserted into a heavily basic region of VP1/2 that serves as nuclear localization signals (23, 24). Therefore, AAV9 and AAV9EU nuclear transgene copy numbers were quantified to assess viral nuclear entry. AAV9-CBA-GFP and AAV9EU-CBA-GFP were directly infused into the rat striatum (1.0E9vg in equal volumes). Striata were harvested 2 weeks post-infusion after which nuclei were isolated, nuclear DNA purified, and GFP nuclear copies quantified with input DNA amount normalized to GAPDH using qPCR TaqMan assays. AAV9EU nuclear GFP copies were significantly reduced (P < 0.05) (~10-fold less) in comparison to AAV9, suggesting that AAV9EU GFP transgenes are present within the nucleus, but in a lower amount than AAV9 (Fig. 1B). Together, these data suggest that AAV9EU is deficient at gaining nuclear entry which contributes to reduced transcription (Fig. 1A) and thus reduced neuronal transduction (Fig. 2).

Separate AAV9 VP1 or VP2 EU insertions do not reproduce AAV9EU reduced neuronal transduction

AAV9EU is a variant of AAV9 with six glutamates (EU) inserted at aa139 (VP1 numbering) resulting in inserts into both VP1 and VP2. We sought to determine the individual contribution the EU insertion in VP1 or VP2 has on AAV9EU oligodendrocyte cellular tropism (28). To this end, vectors were produced using two rep/cap plasmids as previously published (26), instead of a single rep/cap plasmid, to express either VP1 (to produce AAVE99) or VP2 (to produce AAV9E9) with the EU insertion (Fig. S2). It is important to note that producing AAV9EU using two rep/cap plasmid formats (AAVEE9) does not alter the in vivo phenotype (Fig. S3). In addition, the comparable VP1 and VP2 knockout mutations in AAV9 resulted in no VP1 or no VP2 expressed (Fig. S4A) and purified vectors lacked the respective VP subunit (Fig. S4B). Together, these data suggest that producing recombinant AAV9EU with two rep/cap plasmids results in a comparable cellular tropism as when AAV9EU is produced with the standard one rep/cap plasmid and that mutating the start codons of VP1 and VP2 effectively prevents their detection during production and incorporation into purified vectors.

The capsids were packaged with CBA-mCherry and produced to a high titer (Table S1). The vectors were infused (1.0E9vg in equal volumes) into the rat striatum and then harvested 2 weeks post-infusion for fluorescence microscopy image analysis. AAVE99 and AAV9E9 exhibited a dominant neuronal cellular tropism with substantial colocalization of mCherry-positive cells and a neuronal nuclear marker, NeuN (Fig. 2). Thus, AAV9, AAV9E9, and AAVE99 (Fig. 2) exhibit similar neuronal expression that proved far more robust than AAV9EU [Fig. 2 and (28)] suggesting that EU insertions into AAV9 VP1 and VP2 individually cannot account for the lack of neuronal transduction of AAV9EU.

AAV9 VP1 or VP2 EU insertions in combination with MAAP9 or MAAP9EU do not recapitulate AAV9EU reduced neuronal transduction

AAV9EU does not support robust transgene expression and transduces mostly oligodendrocytes, not neurons like AAV9, AAVE99, and AAV9E9 [published data (28) and Fig. 2] suggesting additional factor(s) contribute to the AAV9EU phenotype. The EU insertion into the AAV9 cap at aa139 results in an RRKRRK insertion into the C-terminus of MAAP9 resulting in MAAP9EU (Fig. S5). Although MAAP is not known to have a role outside of vector production, we assessed whether MAAP9EU’s presence during vector production alone could reduce AAV9 neuronal tropism.

AAV9/MAAP9EU-CBA-GFP was produced by mutating the MAAP9 start site (CTG to CCG) while preserving VP1/VP2 protein sequence in the rep/cap plasmid as done previously (9–11) and recapitulated in our hands (Fig. S6). A MAAP9EU rep/cap plasmid that expressed only MAAP9EU and no VPs was generated by mutating the VPs start sites. The two rep/cap plasmids were combined in a 1:2 molar ratio (MAAP: VPs) to provide a higher amount of VP proteins over MAAP for vector production, the vector was harvested after 48 hours from only the cell pellet, and high titer vector was purified (Table S1). AAV9/MAAP9EU-CBA-GFP and AAV9-CBA-mCherry of equal amounts (1.0E9vg/µL) were mixed 1:1 and infused (1.0E9vg total) into the rat striatum to compare reporter gene expression pattern 2 weeks post-infusion. Both vectors exhibited robust transgene expression with a high degree of colocalization in cells morphologically resembling neurons (Fig. 3B, top row). Therefore, MAAP9EU alone does not appear to contribute to the reduced neuronal transduction of AAV9EU.

Fig 3.

