No abstract

Herpes simplex virus type 1 (HSV1) is a major health problem. As for most viral diseases, current antiviral treatments are based on the inhibition of viral replication once it has already started. As a consequence, they impair neither the viral cycle at its early stages nor the latent form of the virus, and thus cannot be considered as real preventive treatments. Latent HSV1 virus could be addressed by rare cutting endonucleases, such as meganucleases. With the aim of a proof of concept study, we generated several meganucleases recognizing HSV1 sequences, and assessed their antiviral activity in cultured cells. We demonstrate that expression of these proteins in African green monkey kidney fibroblast (COS-7) and BSR cells inhibits infection by HSV1, at low and moderate multiplicities of infection (MOIs), inducing a significant reduction of the viral load. Furthermore, the remaining viral genomes display a high rate of mutation (up to 16%) at the meganuclease cleavage site, consistent with a mechanism of action based on the cleavage of the viral genome. This specific mechanism of action qualifies meganucleases as an alternative class of antiviral agent, with the potential to address replicative as well as latent DNA viral forms.

In skeletal muscle, transcription of the gene encoding the mouse type I␣ (RI␣) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (USF) to E1. E2 binds either an unknown protein in a USF͞E1 complex-dependent manner or MyoD. Both E2-bound proteins seem to function as repressors, but with different strengths, of the USF transactivation potential. Previous work has shown localization of the RI␣ protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RI␣ or RII␣ fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RI␣ subunits and requires the amino-terminal residues 1-81. Mutagenesis of Phe-54 to Ala in the full-length RI␣-green fluorescent protein template abolishes localization, indicating that dimerization of RI␣ is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RI␣ at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type I␣ homologue RCE with AKAPCE and for in vitro binding of RI␣ to dual A-kinase anchoring protein 1. We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RI␣ tethering at this site.S ubcellular localization is a crucial mechanism to achieve optimal activation and substrate specificity of the cAMPdependent protein kinase (PKA, EC 2.7.1.37). PKA type II targeting to subcellular structures and organelles or assembly in signaling complexes is a result of tethering of RII regulatory (R) subunits by members of the A-kinase anchoring protein (AKAP) family, a group of structurally divergent proteins possessing a conserved RII-binding site (1, 2).Although a large proportion of type I␣ R subunit (RI␣) is dispersed in the cytosol, it also is associated with the plasma membrane of human erythrocytes (3), recruited to the ''cap site'' of activated T lymphocytes (4) and sequestered along the fibrous sheath of mammalian spermatozoa (5). In addition, we have demonstrated previously the accumulation of RI␣ at the neuromuscular junction (NMJ) of skeletal muscle (6) and its association with microtubules (7). The high affinity RII-binding sites of certain AKAPs, named dual (D)-AKAPs, also sequester RI␣ in vitro but with a 25-500 lower affinity (8, 9). Thus, type I PKA also could be anchored in intact cells through specific AKAP-RI␣ interactions. In fact, Angelo and Rubin (10) have identified AKAP CE from Caenorhabditis elegans, which binds the R CE subunit. Because R CE is closely related to mammalian RI␣, AKAP CE is the first eukaryotic RI-specific teth...