Structural Variability of the Herpes Simplex Virus 1 Genome In Vitro and In Vivo

Abstract

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.

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies

“…Based on the technique of molecular combing, structural variances and non-canonical genomes that incorporate internal duplications, deletions or rearrangements are shown to be present in HHV-1 cultures [ 50 ]. Here we identify these events, based on non-canonical contigs that are generated from the reference-free, de novo assembly of 454 and MinION reads.…”

Recombination Analysis of Herpes Simplex Virus 1 Reveals a Bias toward GC Content and the Inverted Repeat Regions

“…The genomic segments invert with four possible, equivalent arrangements (20); however, a more recent report suggests that the effects of both viral strain and cell type may result in an imbalanced isomeric ratio (21). Replication is initiated from the origins of replication: OriL in the UL segment and two copies of OriS in the inverted repeats flanking the US region (22,23).…”

Recombination between co-infecting herpesviruses occurs where replication compartments coalesce

“…These results suggest that some color mixing can occur without recombining both tag sequences into the same genome. Alternatively they could reflect non-HR events during replication, which were previously observed both in HSV-1 replication (58) and in other herpesviruses (59). These explanations are not mutually exclusive, and both may contribute to the mixing of colors observed without HR.…”

“…This supports the hypothesis that recombination events occur between replicating genomes that are in either branched or circular concatemeric states (7). Since HSV-1 maintains the 4 genome isomers at equivalent distributions (58), the 105Kbp distance between the markers is actually ~50Kbp in half of the concatemeric forms. This may explain the inability to distinguish differences in recombination rates when the markers are not closely linked, as previously suggested (7).…”