AAV9 EU and MAAP9 mutant transgene expression colocalizes with AAV9 transgene expression in cells morphologically resembling neurons in the rat striatum. Vectors were mixed 1:1 with AAV9-CBA-mCherry at equal titers (1.0E9vg/µL) and then infused into the rat striatum. Animals were sacrificed 2 weeks post-infusion and then the brains were paraformaldehyde fixed and sectioned (40 µm). Representative maximum projections of z-stacks captured by confocal microscopy of the native fluorescence signal are shown. The white dashed box indicates the area enlarged in the rightmost image. The percentage of colocalization with AAV9-CBA-mCherry was determined from at least three animals from confocal images spanning the infusion site and depicted with box and whisker plots (whiskers min to max). Statistical significance was determined using the Mann-Whitney test (** indicates P = 0.0065) (***indicates P = 0.0003). (A) Fluorescent confocal images and colocalization quantification of MAAP9 produced AAV9-CBA-GFP, AAV9E9-CBA-GFP, and AAVE99-CBA-GFP mixed with AAV9-CBA-mCherry. (B) Fluorescent confocal images and colocalization quantification of MAAP9EU produced AAV9-CBA-GFP, AAV9E9-CBA-GFP, and AAVE99-CBA-GFP mixed with AAV9-CBA-mCherry. (C) Fluorescent confocal images and colocalization quantification of AAV9/no MAAP-CBA-GFP mixed with AAV9-CBA-mCherry.

Given that our previous experiment assessed AAV9EU VP1 and VP2 individually (Fig. 2) in which vectors were produced from two rep/cap plasmids one with MAAP9 and the other with MAAP9EU, we wanted to assess the neuronal cellular tropism of the individual AAV9EU VP subunit vectors produced with either MAAP9 or MAAP9EU then mixed with AAV9 produced with MAAP9 prior to infusion. In addition, MAAP2 was shown to outcompete mutant MAAP2 during AAV2 production (9) suggesting we could have an uneven influence of either MAAP9 or MAAP9EU in our previous vector preps. To this end, AAV9 with the EU insertion in VP1 (AAVE99) or VP2 (AAV9E9) was produced with either MAAP9 or MAAP9EU and then directly compared to AAV9 (produced with MAAP9) for differences in cellular tropism. In addition, AAV9/noMAAP was also produced and compared for an in vivo activity because MAAP has no known function outside of AAV production. VP1, VP2, and MAAP mutants in AAV9EU and AAV9 were cloned as described above with the relevant MAAP start site mutated that preserved the VP1/VP2 protein sequence (9–11). All vectors (AAVE99/MAAP9-CBA-GFP, AAV9E9/MAAP9-CBA-GFP, AAVE99/MAAP9EU-CBA-GFP, and AAV9E9/MAAP9EU-CBA-GFP) were produced using two rep/cap plasmids (Fig. S2) at equal molar amounts (26), harvested after 48 hours from only the cell pellet, and high titer vector purified (Table S1). Vectors were mixed 1:1 with AAV9-CBA-mCherry in equal amounts/volumes and infused (1.0E9vg total) into the rat striatum to compare cellular tropism at 2 weeks post-infusion by confocal fluorescence microscopy analysis to determine colocalization of native fluorescence (Fig. 3). To determine a baseline of colocalization, AAV9-CBA-GFP and AAV9-CBA-mCherry were mixed and a 77.7% ± 1.696 SEM colocalization determined (Fig. 3A, top row). AAV9E9-CBA-GFP, AAV9E9/MAAP9EU-CBA-GFP, and AAVE99/MAAP9EU-CBA-GFP exhibited robust transgene expression in cells morphologically appearing to be neurons, with no significant difference in colocalization with AAV9-CBA-mCherry reporter expression (Fig. 3A and B). AAVE99-CBA-GFP exhibited 84.7% ± 1.423 SEM colocalization with AAV9-CBA-mCherry (Fig. 3A, bottom row) a statistically significant difference from AAV9-CBA-mCherry (P = 0.0065). AAV9/no MAAP-CBA-GFP exhibited 94.5% ± 1.407 SEM colocalization with AAV9-CBA-mCherry (Fig. 3C), a statistically significant difference from AAV9-CBA-mCherry (P = 0.0003). Importantly, no combination of AAV9EU insertion into VP location or MAAP9/MAAP9 EU resulted in a significantly reduced colocalization with AAV9-CBA-mCherry [Fig. 2B and (28)]. These results suggest that the EU insertion in VP1 or VP2 in combination with MAAP9EU is not responsible for AAV9EU reduced neuronal cellular tropism.

