ABCC1 p.Asp572Asn
| Predicted by SNAP2: | A: D (53%), C: D (53%), E: D (59%), F: D (71%), G: D (53%), H: D (59%), I: D (66%), K: D (71%), L: D (71%), M: D (66%), N: D (53%), P: D (80%), Q: D (53%), R: D (71%), S: N (82%), T: N (53%), V: D (66%), W: D (66%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] AAV exploits subcellular stress associated with in... PLoS Pathog. 2011 May;7(5):e1002053. Epub 2011 May 19. Johnson JS, Gentzsch M, Zhang L, Ribeiro CM, Kantor B, Kafri T, Pickles RJ, Samulski RJ
AAV exploits subcellular stress associated with inflammation, endoplasmic reticulum expansion, and misfolded proteins in models of cystic fibrosis.
PLoS Pathog. 2011 May;7(5):e1002053. Epub 2011 May 19., [PMID:21625534]
Abstract [show]
Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (DeltaF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing DeltaF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded DeltaF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as DeltaF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing DeltaF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, beta-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency.
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283 Materials and Methods Cell culture HeLa cells were maintained at 37uC and 5% CO2 in Dulbecco`s modified Eagle`s medium (DMEM) that was supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. BHK-21 cells were grown at 37uC and 5% CO2 in Dulbecco`s modified Eagle`s medium (DMEM) and Ham`s F12 medium (50:50) that was supplemented with 5% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 g/ml streptomycin. BHK-21 clones expressing wild-type CFTR, CFTR-DF508, CFTR-G551D, CFTR-D572N, CFTR- 1410X, wild-type MRP1, or MRP1-DF728 were maintained in selection medium containing methotrexate (250 mg/ml).
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ABCC1 p.Asp572Asn 21625534:283:540
status: NEW