Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be ␤-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Long non-coding RNA HOTAIR was regarded as an oncogene in multiple cancers. Previous studies have shown that HOTAIR is involved in the proliferation and tumorigenesis of renal carcinoma cells, while microRNA (miR)-217 functions as a tumor suppressor in renal cell carcinoma (Rcc). However, the underlying molecular mechanism of HOTAIR in Rcc, especially in association with miR-217, has not been studied. In this study, we first demonstrated that HOTAIR expression was upregulated, which was correlated with tumor progression, and miR-217 downregulated in Rcc tissues and cells. Importantly, HOTAIR expression was negatively correlated with miR-217 expression in Rcc tissues. Gain- and loss-of-function of HOTAIR revealed that HOTAIR functioned as a ceRNA for miR-217 to facilitate HIF-1α expression and then upregulated AXL level promoting Rcc proliferation, migration, and EMT process, and inhibiting apoptosis. Furthermore, HOTAIR knockdown suppressed tumor growth and reduced the expression of proliferation antigen ki-67, HIF-1α, and AXL, but upregulated the expression of miR-217 in vivo. Finally, with AXL inhibitor BGB324, we confirmed that HOTAIR promoted Rcc activity through AXL signaling both in vitro and in vivo. In conclusion, these results suggest that HOTAIR promotes Rcc tumorigenesis via miR-217/HIF-1α/AXL signaling, which may provide a new target for the diagnosis and therapy of Rcc disease.

As age increases, the risk of developing type 2 diabetes increases, which is associated with senile skeletal muscle dysfunction. During skeletal muscle aging, mitochondrial dysfunction, intramyocellular lipid accumulation, increased inflammation, oxidative stress, modified activity of insulin sensitivity regulatory enzymes, endoplasmic reticulum stress, decreased autophagy, sarcopenia and over-activated renin-angiotensin system may occur. These changes can impair skeletal muscle insulin sensitivity and increase the risk of insulin resistance and type 2 diabetes during skeletal muscle aging. This review of the mechanism of the increased risk of insulin resistance during skeletal muscle aging will provide a more comprehensive explanation for the increased incidence of type 2 diabetes in elderly individuals, and will also provide a more comprehensive perspective for the prevention and treatment of type 2 diabetes in elderly populations.

We examined cytoplasmic trafficking and nuclear translocation of adeno-associated virus type 2 (AAV) by using Alexa Fluor 488-conjugated wild-type AAV, A20 monoclonal antibody immunocytochemistry, and subcellular fractionation techniques followed by DNA hybridization. Our results indicated that in the absence of adenovirus (Ad), AAV enters the cell rapidly and escapes from early endosomes with a t 1/2 of about 10 min postinfection. Cytoplasmically distributed AAV accumulated around the nucleus and persisted perinuclearly for 16 to 24 h. Viral uncoating occurred before or during nuclear entry beginning about 12 h postinfection, when viral protein and DNA were readily detected in the nucleus. Few, if any, intact AAV capsids were found in the nucleus. In the presence of Ad, however, cytoplasmic AAV quickly translocated into the nucleus as intact particles as early as 40 min after coinfection, and this facilitated nuclear translocation of AAV was not blocked by the nuclear pore complex inhibitor thapsigargan. The rapid nuclear translocation of intact AAV capsids in the presence of Ad suggested that one or more Ad capsid proteins might be altering trafficking. Indeed, coinfection with empty Ad capsids also resulted in the appearance of AAV DNA in nuclei within 40 min. Escape from early endosomes did not seem to be affected by Ad coinfection.