MAAP9 mutants alter relative transgene mRNA levels in vivo

Given the increase in transduction and colocalization of AAV9/noMAAP-CBA-GFP with AAV9 (Fig. 3C), 2,000-fold difference in relative transgene mRNA levels between AAV9 and AAV9EU (Fig. 1A) and that AAVE99/MAAP9 (Fig. 3A, bottom row) colocalizes with AAV9 more than when it is produced with MAAP9EU (Fig. 3B, bottom row) we wanted to further assess the relative transgene mRNA levels in the single AAV9 VP1 EU, VP2 EU, and MAAP9 mutants. To quantify the relative transgene mRNA levels, AAV9-CBA-GFP, AAV9/no MAAP-CBA-GFP, AAV9/MAAP9EU-CBA-GFP, AAVE99/MAAP9-CBA-GFP, AAV9E9/MAAP9-CBA-GFP, AAVE99/MAAP9EU-CBA-GFP, and AAV9E9/MAAP9EU-CBA-GFP were produced, harvested after 48 hours from only the cell pellet, and purified to a high titer (Table S1). Each vector was directly infused (1.0E9vg in equal volumes) into the rat striatum contralaterally to AAV9-CBA-GFP, and the striata were harvested at 2 weeks post-infusion. The mRNA was processed and analyzed as described above. Surprisingly, AAV9/no MAAP-CBA-GFP exhibited a significant increase (P < 0.05) in fold change in relative GFP mRNA levels compared to AAV9 from within the same animal (Fig. 4A). The nuclear DNA/transgene copies were assessed and exhibited similar copy numbers, so nuclear vector genome copies did not explain the difference in relative GFP mRNA levels (Fig. 4B). By contrast, the EU insertion in AAV9 VP1, VP2, or MAAP9 reduces the fold change in relative GFP mRNA levels compared to AAV9 (Fig. 4A) but not to the degree as AAV9EU (Fig. 1A). Together, the EU insertion in VP1 or VP2 in combination with MAAP9 does not replicate the transgene mRNA reduction of AAV9EU (Fig. 1A). Furthermore, these findings suggest that MAAP9EU and no MAAP have different, distinct in vivo actions and thus warranted further characterization of the AAV9/no MAAP phenotype in vivo.

Fig 4.

AAV9/no MAAP significantly increases the fold change in the relative GFP mRNA levels in AAV9 mutants. (A) AAV9-CBA-GFP, AAV9/no MAAP-CBA-GFP, AAV9/MAAP9EU-CBA-GFP, AAVE99/MAAP9-CBA-GFP, AAV9E9/MAAP9-CBA-GFP, AAVE99/MAAP9EU-CBA-GFP, and AAV9E9/MAAP9EU-CBA-GFP were infused at an equal amount (1.0E9vg) and volume into the rat striatum. AAV9-CBA-GFP was infused contralaterally to all other vectors to make within animal comparisons. Two weeks post-infusion RNA was isolated and cDNA was synthesized for qPCR Taqman expression assays for GFP and GAPDH. Each animal was run in triplicate and then averaged. The relative GFP mRNA levels to GAPDH mRNA levels were compared to AAV9 values from within the same animal and then graphed as fold change. Values are graphed on box and whisker plots where the whiskers are the min and max with all data points indicated. * indicates significance of P < 0.05 using Wilcoxon test. (B) AAV9-CBA-GFP and AAV9/no MAAP-CBA-GFP were infused at equal titers (1.0E9vg/µL) and amounts into the rat striatum. Two weeks post-infusion nuclear DNA was isolated and the nuclear copies of GFP were determined by qPCR with Taqman assays. Equal amounts of nuclear DNA were included in the assay with DNA concentration determined by GAPDH qPCR Taqman assay using a standard curve. Each animal was run in triplicate and then averaged. The nuclear GFP copies per ng of DNA were graphed on box and whisker plots where the whiskers are the min and max with all data points indicated. No statistically significant difference (ns) was found using the Mann-Whitney test.

A MAAP9-related activity can modulate AAV9 in vivo transgene expression in cis and in trans

Given that AAV9/no MAAP resulted in a significant increase in colocalization with AAV9-CBA-mCherry (Fig. 3C) and the relative GFP mRNA levels (Fig. 4A), we further assessed the potential role of MAAP9 outside of production by directly comparing it to AAV9 in vivo. In addition, we noticed that the microscopy exposure settings in experiments involving mixing AAV9/no MAAP and AAV9 vectors needed to be substantially adjusted to prevent fluorescent signal oversaturation, a result that is not explained by titer or infused amount of vector. This preliminary observation suggested that mixing AAV9 with AAV9/no MAAP increases transgene protein levels from both vectors in vivo. To visualize this observation, AAV9-CBA-mCherry was mixed 1:1 at equal amounts/volumes with either AAV9-CBA-GFP or AAV9/no MAAP-CBA-GFP and infused (1.0E9vg total) bilaterally into the striata of six rats. After 2 weeks, the animals were sacrificed, perfused, and fixed, and the brains were sliced in 40 µm sections sequentially through the striatum. Brightfield and native fluorescence images (4×) were taken through the infusion site to identify the section where the injector pierces the corpus callosum to make analogous comparisons between the vector mixes. Image analysis was performed on 11 sequential sections including and after the corpus callosum break (Fig. S7). The area above a set background threshold of native fluorescence intensity within the striatum was used in the analysis for intra-animal comparisons. Higher transgene protein amounts (average fivefold more) occur when AAV9-CBA-mCherry and AAV9/no MAAP-CBA-GFP are mixed compared to the mix of vectors produced with MAAP9 (Fig. 5A; Table S2). The slide scanner analysis was confirmed using confocal fluorescence microscopy image analysis of the first and last sections to determine and compare fluorescence intensity density (Fig. S8). These results suggest that mixing AAV9/no MAAP with AAV9 results in a significant in trans effect on transgene protein levels.

