Structural variability of the herpes simplex virus 1 genome in vitro and in vivo.

Herpes simplex virus 1 (HSV-1) is a human pathogen that leads to recurrent facial-oral lesions. Its 152-kb genome is organized in two covalently linked segments, each composed of a unique sequence flanked by inverted repeats. Replication of the HSV-1 genome produces concatemeric molecules in which homologous recombination events occur between the inverted repeats. This mechanism leads to four genome isomers (termed P, IS, IL, and ILS) that differ in the relative orientations of their unique fragments. Molecular combing analysis was performed on DNA extracted from viral particles and BSR, Vero, COS-7, and Neuro-2a cells infected with either strain SC16 or KOS of HSV-1, as well as from tissues of experimentally infected mice. Using fluorescence hybridization, isomers were repeatedly detected and distinguished and were accompanied by a large proportion of noncanonical forms (40%). In both cell and viral-particle extracts, the distributions of the four isomers were statistically equivalent, except for strain KOS grown in Vero and Neuro-2a cells, in which P and IS isomers were significantly overrepresented. In infected cell extracts, concatemeric molecules as long as 10 genome equivalents were detected, among which, strikingly, the isomer distributions were equivalent, suggesting that any such imbalance may occur during encapsidation. In vivo, for strain KOS-infected trigeminal ganglia, an unbalanced distribution distinct from the one in vitro was observed, along with a considerable proportion of noncanonical assortment.

Meganuclease-mediated Inhibition of HSV1 Infection in Cultured Cells.

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.

Intracellular targeting of the type-I alpha regulatory subunit of cAMP-dependent protein kinase.

The specificity of cyclic adenosine monophosphate (cAMP)-mediated signaling events is achieved by the composition and biochemical properties of the different cAMP-dependent protein kinase holoenzymes (PKAI and II) and by compartmentalization of PKA to discrete subcellular locations. Intracellular localization is mediated by interaction with A-kinase anchoring proteins (AKAPs) that recruit PKAII close to its substrates and to sites where it can respond optimally to local changes in intracellular cAMP concentration, thereby directing and amplifying the effects of cAMP. This review presents recent evidence that indicates that specific AKAPs mediate PKAI anchoring through interaction with its regulatory subunit RI alpha, notably at the neuromuscular junction of skeletal muscle.