Respiratory syncytial virus infection alters surfactant protein A expression in human pulmonary epithelial cells by reducing translation efficiency

Abstract

, a collectin that is an important component of the pulmonary innate immune system. SP-A binds to the virus, targeting the infectious agent for clearance by host defense mechanisms. We have previously shown that while the steady-state level of SP-A mRNA increases approximately threefold after RSV infection, steady-state levels of cellular and secreted SP-A protein decrease 40 -60% in human type II cells in primary culture, suggesting a mechanism where the virus alters components of the innate immune response in infected cells. In these studies, we find that changes in SP-A mRNA and protein levels in RSV-infected NCI-H441 cells (a bronchiolar epithelial cell line) recapitulate the results in SP-A expression observed in primary lung cells. While SP-A protein is normally ubiquitinated, there is no change in the level of SP-A protein ubiquitination or proteasome activity during RSV infection, suggesting that the reduced levels of SP-A protein are not due to degradation by activated proteasomes. SP-A mRNA is appropriately processed and exported from the nucleus to the cytoplasm during RSV infection. As evidenced by polysome analysis of SP-A mRNA and pulse-chase analysis of newly synthesized SP-A protein, we find a decrease in translational efficiency that is specific for SP-A mRNA. Therefore, the decrease in SP-A protein levels observed after RSV infection of pulmonary bronchiolar epithelial cells results from a mechanism that affects SP-A mRNA translation efficiency.

An 11-nt sequence polymorphism at the 3′UTR of human SFTPA1 and SFTPA2 gene variants differentially affect gene expression levels and miRNA regulation in cell culture

“…The opposite effect was found when this sequence element was deleted from the variants where it naturally occurs, i.e., 6A 2 , and all SFTPA2 variants (1A, 1A 0 , 1A1, 1A 2 , 1A 3 , and 1A 5 ). Together, these results indicate that the SP-A 11-nt element has a negative effect on gene expression under normal conditions and are consistent with our previous work where variants 6A 2 , 6A 3 , 6A 4 , and their corresponding mutants were studied for their impact of this element on translation and mRNA stability (60).…”

“…Reporter constructs were transfected in NCI-H441 cells in the presence or absence of miRNA mimics, and reporter gene expression was analyzed. We found that human miRNA mir-767 negatively affected expression of constructs containing SFTPA1 and SFTPA2 variants, whereas mir-4507 affected only constructs with 3=UTRs of SFTPA1 variants 6A, 6A 3 , and 6A 4 (not containing the 11-nt element). Three miRNAs (mir-183, mir-449b, and mir-612) inhibited expression of recombinants of SFTPA2 variants and the SFTPA1 variant 6A 2 , all containing the 11-nt element.…”

“…miRNA 4507 is a selective inhibitor of SFTPA1 variants 6A, 6A 3 , and 6A 4 . When we cotransfected miRNA mimic hsa-mir-4507 with recombinant reporter vectors containing 3=UTRs of SFTPA2 variants and mutants of the 11-nt element, we found that this miRNA mimic selectively affected expression of constructs with the 3=UTRs of SFTPA1 variants 6A, 6A 3 , and 6A 4 ( Figs.…”

“…These are found in the population with variable frequency, and some of them have been found to be risk factors for lung disease, including tuberculosis, idiopathic pulmonary fibrosis, lung cancer, bronchopulmonary dysplasia, respiratory distress syndrome, and various alveolar and airway infections (15,24,51,55,58). The most commonly found variants (Ͼ1% in the general population) are 6A, 6A 2 , 6A 3 , and 6A 4 for SFTPA1, and 1A, 1A 0 , 1A 1 , 1A 2 , 1A 3 , and 1A 5 for SFTPA2 (Fig. 1).…”

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…H441 cells have also been evaluated as models of the peripheral lung. These cells, which were derived from a papillary adenocarcinoma of the lung, have features characteristic of Club cells (Gazdar et al, 1990) and have been used as a model of bronchiolar epithelium (Bruce et al, 2009). However, more recently, it has been found that culture of H441 cells on semi-permeable filter supports results in formation of electrically tight monolayers (peak TEER values of approximately 1000 Ω cm 2 after 8-12 days in culture) which are able to differentiate paracellularly transported substrates according to their molecular weight (Salomon et al, 2014).…”