Fig 5.

AAV9/no MAAP and AAV9 vectors exhibit enhanced fluorescence intensity in the rat striatum when mixed. AAV9-CBA-mCherry was mixed 1:1 with either AAV9-CBA-GFP or AAV9/no MAAP-CBA-GFP of equal titers and infused bilaterally in an equal amount (1.0E9vg) into the rat striatum. 2 weeks post-infusion the brains were harvested. (A) Paraformaldehyde fixed brains were sequentially sectioned (40 µm) through the striatum infusion site and imaged at 4× in brightfield and native fluorescence using a slide scanner (Fig. S7). The fluorescence intensity of each section was quantified and compared within animals and then graphed as fold difference in a box and whisker plot where the whiskers are the min and max values. Statistical significance was determined using the Wilcoxon test where * indicates P < 0.05. (B) RNA was isolated from each striatum and cDNA was synthesized for GFP and GAPDH qPCR with Taqman expression assays. Each sample was run in triplicate and then averaged. The relative GFP mRNA levels to GAPDH mRNA levels were compared to AAV9 from within the same animal and then graphed as fold change. Values are graphed on box and whisker plots where the whiskers are the min and max with all data points indicated. Statistical significance was determined using the Wilcoxon test where no statistical significance (ns) was found. (C) Nuclear DNA was isolated from each striatum and the nuclear copies of GFP were determined by qPCR with Taqman assays. Equal amounts of nuclear DNA were included in the assay with DNA concentration determined by GAPDH qPCR Taqman assay using a standard curve. Each sample was run in triplicate and then averaged. The nuclear GFP copies per ng of DNA were graphed on box and whisker plots where the whiskers are the min and max with all data points indicated. No statistically significant difference was found using the Mann-Whitney test (ns).

While the data from all animals showed the same trend, we wanted to determine the nuclear transgene copies and relative transgene mRNA levels between the experimental conditions. Vectors were mixed and infused as above. After 2 weeks, nuclear DNA and RNA were isolated from each striatum and then processed and analyzed as above. The relative levels of both GFP and mCherry mRNA compared to GAPDH were similar in both vector mixtures (Fig. 5B) suggesting that the difference in transgene protein levels (Fig. 5A) is not due to differences in mRNA levels. Furthermore, comparable nuclear vector copies of GFP and mCherry (Fig. 5C) were obtained from both comparisons suggesting that the difference in transgene protein levels (Fig. 5A) is also not due to differences in nuclear entry or transduction. These results suggest that MAAP9 exhibits an activity that acts both in cis and in trans to modulate viral vector transgene protein levels in vivo. This activity is independent of altering nuclear vector copies or transgene mRNA levels. Clearly, MAAP9 exerts a role distinct from that during AAV production involving association with the cellular membrane and exosomes.

DISCUSSION

Over the years, significant efforts have focused on manipulating AAV capsid structure to create new AAVs that exhibit novel, clinically beneficial properties. The majority of AAV9-based studies have utilized either 7 or 21 amino acid insertions between AAV9 aa588 and 589, part of a hypervariable region that allows the display of peptides on the surface of the capsid (30–32). Recently, Powell et al. showed that a six glutamate insertion (EU insertion) into AAV9 VP1/2 at aa139 resulted in dramatic alterations in in vivo transgene expression, leading to the novel concept of a heretofore unknown AAV9 capsid-promoter interaction (28). To further define the nature of this AAV9 capsid/promoter interaction, the present studies investigated the singular influence of the EU insertion on VP1, VP2, or MAAP.

The present investigations found that the EU insertion into AAV9 VP1 alone or VP2 alone did influence the relative mRNA levels in the rat striatum (Fig. 4A) but did not recapitulate the dramatic reduction in transgene expression of AAV9EU (Fig. 1A). By contrast, the EU insertion into both VP1 and VP2 (AAV9EU) resulted in a 2,000-fold reduction in in vivo relative transgene mRNA levels and a 10-fold reduction in nuclear transgene DNA (Fig. 1B). Furthermore, AAV9EU contains a mutant MAAP that contributes to the reduction in transduction given that MAAP9EU alone exhibited a reduction in relative mRNA levels (Fig. 4A). Although both DNA and mRNA levels could contribute to the reduction in transduction and transgene expression for AAV9EU, the underlying mechanisms remain unclear. Recent studies have shown that the capsid can alter the epigenetic state of its genome (33, 34). Furthermore, in vitro studies revealed that protein levels and mRNA levels are not necessarily correlated (27). While ours and others’ observations were in experiments using different AAV serotypes and tissues, together, they all support the conclusion that AAV VP1/2 exhibits activity to influence transduction and transgene expression beyond the previously established roles in endosome escape and nuclear localization. The nature of this contribution of VP1, VP2, and MAAP to AAV9 transduction remains to be defined. However, it is striking that when EU insertions into VP1 or VP2 were combined with MAAP9EU, the cellular tropism (Fig. 3B) and relative mRNA levels (Fig. 4A) were similar. These results suggest that VP1 or VP2 can compensate for the EU insertion in the other.

