The Golgi Calcium ATPase Pump Plays an Essential Role in Adeno-associated Virus Trafficking and Transduction

Abstract

Adeno-associated viruses (AAVs) are dependoparvoviruses that have proven useful for therapeutic gene transfer; however, our understanding of host factors that influence AAV trafficking and transduction is still evolving. Here, we investigate the role of cellular calcium in the AAV infectious pathway. First, we demonstrate a critical role for the host Golgi-resident ATP-powered calcium pump (secretory pathway calcium ATPase 1; SPCA1) encoded by the ATP2C1 gene in AAV infection. CRISPR-based knockout (KO) of ATP2C1 decreases transduction by different AAV serotypes. ATP2C1 KO does not appear to inhibit AAV binding, cellular uptake or nuclear entry; however, capsids within ATP2C1 KO cells demonstrate disperse and punctate trafficking distinct from the perinuclear, trans-Golgi pattern observed in normal cells. In addition, we observed a defect in the ability of AAV capsids to undergo conformational changes and support efficient vector genome transcription in ATP2C1 KO cells. The calcium chelator, BAPTA-AM, which reduces cytosolic calcium, rescues the defective ATP2C1 KO phenotype and AAV transduction in vitro. Conversely, the calcium ionophore, Ionomycin, which disrupts calcium gradients, blocks AAV transduction. Further, we demonstrate that modulating calcium in the murine brain using BAPTA-AM augments AAV gene expression in vivo. Taken together, we postulate that the maintenance of an intracellular calcium gradient by the calcium ATPase and processing within the Golgi compartment are essential for priming the capsid to support efficient AAV genome transcription. IMPORTANCE Adeno-associated viruses (AAVs) have proven to be effective gene transfer vectors. However, our understanding of how the host cell environment influences AAV transduction is still evolving. In the present study, we investigate the role of ATP2C1, which encodes a membrane calcium transport pump, SPCA1, essential for maintaining cellular calcium homeostasis on AAV transduction. Our results indicate that cellular calcium is essential for efficient intracellular trafficking and conformational changes in the AAV capsid that support efficient genome transcription. Further, we show that pharmacological modulation of cellular calcium levels can potentially be applied to improve the AAV gene transfer efficiency.

Single amino acid insertion allows functional transduction of murine hepatocytes with human liver tropic AAV capsids

“…This is not in contradiction to published data, given that only functional tests, specifically luciferase expression assays, were performed. 17 However, our data are more consistent with a model where AAVR plays a similar role in AAV transduction to the Golgi calcium transport pump SPCA1, 28 which has recently been described as an essential host factor for intracellular trafficking. Productive intracellular trafficking would then lead to capsid conformational changes that support efficient transduction.…”

“…Productive intracellular trafficking would then lead to capsid conformational changes that support efficient transduction. 28 …”

Adeno-associated virus (AAV) cell entry: structural insights

“…High among those implicated by gene trap and CRISPR screens is ATP2C1 (Table 2), encoding the SPCA1 calcium pump [4,51]. Knockout of ATP2C1 reduces transduction by multiple AAV serotypes but can be rescued by an intracellular Ca 2+ -chelating agent [74]. The impact might be mostly indirect: low Ca 2+ appears to be needed to trigger exposure of VP1u, but there might be pleiotropic effects [74].…”

Answered and Unanswered Questions in Early-Stage Viral Vector Transduction Biology and Innate Primary Cell Toxicity for Ex-Vivo Gene Editing

“…While several studies have demonstrated the importance of TLR signaling in vivo for hematopoiesis in response to microbial infection, ex vivo studies highlight the innate TLR functionality present in HSPCs. Ex vivo TLR9 activation by CpG DNA can induce mouse common lymphoid progenitors (CLPs) to differentiate to dendritic cells in response to herpesvirus infection (72). Additionally, TLR2 activation of human CD34+ cells in vitro with Pam 3 CSK 4 pushes cells towards a myeloid-biased lineage, generating CD11c+ monocytes and dendritic cells (71,73,74).…”