RESUMO
Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.
Assuntos
Adenosina , Replicação do DNA , DNA Viral , DNA Polimerase Dirigida por DNA , RNA Viral , Replicação Viral , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Replicação Viral/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Viral/genética , DNA Viral/metabolismo , Células HEK293 , RNA Viral/genética , RNA Viral/metabolismo , Bocavirus Humano/genética , Bocavirus Humano/metabolismo , Genoma Viral/genética , Infecções por Parvoviridae/virologiaRESUMO
The dosing interval for effective recombinant adeno-associated virus (rAAV)-mediated gene therapy of cystic fibrosis lung disease remains unknown. Here, we assessed the durability of rAAV2.5T-fCFTRΔR-mediated transgene expression and neutralizing antibody (NAb) responses in lungs of adult wild-type ferrets. Within the first 3 months following rAAV2.5T-fCFTRΔR delivery to the lung, CFTRΔR transgene expression declined â¼5.6-fold and then remained stable to 5 months at â¼26% the level of endogenous CFTR. rAAV NAbs in the plasma and bronchoalveolar lavage fluid (BALF) peaked at 21 days, coinciding with peak ELISpot T cell responses to AAV capsid peptides, after which both responses declined and remained stable at 4-5 months post dosing. Administration of reporter vector rAAV2.5T-gLuc (gaussia luciferase) at 5 months following rAAV2.5T-fCFTRΔR dosing gave rise to similar levels of gLuc expression in the BALF as observed in age-matched reporter-only controls, demonstrating that residual BALF NAbs were functionally insignificant. Notably, the second vector administration led to a 2.6-fold greater ELISpot T cell response and â¼2.3-fold decline in fCFTRΔR mRNA and vector genomes derived from the initial rAAV2.5T-fCFTRΔR administration, suggesting selective destruction of transduced cells from the first vector dose. These findings provide insights into humoral and cellular immune response to rAAV that may be useful for optimizing gene therapy to the cystic fibrosis lung.
RESUMO
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.
Assuntos
Aminofenóis , Aminopiridinas , Benzodioxóis , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Furões , Quinolonas , Animais , Feminino , Gravidez , Aminofenóis/uso terapêutico , Aminofenóis/farmacologia , Aminopiridinas/farmacologia , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Benzodioxóis/farmacologia , Agonistas dos Canais de Cloreto/uso terapêutico , Agonistas dos Canais de Cloreto/farmacologia , Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Modelos Animais de Doenças , Combinação de Medicamentos , Mutação , Quinolonas/farmacologia , Quinolonas/uso terapêuticoRESUMO
IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) 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.
Assuntos
Brônquios , Dependovirus , Epitélio , Técnicas de Transferência de Genes , Vetores Genéticos , Transdução Genética , Humanos , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , DNA , Epitélio/metabolismo , Epitélio/virologia , Técnicas de Transferência de Genes/tendências , Terapia Genética/métodos , Vetores Genéticos/genética , Brônquios/metabolismo , Brônquios/virologia , Transporte Ativo do Núcleo Celular , Edição de Genes/tendênciasRESUMO
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.
RESUMO
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.
RESUMO
Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.
