Inhibition of DNA-dependent protein kinase catalytic subunit boosts rAAV transduction of polarized human airway epithelium.

Adeno-associated virus 2.5T (AAV2.5T) was selected from the directed evolution of AAV capsid library in human airway epithelia. This study found that recombinant AAV2.5T (rAAV2.5T) transduction of well-differentiated primary human airway epithelia induced a DNA damage response (DDR) characterized by the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all three phosphatidylinositol 3-kinase-related kinases: ataxia telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). While suppressing the expression of ATR by a specific pharmacological inhibitor or targeted gene silencing inhibited rAAV2.5T transduction, DNA-PKcs inhibition or targeted gene silencing significantly increased rAAV2.5T transgene expression. Notably, DNA-PKcs inhibitors worked as a "booster" to further increase rAAV2.5T transgene expression after treatment with doxorubicin and did not compromise epithelial integrity. Thus, our study provides evidence that DDR is associated with rAAV transduction in well-differentiated human airway epithelia, and DNA-PKcs inhibition has the potential to boost rAAV transduction. These findings highlight that the application of DDR inhibition-associated pharmacological interventions has the potential to increase rAAV transduction and thus to reduce the required vector dose.

Identification of Host Restriction Factors Critical for Recombinant AAV Transduction of Polarized Human Airway Epithelium.

Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L , TM9SF2 , VPS51 , and VPS54 , as well as a novel gene WDR63 . We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T. Significance: The essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63 , whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication.

Adeno-associated virus (AAV) belongs to the Dependoparvovirus genus of the Parvoviridae family. AAV replication relies on a helper virus, such as adenovirus (Ad). Co-infection of AAV and Ad induces a DNA damage response (DDR), although its function in AAV DNA replication remains unknown. In this study, monoinfection of AAV2 in HEK293T cells expressing a minimal set of Ad helper genes was used to investigate the role of the DDR solely induced by AAV. We found that AAV2 DNA replication, but not single stranded (ss)DNA genome accumulation and Rep expression only, induced a robust DDR in HEK293T cells. The induced DDR featured the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all 3 phosphatidylinositol 3-kinase-related kinases (PIKKs). We also found that the kinase ataxia telangiectasia and Rad3-related protein (ATR) plays a major role in AAV2 DNA replication and that Y family DNA repair DNA polymerases η (Pol η) and Pol κ contribute to AAV2 DNA replication both in vitro and in HEK293T cells. Knockout of Pol η and Pol κ in HEK293T cells significantly decreased wild-type AAV2 replication and recombinant AAV2 production. Thus, our study has proven that AAV2 DNA replication induces a DDR, which in turn initiates a DNA repairing process that partially contributes to the viral genome amplification in HEK293T cells. IMPORTANCE Recombinant AAV (rAAV) has emerged as one of the preferred delivery vectors for clinical gene therapy. rAAV production in HEK293 cells by transfection of a rAAV transgene plasmid, an AAV Rep and Cap expression packaging plasmid, and an Ad helper plasmid remains the popular method. Here, we demonstrated that the high fidelity Y family DNA repair DNA polymerase, Pol η, and Pol κ, plays a significant role in AAV DNA replication and rAAV production in HEK293T cells. Understanding the AAV DNA replication mechanism in HEK293T cells could provide clues to increase rAAV vector yield produced from the transfection method. We also provide evidence that the ATR-mediated DNA repair process through Pol η and Pol κ is one of the mechanisms to amplify AAV genome, which could explain AAV replication and rAAV ssDNA genome conversion in mitotic quiescent cells.

Eight years' advances on Bourbon virus, a tick-born Thogotovirus of the Orthomyxovirus family.

Bourbon virus (BRBV) was first isolated from a blood sample collected from a male patient living in Bourbon county, Kansas, during the spring of 2014. The patient later died due to complications associated with multiorgan failure. Currently, several BRBV infection-caused deaths have been reported in the United States, and misdiagnosed cases are often undercounted. BRBV is a member of the genus Thogotovirus of the Orthomyxoviridae family, and is transmitted through the Lone Star tick, Amblyomma Americanum, in North America. Currently, there are no specific antivirals or vaccinations available to treat or prevent BRBV infection. Several small molecular compounds have been identified to effectively inhibit BRBV infection of in vitro cell cultures at a single- or sub-micromolar level. Favipiravir, an RNA-dependent RNA polymerase inhibitor, prevented the death of Type I interferon receptor knockout mice infected with BRBV infection.

Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.

Long Period Modeling SARS-CoV-2 Infection of in Vitro Cultured Polarized Human Airway Epithelium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. However, previous studies only characterized short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7-10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed Zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of 2.5 × 10 5 virions per cm 2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.