Cytotoxic T lymphocytes (CTLs) specific for conserved viral antigens can respond to different strains of virus, in contrast to antibodies, which are generally strain-specific. The generation of such CTLs in vivo usually requires endogenous expression of the antigen, as occurs in the case of virus infection. To generate a viral antigen for presentation to the immune system without the limitations of direct peptide delivery or viral vectors, plasmid DNA encoding influenza A nucleoprotein was injected into the quadriceps of BALB/c mice. This resulted in the generation of nucleoprotein-specific CTLs and protection from a subsequent challenge with a heterologous strain of influenza A virus, as measured by decreased viral lung titers, inhibition of mass loss, and increased survival.

Adeno-associated virus 2 (AAV)-based vectors have gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy. Although AAV uses the ubiquitously expressed cell surface heparan sulfate proteoglycan (HSPG) as a receptor, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We demonstrate here that cell surface expression of HSPG alone is insufficient for AAV infection, and that AAV also requires human fibroblast growth factor receptor 1 (FGFR1) as a co-receptor for successful viral entry into the host cell. We document that cells that do not express either HSPG or FGFR1 fail to bind AAV and, consequently, are resistant to infection by AAV. These non-permissive cells are successfully transduced by AAV vectors after stable transfections with cDNAs encoding the murine HSPG and the human FGFR1. Furthermore, AAV infection of permissive cells, known to express both FGFR1 and the epidermal growth factor receptor, is abrogated by treatment of cells with basic fibroblast growth factor, but not with epidermal growth factor. The identification of FGFR1 as a co-receptor for AAV should provide new insights not only into its role in the life cycle of AAV, but also in the optimal use of AAV vectors in human gene therapy.

We focus on the mechanism by which MyoD activates transcription. Previous experiments showed that when the 13-amino-acid basic region of El2 replaced the corresponding basic region of MyoD, the resulting MyoD-E12Basic chimeric protein could bind specifically to muscle-specific enhancers in vitro and form dimers with El2, but could not activate a cotransfected reporter gene or convert 10T1A cells to muscle. Here we show that back mutation of this chimeric protein (with the corresponding residues in MyoD) re-establishes activation, and we identify a specific alanine involved in increasing DNA binding and a specific threonine required for activation. Using a reporter gene containing MyoD-binding sites located downstream from the transcription start site, we show that MyoD-E12Basic can bind in vivo and thereby inhibit expression of the reporter. In vivo binding is also supported by the fact that the addition of the "constitutive" VPI6 activation domain to MyoD-E12Basic restores full trans-activation potential. The normal MyoD-activation domain maps within the amino-terminal 53 residues and can be functionally replaced by the activation domain of VP16. The activity of the MyoD-activation domain is dramatically elevated when deletions are made almost anywhere in the rest of the MyoD molecule, suggesting that the activation domain in MyoD is usually masked. Surprisingly, MyoD-E12Basic can activate transcription in CV1 and B78 cells (but not in 10T1A or 3T3 cells), suggesting that the activation function of the basic domain requires a specific factor present in CV1 and B78 cells. We propose that to function, the masked MyoD-activation domain requires the participation of a second factor that recognizes the basic region. We refer to such a factor as a recognition factor.

No abstract

Direct injection of nonviral, covalently closed circular plasmid DNA into muscle results in expression of the DNA in myofiber cells. We have examined the expression of firefly luciferase DNA constructs injected into adult murine skeletal muscle. Considerable variation in luciferase enzyme expression was noted among constructs with different regulatory elements, among different batches of the same DNA construct, and among similar transfection experiments performed at different times. This variation was minimized by using single batches of plasmid DNA and by performing comparable sets of experiments concurrently. A quantitative experimental protocol was defined for comparing various aspects of the transfection process. We report that a luciferase construct containing the human cytomegalovirus immediate-early gene promoter plus intron A (a construct termed "p-CMVint-lux") showed the highest expression among several constructs tested. Dose-response and time course analyses of p-CMVint-lux DNA injections showed that maximal luciferase expression was achieved with 25 micrograms of DNA at 7-14 days post-injection. Selected manipulations of the transfection process were examined for their influence on luciferase expression. Variations in the rate of DNA injection, needle size, injection volume, and vehicle temperature had no significant effect on luciferase expression. The presence of endotoxin, cationic peptide, muscle stimulants or relaxants, vasoconstrictors, metal chelators, or lysosomal lytic reagents had no significant effect on expression. However, linearization of the DNA, injection of the DNA in water rather than saline, or inclusion of a DNA intercalating agent nearly abolished luciferase expression. And finally, increasing the injection dose by giving multiple injections over a 10-day period increased expression proportionally to the number of injections.