SUMMARY Stress granules are mRNA-protein granules that form when translation initiation is limited and are related to pathological granules in various neurodegenerative diseases. Super-resolution microscopy reveals stable substructures referred to as cores within stress granules that can be purified. Proteomic analysis of stress granule cores reveals a dense network of protein-protein interactions, links between stress granules and human diseases, and identifies ATP-dependent helicases and protein remodelers as conserved stress granule components. ATP is required for stress granule assembly and dynamics. Moreover, multiple ATP-driven machines affect stress granules differently; with the CCT complex inhibiting stress granule assembly, while the MCM and RVB complexes promote stress granule persistence. Our observations suggest that stress granules contain a stable core structure surrounded by a dynamic shell with assembly, disassembly and transitions between the core and shell modulated by numerous protein and RNA remodeling complexes.

Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I ␣-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency.The mechanism of infection by type 2 and type 5 adenovirus has been extensively studied. However, most of the knowledge on adenoviral infection has been obtained from studies done on immortalized cell lines. The first steps in adenovirus infection are thought to involve primarily two proteins in the capsid, fiber and penton base (1-3). The fiber protein is important for binding to a high affinity fiber receptor. In a human oral epidermoid carcinoma cell line (KB cells), A549 cells, and HeLa cells, this receptor is thought to be present in the range of 3,000 -10,000 receptors/cell (4 -6). NIH 3T3 cells, which are resistant to adenovirus infection, have less than 100 receptors/ cell (7). After binding to the fiber receptor, penton base interaction with ␣ V ␤ 3 and ␣ V ␤ 5 integrins facilitates internalization via receptor-mediated endocytosis (2,8,9). The acidic pH the virus encounters in the endosome may trigger a conformational change that releases the virus into the cytoplasm (10 -12) and allows the adenovirus capsid to travel to the nucleus (2, 13). Then viral proteins and DNA bind to the nuclear pore complex, capsid disassembly continues, and DNA enters the nucleus accompanied by DNA-associated protein 7 (1, 2, 14). These studies have concluded that a high affinity fiber receptor is required for binding and infection and that an ␣ V ␤ integrin acts as a co-receptor.We and others (15-22) have found infection of ciliated airway epithelia by adenovirus to be inefficient. In an in vitro model of human airway epithelia, we found that unlike infections of HeLa cells adenovirus infection of ciliated airway epithelia was quite limited and that which did occur w...

Adeno-associated virus serotype 4 (AAV4) and AAV5 have different tropisms compared to AAV2 and to each other. We recently reported that ␣2-3 sialic acid is required for AAV5 binding and transduction. In this study, we characterized AAV4 binding and transduction and found it also binds sialic acid, but the specificity is significantly different from AAV5.

Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.

In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring ␤-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and G i /G o proteins, here we dissected the roles of G proteins and the adaptor proteins, β-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of β-arrestins to the cell membrane, an activating conformational change in β-arrestin, and β-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of β-arrestin1 to activated cytosolic phospholipase A 2 as well as β-arrestin1-dependent activation of cytosolic phospholipase A 2 and release of arachidonate, the precursor of prostaglandin D 2 and the vasodilator responsible for the flushing response. Moreover, β-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by β-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a β-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

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