Assuntos
Fibrose Cística , Modelos Animais de Doenças , Furões , Pulmão , Transgenes , Animais , Humanos , Animais Geneticamente Modificados , Linhagem da Célula , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Furões/genética , Furões/fisiologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Pulmão/patologia , Traqueia/citologia , Transgenes/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause the ongoing pandemic of coronavirus disease 2019 (COVID19). One key feature associated with COVID-19 is excessive pro-inflammatory cytokine production that leads to severe acute respiratory distress syndrome. Although the cytokine storm induces inflammatory cell death in the host, which type of programmed cell death mechanism that occurs in various organs and cells remains elusive. Using an in vitro culture model of polarized human airway epithelium (HAE), we observed that necroptosis, but not apoptosis or pyroptosis, plays an essential role in the damage of the epithelial barrier of polarized HAE infected with SARS-CoV-2. Pharmacological inhibitors of necroptosis, necrostatin-2 and necrosulfonamide, efficiently prevented cell death and epithelial barrier dysfunction caused by SARS-CoV-2 infection. Moreover, the silencing of genes that are involved in necroptosis, RIPK1, RIPK3, and MLKL, ameliorated airway epithelial damage of the polarized HAE infected with SARS-CoV-2. This study, for the first time, confirms that SARS-CoV-2 infection triggers necroptosis that disrupts the barrier function of human airway epithelia in vitro.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Necroptose , Apoptose , EpitélioRESUMO
The efficacy of redosing the recombinant adeno-associated virus (rAAV) vector rAAV2.5T to ferret lung is limited by AAV neutralizing antibody (NAb) responses. While immunosuppression strategies have allowed for systemic rAAV repeat dosing, their utility for rAAV lung-directed gene therapy is largely unexplored. To this end, we evaluated two immunosuppression (IS) strategies to improve repeat dosing of rAAV2.5T to ferret lungs: (1) a combination of three IS drugs (Tri-IS) with broad coverage against cellular and humoral responses (methylprednisolone [MP], azathioprine, and cyclosporine) and (2) MP alone, which is typically used in systemic rAAV applications. Repeat dosing utilized AAV2.5T-SP183-fCFTRΔR (recombinant ferret CFTR transgene), followed 28 days later by AAV2.5T-SP183-gLuc (for quantification of transgene expression). Both the Tri-IS and MP strategies significantly improved transgene expression following repeat dosing and reduced AAV2.5T NAb responses in the bronchioalveolar lavage fluid (BALF) and plasma, while AAV2.5T binding antibody subtypes and cellular immune responses by ELISpot were largely unchanged by IS. One exception was the reduction in plasma AAV2.5T binding immunoglobulin G (IgG) in both IS groups. Only the Tri-IS strategy significantly suppressed splenocyte expression of IFNA (interferon α [IFN-α]) and IL4. Our studies suggest that IS strategies may be useful in clinical application of rAAV targeting lung genetic diseases such as cystic fibrosis.
RESUMO
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.
Assuntos
Histonas , Replicação Viral , Humanos , Histonas/genética , Dependovirus/genética , Replicação do DNA , Células HEK293 , DNA Viral/genética , Reparo do DNA , Dano ao DNA , Vetores GenéticosRESUMO
Human bocavirus 1 (HBoV1), a member of the genus Bocaparvovirus of the family Parvoviridae, causes acute respiratory tract infections in young children. Well-differentiated pseudostratified human airway epithelium cultured at an air-liquid interface (HAE-ALI) is an ideal in vitro culture model to study HBoV1 infection. Unique to other parvoviruses, bocaparvoviruses express a small nonstructured protein NP1 of ~25 kDa from an open reading frame (ORF) in the center of the viral genome. NP1 plays an important role in viral DNA replication and pre-mRNA processing. In this study, we performed an affinity purification assay to identify HBoV1 NP1-inteacting proteins. We identified that Ku70 and RPA70 directly interact with the NP1 at a high binding affinity, characterized with an equilibrium dissociation constant (KD) of 95 nM and 122 nM, respectively. Furthermore, we mapped the key NP1-interacting domains of Ku70 at aa266-439 and of RPA70 at aa181-422. Following a dominant negative strategy, we revealed that the interactions of Ku70 and RPA70 with NP1 play a significant role in HBoV1 DNA replication not only in an in vitro viral DNA replication assay but also in HBoV1-infected HAE-ALI cultures. Collectively, our study revealed a novel mechanism by which HBoV1 NP1 enhances viral DNA replication through its direct interactions with Ku70 and RPA70.
