dHerpes 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.

Although important shifts in the isoelectric point of prokaryotic proteins, mainly due to adaptation to environmental pH, have been widely reported, such studies have not covered mammalian proteins, where pH changes may relate to changes in subcellular or tissue compartmentalization. We explored the isoelectric point of the proteome of 13 mammalian species. We detected proteins that have shifted their pI the most among 13 mammalian species, and investigated if these differences reflect adaptations of the orthologous proteins to different conditions. We find that proteins exhibiting a high isoelectric point change are enriched in certain GO terms, including immune defense, and mitochondrial proteins. We show that the shift in pI between orthologous proteins is not strongly associated with the overall rate of protein evolution, nor with protein length. Our results reveal that insertions/deletions are the main reason behind the shift of pI. However, for some proteins we find evidence of selection shifting the pI of the protein through amino acid replacement. Finally, we argue that shifts in pI might relate to the gain of additional activities, such as new interacting partners, in one ortholog as opposed to the other, and may potentially relate to functional differences between mammals.