Ivacaftor is the first clinically approved monotherapy potentiator to treat CFTR channel dysfunction in people with cystic fibrosis. Ivacaftor (Iva) is a critical component for all current modulator therapies, including highly effective modulator therapies. Clinical studies show that CF patients on ivacaftor-containing therapies present various clinical responses, off-target effects, and adverse reactions, which could be related to metabolites of the compound. In this study, we reported the concentrations of Iva and two of its major metabolites (M1-Iva and M6-Iva) in capillary plasma and estimated M1-Iva and M6-Iva metabolic activity via the metabolite parent ratio in capillary plasma over 12 h. We also used the ratio of capillary plasma versus human nasal epithelial cell concentrations to evaluate entry into epithelial cells in vivo. M6-Iva was rarely detected by LC-MS/MS in epithelial cells from participants taking ivacaftor, although it was detected in plasma. To further explore this discrepancy, we performed in vitro studies, which showed that M1-Iva, but not M6-Iva, readily crossed 16HBE cell membranes. Our studies also suggest that metabolism of these compounds is unlikely to occur in airway epithelia despite evidence of expression of metabolism enzymes. Overall, our data provide evidence that there are differences between capillary and cellular concentrations of these compounds that may inform future studies of clinical response and off-target effects.

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 ∼5 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.

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.

Human bocavirus 1 (HBoV1) infects well-differentiated (polarized) human airway epithelium (HAE) cultured at an air-liquid interface (ALI). In the present study, we applied next-generation RNA sequencing to investigate the genome-wide transcription profile of HBoV1, including viral mRNA and small RNA transcripts, in HBoV1-infected HAE cells. We identified novel transcription start and termination sites and confirmed the previously identified splicing events. Importantly, an additional proximal polyadenylation site (pA)p2 and a new distal polyadenylation site (pA)dREH lying on the right-hand hairpin (REH) of the HBoV1 genome were identified in processing viral pre-mRNA. Of note, all viral nonstructural proteins-encoding mRNA transcripts use both the proximal polyadenylation sites [(pA)p1 and (pA)p2] and distal polyadenylation sites [(pA)d1 and (pA)dREH] for termination. However, capsid proteins-encoding transcripts only use the distal polyadenylation sites. While the (pA)p1 and (pA)p2 sites were utilized at roughly equal efficiency for proximal polyadenylation of HBoV1 mRNA transcripts, the (pA)d1 site was more preferred for distal polyadenylation. Additionally, small RNA-seq analysis confirmed there is only one viral noncoding RNA (BocaSR) transcribed from nt 5199⁻5340 of the HBoV1 genome. Thus, our study provides a systematic and unbiased transcription profile, including both mRNA and small RNA transcripts, of HBoV1 in HBoV1-infected HAE-ALI cultures.

Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 hours did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae, the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.