As importantly, further investigations of other potential factors that underlie the AAV9EU phenotype led to the discovery of a MAAP9 influence on in vivo transgene expression. Following the initial discovery of the MAAP sequence in the AAV capsid coding sequence (9), subsequent studies established that MAAP facilitated the movement of AAV particles into exosomes/extracellular vesicles, an event that resulted in recombinant virus secretion into the media during virus production (9–11). Having the sequence and location of MAAP, we noted that the EU insertion into AAV9 VP1 and VP2 resulted in an RRKRRK insertion into the C-terminus of MAAP9 (MAAP9EU)(Fig. S5). Therefore, our studies inserted basic amino acids in an already highly basic region (NLS signals) and into the proposed membrane-binding domain of MAAPs. MAAP2 truncations immediately before these insertions were previously shown to affect titer and capsid protein levels (11), but C-terminal tags, 3xFlag and GFP, did allow for several serotypes of MAAP to localize to membranes (9, 10). Together, how the EU insertion altered MAAP9 function remains an outstanding question, but the absence of MAAP9 increased relative transgene mRNA levels in the rat striatum (Fig. 4A).

In our earlier VP1 and VP2 insertion studies (Fig. 2), vectors were presumably produced with both MAAP9 and MAAP9EU on their respective rep/cap plasmids. However, it is unknown if mutations to the VP1 start site or VP2 start site altered the expression levels of MAAP9 and MAAP9EU during production. Recent studies suggest that MAAP is translated from the polycistronic mRNA that contains VP2, AAP, and VP3 (13); therefore, it is possible that VP1 and VP2 mutated start sites could differentially alter MAAP translation. In terms of two different MAAPs present during production, studies from the Church lab (9) found that when a MAAP and no MAAP AAV2 virus were produced together, the MAAP virus out competes with the no MAAP virus in terms of titer. Although our studies involved different capsids and MAAP configurations, it is unlikely in our present studies that one MAAP configuration out competes another given similar titers across the different manipulations (Table S1). Also, our results show that the different configurations of glutamate inserts and MAAPs exhibit a similar cellular tropism (neuronal) (Fig. 2) as the vectors produced with only MAAP9 or MAAP9EU then mixed prior to infusion (Fig. 3A and 3B). While our experiments are unable to discern the amounts of each MAAP during production and how that translates to in vivo outcomes, the in vivo neuronal cellular tropism is independent of MAAP9 and MAAP9EU during production (Fig. 2 and 3). AAV2 and MAAP2 in vitro studies indicate that mutations in MAAP2 can alter the titer and the authors further suggest that levels of MAAP2, AAP, and VPs are required in a certain ratio (11). While these results suggest that mutations in MAAP or an altered molar amount can affect titers, purified AAV9/no MAAP and AAV9 have similar titers and VP ratios (Table S1; Fig. S1).

The removal of MAAP9 from AAV9 vector production clearly influenced the cellular environment involved in vector transgene expression. The absence of MAAP9 during vector production resulted in an increase in relative GFP mRNA levels in vivo (Fig. 4A). When assessing a mixture of AAV9 vectors produced with and without MAAP, there was no discernable difference in relative transgene mRNA levels (Fig. 5B). However, surprisingly this mixture of MAAP9 and no MAAP produced recombinant AAV9 resulted in a significant increase in in vivo transgene expression for not only the no MAAP vector but also for the MAAP9 produced vector (Fig. 5A; Table S2). Previous studies only showed that the lack of MAAP during production results in more Rep and VP protein levels (11), an increase in aberrant AAV genome packaging (11), and a reduced titer, when produced in combination with an MAAP-containing virus (9). Although we did not assess the packaged genomes, aberrant genomic packaging could provide an explanation for the no MAAP-produced vector, but not for the in vivo enhancement of gene expression from the MAAP9-produced vector. Clearly, some element of the vector exerts an in trans influence on cellular processes that support transgene expression. Furthermore, this finding provides a cautionary tale when assessing multiple AAV variants in a single cellular population as is commonly done with AAV capsid library screening.

The ability of MAAP to traffic AAVs into production media suggests that MAAP and VPs interact. An interaction of MAAP and VPs during production is certainly reasonable and attractive, yet studies looking for binding evidence proved unsuccessful (10). Studies using in vitro localization have shown that AAV2 capsid proteins remain in the cytoplasm with an MAAP2 mutant which suggests a potential binding interaction between MAAP and VPs (11). While MAAP activity during production has a more straightforward explanation, an in vivo influence on transgene expression is not straightforward. It is possible that MAAP remains with the purified vector or facilitates capsid manipulation during production in such a way to influence transgene protein expression on a cellular level. Work from our laboratory and others have highlighted that the AAV capsid can modify in vivo transgene expression (28) and that capsid/subunits can direct the epigenetic state of the transgene that determines its ability to express (33, 34). The mechanisms of MAAP activities are not well studied and require further experimentation, but it is also intriguing as to why MAAP evolved to modulate transgene protein levels. Considering AAVs unique life cycle, MAAP could be an integral component in AAV latency. Our current studies show that no MAAP increases protein expression, so it is possible that MAAP9 functions to lower protein expression in vivo as a means to tailor AAV genome expression for latency. It has been shown that the AAV2 ITRs can support a low level of expression in the absence of a promoter in vivo (35), so MAAP could act to further control AAV genome expression. In conclusion, it is uncertain if MAAP is acting directly or indirectly to influence AAV in vivo transduction. However, MAAP comprises a novel factor that can be manipulated to alter AAV expression that does not require space on the transgene. Furthermore, given the limited research in this region of the AAV capsid, these in vivo effects should prompt further investigations which may reveal additional novel functions within this capsid region.

