Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides

Nuclear domain 10 as pre-existing potential replication start sites of herpes simplex virus type-1

The herpes simplex virus type 1 (HSV-1) nuclear replication cycle begins at localized sites, but it has remained unclear whether these sites are associated with any defined nuclear structure. We have previously shown that during infection, the HSV-1 immediate-early protein ICP0 dispersed proteins associated with ND10, nuclear sites that contain high concentrations of PML and other potentially regulatory proteins and correspond to the ultrastructurally defined nuclear bodies. Usingin situhybridization and immunohistochemical techniques, we found that ICP0 mutants of HSV-1 replicate in the close proximity with ND10, but increasing replication sites develop away from these nuclear structures. Input wild-type HSV-1 DNA was found preferentially adjacent to ND10 before ICP0 modified these nuclear structures and did not colocalize with ICP8 containing so-called prereplication sites. The sites where HSV-1 can begin replication then need to be redefined as preexisting potential replication sites. Viral RNA was also found associated with ND10 before early protein synthesis (ICP8), suggesting that input virus genomes are deposited at ND10 before they start replication. The deposition of input viral DNA at ND10 is virus gene expression independent, probably indicating cell regulation of this process. Taken together, these data demonstrate that some very early processes of the nuclear viral replication cycle happen in close proximity or at the periphery of ND10. The localization of input HSV-1 to ND10 represents a new host–virus interaction and provides an unexpected functional property for this large nuclear site