The Coxsackievirus and Adenovirus Receptor Has a Short Half-Life in Epithelial Cells

The coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell adhesion, cell signaling, and viral infection. The 8-exon encoded isoform (CAREx8) resides at the apical surface of polarized epithelia, where it is accessible as a receptor for adenovirus entering the airway lumen. Given its pivotal role in viral infection, it is a target for antiviral strategies. To understand the regulation of CAREx8 and determine the feasibility of receptor down regulation, the half-life of total and apical localized CAREx8 was determined and correlated with adenovirus transduction. Total and apical CAREx8 has a relatively short half-life of approximately 2 h. The half-life of apical CAREx8 correlates well with adenovirus transduction. These results suggest that antiviral strategies that aim to degrade the primary receptor for apical adenovirus infection will be effective within a relatively short time frame after application

The Regulation of the Eight-Exon Isoform of the Coxsackievirus and Adenovirus Receptor (CAR\u3csup\u3eEX8\u3c/sup\u3e) and Its Biological Relevance

The airway epithelium poses a formidable barrier for the entry of pathogenic viruses due to the formation of tight junctions between adjacent epithelial cells. The coxsackievirus and adenovirus receptor (CAR), a member of the Ig superfamily of cell junction adhesion proteins, is the primary receptor for adenovirus entry and infection. As a result of alternative splicing, two transmembrane isoforms of CAR are generated. While the seven-exon isoform of CAR (CAREX7) is hidden on the basolateral surface of polarized epithelia, the eight-exon isoform of CAR (CAREX8) localizes within the sub-apical region and at the air-exposed apical surface. Apical localization of CAREX8 makes it accessible to invading adenovirus entering the lumen of the airway and able to facilitate viral entry into the epithelium. Previous studies have shown that Interleukin-8 (IL-8), a proinflammatory cytokine and a neutrophil chemoattractant, increases the susceptibility of the airway epithelium to adenoviral infection. I hypothesized that the apical CAREX8 protein expression level and localization are responsible for the susceptibility of a polarized epithelium to viral infection. Moreover, I hypothesized that CAREX8expression is tightly regulated by mediators of IL-8 signaling and the endogenous function CAREX8is to tether neutrophils at the apical surface of the polarized epithelium. Finally, I hypothesized that adenovirus has co-opted CAREX8 and neutrophil transmigration to enhance infection of the polarized epithelium. Consistent with these hypotheses, I demonstrate that IL-8 increases the expression and the apical localization of CAREX8 in polarized airway epithelial cells. In addition, IL-8 differentially activates AKT/S6K and inactivates GSK3ß to augment the protein synthesis of CAREX8. Increased CAREX8 is able to mediate increased neutrophil binding at the apical surface of the epithelium that is completely abolished by competition with CAR-binding adenovirus fiber-knob. Finally, I also demonstrate that neutrophils adhering to the epithelial apical surface are able to promote adenoviral infection. Taken together, these data suggest that adenovirus has evolved to co-opt the host innate-immune response to the inflammation caused by molecules within inhaled droplets, pre-existing inflammation, or even adenovirus itself, in order to gain entry into the polarized epithelium by inducing the increased expression of endogenous apically localized CAREX8

Rev Interacts with Tubulin Heterodimers to Cause Cell Cycle Defects

Rev is a regulatory protein that plays an important role in the replication of HIV virus by post-transcriptionally promoting expression of viral proteins late in infection. Rev expression also slows cell growth, leads to an accumulation of cells in G2/M specifically before the spindle checkpoint, and can produce changes in ploidy. Because Rev is capable of depolymerizing microtubules (MTs) in vitro, possibly by a mechanism shared with Kinesin-13 proteins, themselves potent cellular MT depolymerases, I tested the hypothesis that these cellular defects were due to an interaction between Rev and tubulin. To this end, Rev and select Rev mutants defective in RNA binding and nuclear import (M6), nuclear export (M10), and Rev multimerization (M4) were expressed in HeLa cells. Rev\u27s ability to interact with tubulin was monitored by reciprocal co-immunoprecipitation experiments using antibodies specific for tubulin and the Rev transgene. Results from these experiments are consistent with this hypothesis as Rev and tubulin can be detected in the same immunoprecipitates. To extend these results, deconvolution microscopy was used to colocalize Rev and spindle microtubules. Whereas Rev, M4, M6, and M10 fused to green or yellow fluorescent protein are largely dispersed throughout the cytoplasm of mitotic cells, the use of colocalization software indicates there is a shallow gradient of Rev accumulation proximal to the spindle. Some M6 appears to colocalize at or near spindle poles although this is also seen in control cells. However, while these data suggest there is a potential for substantial colocalization between Rev and tubulin, visual inspection shows there is little compelling colocalization with spindle MTs. However, because immunostaining readily detects tubulin polymerized into MTs and not soluble tubulin heterodimers, the results of the colocalization and co-immunoprecipitation assays are both consistent with the hypothesis that Rev and Rev mutants are interacting with the heterodimer and not the polymerized tubulin. Intriguingly, significant amounts of wild-type Rev, M4 and M10 accumulate perichromosomally where a large fraction of spindle MTs nucleates early in mitosis. Thus Rev is spatially positioned within the cell to affect spindle assembly during early mitosis. Indeed, the previously discovered cell cycle defects of wild-type Rev, M4, M6, and M10 are all consistent with this hypothesis. Taken together, these results suggest that cells have the ability to correct spindle defects that occur during prometaphase. In conclusion, these results suggest that Rev and Rev mutants interact with tubulin heterodimers and might interfere with cell cycle progression. Since Rev expressing cells accumulate in G2/M phase, the mitotic defects in cells expressing Rev and Rev mutants were examined. Previous research has suggested that expression of Rev and Rev mutants alters progression through mitosis with cells accumulating before the spindle assembly checkpoint. These results suggest that Rev expression may interfere with chromosomal congression and therefore alter tension across the spindle and between kinetochores. To investigate this, the distances between spindle poles and interkinetchore distances were measured in metaphase cells. No significant differences were found between cells expressing Rev or Rev mutants and control cells

A Directed Evolution Approach to Select for Novel Adeno-Associated Virus Capsids on an HIV-1 Producer T Cell Line

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1

A Directed Evolution Approach to Select for Novel Adeno-Associated Virus Capsids on an HIV-1 Producer T Cell Line

A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1