MATERIALS AND METHODS

Plasmid cloning

The CBA-mCherry construct was previously described (28) with the CBA-GFP version differing in only the reporter gene. GFP was swapped for mCherry into the CBA-mCherry backbone using EcoRI and HindIII restriction sites.

To create hybrid VP viruses, the respective start codons were mutated as published (25, 26). Briefly, the VP1 start site was mutated to M1L, the VP2 start site was mutated to T138A, and the VP3 start site mutated to M209L (AAV9EU) (Fig. S2). Mutations were introduced using mutagenic PCR primers (Table S3) (IDT, Coralville, IA) with two rounds of PCR using PfuUltra II Hotstart PCR Master Mix (Agilent, Santa Clara, CA., cat. no.: 600850), the appropriately sized PCR products were then assembled into SalI and BsiWI sites in rep/cap (pXR) plasmid using NEBuilder HiFi DNA assembly master mix (NEB, Ipswich, MA, cat. no.: E2621S) following the manufacturer’s recommendations.

Cloning of MAAP9EU-only plasmid was partially synthesized (IDT, Coralville, IA) to include mutated VP1 and VP2 start sites (did not alter the VP protein sequence), a stop codon before the MAAP start site, a stop site after MAAP, and VP3 was truncated at aa156 (VP1 numbering). The synthesized DNA was cloned into rep/cap (pXR9) plasmid backbone between SwaI and XbaI sites using NEBuilder HiFi DNA assembly master mix (NEB, Ipswich, MA, cat. no.: E2621S) following the manufacturer’s recommendations.

AAV vector production

All transgenes used the same Chicken Beta-actin promoter (CBA) with either GFP or mCherry as a fluorescent reporter. The vector was produced in HEK293 cells as previously described (24). Briefly, polyethylenimine max (PEI) was used for the triple transfection of the rep/cap (pXR) plasmid(s), the pXX680 helper plasmid, and the transgene pTR-CBA-GFP-hGHpolyA or pTR-CBA-mCherry-hGHpolyA with AAV2 inverted terminal repeats. For chimera/hybrid capsids, the two rep/cap plasmids (Fig. S2) were used at equal molar amounts. For AAV9 with MAAP9EU, the MAAP9EU plasmid was used at ½ the molar amount of AAV9. Cells were harvested 48 hours post-transfection and purified as previously described (28) with triple-phase partitioning (36) followed by cesium chloride ultracentrifugation. The relative capsid subunit ratio was assessed by SDS-PAGE gel analysis as follows: boiled samples in a final 1× concentration of Novex Tris-Glycine SDS Sample buffer (Invitrogen, Waltham, MA, cat. no.: LC2676) and 1× NuPAGE sample reducing agent (Invitrogen, Waltham, MA, cat no: NP0004) then loaded onto Novex 4%–12% Tris-Glycine gel (Invitrogen, Waltham, MA, cat. no.: XP4120) with PageRuler protein weight marker (Invitrogen, Waltham, MA, cat. no.: 25616). The gel was run at 100 volts for 2 hours in 1× Tris-Glycine SDS Running Buffer (Invitrogen, Waltham, MA, cat. no.: LC2675) and then stained with PageBlue Protein Staining Solution (Invitrogen, Waltham, MA, cat. no.: 24620) per the manufacturer’s recommendations. The gels were imaged on an Amersham Imager 600 (Cytiva, Marlborough, MA).