Assuntos
Bocavirus Humano , Infecções por Parvoviridae , Criança , Pré-Escolar , Replicação do DNA , DNA Viral/genética , DNA Viral/metabolismo , Genoma Viral , Bocavirus Humano/genética , Bocavirus Humano/metabolismo , Humanos , Replicação Viral/genéticaRESUMO
Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), a chronic disease that affects multiple organs, including the lung. We developed a CF ferret model of a scarless G551âD substitution in CFTR (CFTRG551D-KI), enabling approaches to correct this gating mutation in CF airways via gene editing. Homology-directed repair (HDR) was tested in Cas9-expressing CF airway basal cells (Cas9-GKI) from this model, as well as reporter basal cells (Y66S-Cas9-GKI) that express an integrated nonfluorescent Y66S-EGFP (enhanced green fluorescent protein) mutant gene to facilitate rapid assessment of HDR by the restoration of fluorescence. Recombinant adeno-associated virus (rAAV) vectors were used to deliver two DNA templates and sgRNAs for dual-gene editing at the EGFP and CFTR genes, followed by fluorescence-activated cell sorting of EGFPY66S-corrected cells. When gene-edited airway basal cells were polarized at an air-liquid interface, unsorted and EGFPY66S-corrected sorted populations gave rise to 26.0% and 70.4% CFTR-mediated Cl- transport of that observed in non-CF cultures, respectively. The consequences of gene editing at the CFTRG551D locus by HDR and nonhomologous end joining (NHEJ) were assessed by targeted gene next-generation sequencing (NGS) against a specific amplicon. NGS revealed HDR corrections of 3.1% of G551 sequences in the unsorted population of rAAV-infected cells, and 18.4% in the EGFPY66S-corrected cells. However, the largest proportion of sequences had indels surrounding the CRISPR (clustered regularly interspaced short palindromic repeats) cut site, demonstrating that NHEJ was the dominant repair pathway. This approach to simultaneously coedit at two genomic loci using rAAV may have utility as a model system for optimizing gene-editing efficiencies in proliferating airway basal cells through the modulation of DNA repair pathways in favor of HDR.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Furões/genética , Furões/metabolismo , Vetores Genéticos/genética , Fibrose Cística/genética , Fibrose Cística/terapia , Mutação , Pulmão/metabolismo , DNARESUMO
Alpha-1 antitrypsin deficiency (AATD) is the most common genetic cause and risk factor for chronic obstructive pulmonary disease, but the field lacks a large-animal model that allows for longitudinal assessment of pulmonary function. We hypothesized that ferrets would model human AATD-related lung and hepatic disease. AAT-knockout (AAT-KO) and PiZZ (E342K, the most common mutation in humans) ferrets were generated and compared with matched controls using custom-designed flexiVent modules to perform pulmonary function tests, quantitative computed tomography (QCT), bronchoalveolar lavage (BAL) proteomics, and alveolar morphometry. Complete loss of AAT (AAT-KO) led to increased pulmonary compliance and expiratory airflow limitation, consistent with obstructive lung disease. QCT and morphometry confirmed emphysema and airspace enlargement, respectively. Pathway analysis of BAL proteomics data revealed inflammatory lung disease and impaired cellular migration. The PiZ mutation resulted in altered AAT protein folding in the liver, hepatic injury, and reduced plasma concentrations of AAT, and PiZZ ferrets developed obstructive lung disease. In summary, AAT-KO and PiZZ ferrets model the progressive obstructive pulmonary disease seen in AAT-deficient patients and may serve as a platform for preclinical testing of therapeutics including gene therapy.
Assuntos
Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Deficiência de alfa 1-Antitripsina , Animais , Furões , Humanos , Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/terapiaRESUMO
Parvoviruses package a linear single-stranded DNA genome with hairpin structures at both ends. It has been thought that terminal hairpin sequences are indispensable for viral DNA replication. Here, we provide evidence that the hairpin-deleted duplex genomes of human bocavirus 1 (HBoV1) replicate in human embryonic kidney 293 (HEK293) cells. We propose an alternative model for HBoV1 DNA replication in which the leading strand can initiate strand displacement without hairpin transfer. The transfection of the HBoV1 duplex genomes that retain a minimal replication origin at the right end (OriR) but with extensive deletions in the right-end hairpin (REH) generated viruses in HEK293 cells at a level 10 to 20 times lower than that of the wild-type (WT) duplex genome. Importantly, these viruses that have a genome with various deletions after the OriR but not the one retaining only the OriR replicated in polarized human airway epithelia. We discovered that the 18-nucleotide (nt) sequence (nt 5403 to 5420) beyond the OriR was sufficient to confer virus replication in polarized human airway epithelia, although its progeny virus production was â¼5 times lower than that of the WT virus. Thus, our study demonstrates that hairpin transfer-independent productive parvovirus DNA replication can occur. IMPORTANCE Hairpin transfer-independent parvovirus replication was modeled with human bocavirus 1 (HBoV1) duplex genomes whose 5' hairpin structure was ablated by various deletions. In HEK293 cells, these duplex viral genomes with ablated 5' hairpin sequence replicated efficiently and generated viruses that productively infected polarized human airway epithelium. Thus, for the first time, we reveal a previously unknown phenomenon that productive parvovirus DNA replication does not depend on the hairpin sequence at REH to initiate rolling-hairpin DNA replication. Notably, the intermediates of viral DNA replication, as revealed by two-dimensional electrophoresis, from transfections of hairpin sequence-deleted duplex genome and full-length genome in HEK293 cells as well as from virus infection of polarized human airway epithelia are similar. Thus, the establishment of the hairpin transfer-independent parvoviral DNA replication deepens our understanding of viral DNA replication and may have implications in the development of parvovirus-based viral vectors with alternative properties.