MAAP deletion validation

To validate that MAAP was deleted (CTG mutated to CCG) (9, 10) and not expressed during production, rep/cap (pXR9) plasmids were generated to include a C-terminal 3xFlag (DYKDHDGDYKDHDIDYKDDDDK) tag on MAAP9 with VP1, VP2, VP3, and AAP start codons mutated (pXR9-MAAP9-3xFlag or pXR9-noMAAP-3xFlag). The MAAP-3xFlag sequence portions were synthesized by Genscript (Piscataway, NJ) and cloned into the pXR9 backbone using SwaI and BsiWI. MAAP expression and lack of expression were validated using freshly split HEK293 cells transfected as performed for virus production, but with 12 µg of XX-680, 6 µg of pTR-CBA-GFP, 6 µg of pXR9, and 4 µg of pXR9-MAAP9-3xFLAG or pXR9-noMAAP-3xFlag per plate. Cells were washed and pelleted 48 hours later and resuspended in denaturing lysis buffer (50 mM Tris pH 7.5, 1% SDS, 5 mM EDTA, 1% BME, and protease inhibitor tablet (Roche, Basel Switzerland, cat. no.: 11836170001) followed by a 5-minute incubation at 95°C to denature the chromatin. Non-denaturing lysis buffer was added (20 mM Tris-HCl pH 8, 137 mM NaCl, 1% Igepal, 2 mM EDTA, and protease inhibitor tablet (Roche, Basel Switzerland, cat. no.: 11836170001) and the chromatin further sheared by passaging through an 18-gauge needle attached to a syringe. For immunoprecipitation, half the volume of the lysate (250 µL) was added to 16 µg anti-FLAG (Millipore-Sigma, Burlington, MA, cat. no.: F3165) and then rocked at 4°C overnight. The lysate-antibody mixture was added to Protein G beads (Thermo-Fisher Waltham, MA, cat. no.: 10004D) washed in non-denaturing lysis buffer and then incubated at 4°C with rocking for 2 hours. The beads were washed with non-denaturing lysis buffer and proteins were eluted with 2×SDS loading buffer (Invitrogen, Waltham, MA, cat. no.: LC2676] and 1× NuPAGE sample reducing agent (Invitrogen, Waltham, MA, cat. no.: NP0004) followed by SDS-PAGE gel analysis as described above. For western blot analysis, proteins were transferred to PVDF membrane (Invitrogen, Waltham, MA, cat. no.: LC2002) in 1× transfer buffer with 20% methanol (Invitrogen, Waltham, MA, cat. no.: LC3675) for 1 hour at 100 V at room temperature. The membrane was blocked with 1× Blocking buffer (Thermo-Fisher, Waltham, MA, cat. no.: 37565) for 1 hour at room temperature followed by primary antibody anti-Flag (1:250) (Millipore-Sigma, Burlington, MA, cat. no.: F7425) diluted in 1× blocking buffer overnight at 4°C with rotation. The membrane was washed with TBST (Tris 20 mM, NaCl 150 mM, and 0.1% Tween-20) three times for 5 minutes each with rotation at room temperature. The membrane was then incubated with the Cy3-conjugated (anti-rabbit) secondary antibody at 1:10000 (Millipore-Sigma, Burlington, MA, cat. no.: AP187C) diluted in 1× blocking buffer for 1 hour at room temperature. The membrane was washed as before and then imaged as described above. For IP input controls, lysates were subjected to western blot for GAPDH by incubation with (1:250) anti-GAPDH (Millipore-Sigma, Burlington, MA, cat. no.: CB1001) at 4°C overnight and then with Cy5-conjugated secondary antibody (anti-mouse) (Rockland Immunochemicals, Inc. Limerick, PA, cat: 610-110-121) as described above. The membrane was washed and then imaged as described above.

VP null validation

To validate that the M1L and T138A mutations in AAV9 replicated those published for AAV2, the corresponding mutations were generated in rep/cap (pXR9) plasmids as published (26). Each pXR9 plasmid (13 µg) and XX680 (15 µg) was transfected with 0.1 mg PEI in each 150 cm plate containing low passage number HEK293 cells (as described in vector production). After 48 hours, cell lysates were prepared using a denaturing lysis buffer (described above). For immunoprecipitation, the procedure described above was followed with following differences, each cell lysate (250 µL) was added to (150 µL) Protein G beads (Thermo-Fisher Waltham, MA, cat. no.:10004D) bound with (1:100) clone B1 antibody (anti-AAV VP1/VP2/VP3) (Progen, Heidelberg, Germany, cat. no.:61058) and incubated overnight with rotation at 4°C. The beads were washed with non-denaturing lysis buffer and proteins were eluted and mixed with 2×SDS loading buffer for western blot analysis (described above). The blot was incubated with (1:200) clone B1 antibody (anti-AAV VP1/VP2/VP3) (Progen, Heidelberg, Germany, cat. no.:61058) overnight at 4°C with rotation with Cy5-conjugated secondary antibody (anti-mouse) (Rockland Immunochemicals, Inc. Limerick, PA., cat. no.: 610-110-121) then washed and imaged as described above.

Nuclear DNA isolation and qPCR analysis

Nuclei were isolated from 50 mg of resected rat striata tissue using an NE-PER reagent kit (Thermo-Fisher, Waltham, MA, cat. no.: 78833) with modifications. After nuclei were obtained, they were gently washed three times with ice-cold 1×PBS (phosphate-buffered saline pH 7.4) and centrifuged for 1 minute at 16,000 × g. The supernatant was removed from the pelleted nuclei, and then DNA was extracted using DNeasy blood and tissue kit (Qiagen, Hilden, Germany, cat. no.: 69504) with RNaseA treatment as per the protocol. The nuclear DNA was eluted with molecular biology grade water (Corning, Manassas, VA., cat. no.: 46-000-CM) for use in qPCR. 10 ng of input nuclear DNA was used in each reaction. Taqman expression assay (Thermo-Fisher, Waltham, MA) for endogenous control GAPDH (rn01775763_g1) and transgenes GFP (mr04329676_mr) and mCherry (mr07319438_mr) using TaqMan fast advanced mastermix (Thermo-Fisher, Waltham, MA, cat. no.: 4444556) per instructions on Quantstudio3 (AppliedBiosystems, Waltham, MA). A transgene standard curve was made using linearized double-stranded transgene from plasmid then diluted from 1.0E10 copies-1.0E2 copies in uninjected striatum nuclear DNA. Uninjected nuclear DNA was included in a standard curve to determine GAPDH values to calculate the genome ng amount in each sample. Each sample was run in triplicate and then averaged. The transgene copies and amount of total DNA were calculated using the appropriate standard curve run in parallel. qPCR data were exported and tabulated in Excel (Microsoft, Redmond, WA). All statistics (Mann-Whitney) and graphing were completed in Prism Software (Irvine, CA).