Assuntos
Replicação do DNA/genética , Bocavirus Humano/genética , Sequências Repetidas Invertidas/genética , DNA Viral/genética , Células Epiteliais/virologia , Genoma Viral/genética , Células HEK293 , Humanos , Parvovirus/genética , Origem de Replicação , Mucosa Respiratória/virologia , Proteínas não Estruturais Virais/genética , Viroses/genética , Replicação Viral/genéticaRESUMO
The spike (S) polypeptide of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) consists of the S1 and S2 subunits and is processed by cellular proteases at the S1/S2 boundary that contains a furin cleavage site (FCS), 682RRAR↓S686 Various deletions surrounding the FCS have been identified in patients. When SARS-CoV-2 propagated in Vero cells, it acquired deletions surrounding the FCS. We studied the viral transcriptome in Vero cell-derived SARS-CoV-2-infected primary human airway epithelia (HAE) cultured at an air-liquid interface (ALI) with an emphasis on the viral genome stability of the FCS. While we found overall the viral transcriptome is similar to that generated from infected Vero cells, we identified a high percentage of mutated viral genome and transcripts in HAE-ALI. Two highly frequent deletions were found at the FCS region: a 12 amino acid deletion (678TNSPRRAR↓SVAS689) that contains the underlined FCS and a 5 amino acid deletion (675QTQTN679) that is two amino acids upstream of the FCS. Further studies on the dynamics of the FCS deletions in apically released virions from 11 infected HAE-ALI cultures of both healthy and lung disease donors revealed that the selective pressure for the FCS maintains the FCS stably in 9 HAE-ALI cultures but with 2 exceptions, in which the FCS deletions are retained at a high rate of >40% after infection of ≥13 days. Our study presents evidence for the role of unique properties of human airway epithelia in the dynamics of the FCS region during infection of human airways, which is likely donor dependent.IMPORTANCE Polarized human airway epithelia at an air-liquid interface (HAE-ALI) are an in vitro model that supports efficient infection of SARS-CoV-2. The spike (S) protein of SARS-CoV-2 contains a furin cleavage site (FCS) at the boundary of the S1 and S2 domains which distinguishes it from SARS-CoV. However, FCS deletion mutants have been identified in patients and in vitro cell cultures, and how the airway epithelial cells maintain the unique FCS remains unknown. We found that HAE-ALI cultures were capable of suppressing two prevalent FCS deletion mutants (Δ678TNSPRRAR↓SVAS689 and Δ675QTQTN679) that were selected during propagation in Vero cells. While such suppression was observed in 9 out of 11 of the tested HAE-ALI cultures derived from independent donors, 2 exceptions that retained a high rate of FCS deletions were also found. Our results present evidence of the donor-dependent properties of human airway epithelia in the evolution of the FCS during infection.
Assuntos
Brônquios/virologia , Furina/metabolismo , Mucosa Respiratória/virologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Transcriptoma , Animais , Brônquios/citologia , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/virologia , Humanos , RNA-Seq , Mucosa Respiratória/citologia , Deleção de Sequência , Glicoproteína da Espícula de Coronavírus/metabolismo , Células VeroRESUMO
Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing-antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures and complemented CFTR Cl- currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200%-300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat dosing significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.
RESUMO
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.
Assuntos
Betacoronavirus/fisiologia , Modelos Biológicos , Mucosa Respiratória/virologia , Brônquios/citologia , Células Cultivadas , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Cinética , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , SARS-CoV-2 , Carga Viral , Tropismo Viral , Liberação de Vírus , Replicação ViralRESUMO
Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.