RNA isolation and qPCR analysis

RNA was isolated from 40 to 50 mg resected striata tissue using Qiagen RNeasy lipid kit (Qiagen, Hilden, Germany, cat. no.:74804) with on-column DNaseI treatment as per the manual. RNA was eluted in RNase-free water. 2.5 µg of RNA was used for cDNA synthesis with Superscript IV VILO master mix with ezDNase enzyme (Thermo-Fisher, Waltham, MA, cat. no.: 11766050) following the manufacturer’s recommendations. cDNA was then used in qPCR Taqman Expression assays (Thermo-Fisher, Waltham, MA) for control GAPDH (rn01775763_g1) and transgenes GFP (mr04329676_mr) and mCherry (mr07319438_mr) using TaqMan fast advanced mastermix (Thermo-Fisher, Waltham, MA, cat. no.: 4444556) per instructions on Quantstudio3 (AppliedBiosystems, Waltham, MA). Each sample was run in triplicate and repeated at least twice. The relative GFP or mCherry mRNA levels were related to GAPDH and then compared to the control within the same animal to determine fold change. qPCR data were exported and tabulated in Excel (Microsoft, Redmond, WA). All statistics (Wilcoxon) and graphing were completed in Prism (Irvine, CA).

Animals and stereotactic infusions

Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were randomly assigned to each experiment based on sex and weighed between 175 and 250 g at the time of intracranial infusion. The animals were maintained on a 12-hour light-dark cycle and had free access to water and food. For all animal studies, care and procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and all procedures received prior approval by the University of North Carolina Institutional Animal Care and Usage Committee.

For the virus vector infusions, first, the animals were anesthetized with inhalational isoflurane (3% induction, 2.5% maintenance) and placed into a stereotactic frame. Using a 32G stainless steel injector and an infusion pump, animals received 1 µL of each vector/mix (1.0E9vg/µL) into the striatum over 5 minutes either unilaterally or bilaterally (0.5–1.0 mm anterior to bregma, 3.0 mm lateral, and 5.5 mm vertical, according to the atlas of Paxinos and Watson (37). The injector was left in place for 1 minute post-infusion to allow diffusion from the injector.

Fluorescence visualization and analysis

Two weeks after AAV vector infusion, animals were euthanized for confocal microscopy evaluation or tissue collection using the drop isoflurane method. For the confocal microscopy, the animals were perfused transcardially with ice-cold 100 mM PBS (pH 7.4), followed by 4% paraformaldehyde in PB (pH 7.4). After brains were post-fixed for 12–48 hours at 4°C in the paraformaldehyde-PB, 40 µm coronal sections were cut using a vibrating blade microtome for confocal or slide scanning determination of mCherry or GFP native fluorescence. For NeuN detection, NeuN primary antibody (Millipore, Bedford, MA, cat. No.: MAB377) at 1:500 followed by secondary goat anti-mouse conjugated with Alexa 488 (Invitrogen Waltham, MA, cat. No.: A11032).

For native fluorescence visualization, sections were mounted, and native fluorescence was visualized using an Olympus FV3000RS confocal microscope in the UNC Neuroscience Center Confocal and Multiphoton Imaging Core. Images were taken using 20× objective and displayed as maximum projections. For confocal imaging, fluorescence density IMARIS software (Oxford Instruments, UK) was used.

For slide scanner analysis, sections were sequentially mounted and visualized with brightfield and fluorescence at 4× on the Evident/Olympus VS200 (Waltham, MA) at the UNC Hooker Imaging Core. A total of six animals were imaged through the entire infusion site. The average area of native fluorescence intensity signal above 20,000 was determined for 11 sequential sections for each condition using QuPath (38). The fold difference was determined by averaging the values from 11 sections followed by intra-animal comparisons. Statistical analysis was performed in Prism Software (Irvine, CA).

DATA AVAILABILITY

All data sets are deposited in the UNC Dataverse, a trustworthy, generalist data repository managed by the Research Data Management Core at the University of North Carolina at Chapel Hill. The raw data files related to this paper are publicly accessible at https://dataverse.unc.edu/dataverse/JVI01681-24.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.01681-24.

Supplemental material. jvi.01681-24-s0001.docx.

Fig. S1 to S8; Tables S1 to S3.

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.