Herpes Simplex Virus Type 1 Genomes Are Associated with ND10 Nuclear Substructures in Quiescently Infected Human Fibroblasts

Abstract

Herpes simplex virus type 1 (HSV-1) genomes become associated with structures related to cellular nuclear substructures known as ND10 or promyelocytic leukemia nuclear bodies during the early stages of lytic infection. This paper describes the relationship between HSV-1 genomes and ND10 in human fibroblasts that maintain the viral genomes in a quiescent state. We report that quiescent HSV-1 genomes detected by fluorescence in situ hybridization (FISH) are associated with enlarged ND10-like structures, frequently such that the FISH-defined viral foci are apparently enveloped within a sphere of PML and other ND10 proteins. The number of FISH viral foci in each quiescently infected cell is concordant with the input multiplicity of infection, with each structure containing no more than a small number of viral genomes. A proportion of the enlarged ND10-like foci in quiescently infected cells contain accumulations of the heterochromatin protein HP1 but not other common markers of heterochromatin such as histone H3 di-or trimethylated on lysine residue 9. Many of the virally induced enlarged ND10-like structures also contain concentrations of conjugated ubiquitin. Quiescent infections can be established in cells that are highly depleted for PML. However, during the initial stages of establishment of a quiescent infection in such cells, other ND10 proteins (Sp100, hDaxx, and ATRX) are recruited into virally induced foci that are likely to be associated with HSV-1 genomes. These observations illustrate that the intimate connections between HSV-1 genomes and ND10 that occur during lytic infection also extend to quiescent infections.Herpes simplex virus type 1 (HSV-1) is an important human pathogen that establishes a latent state in neuronal cells after a primary infection in epithelial tissues (for a general review, see reference 44). During latency, transcription of the great majority of the viral genome is repressed and only a family of related latency-associated transcripts is detectable (39,58). This contrasts with lytic infection, during which the whole viral genome is transcriptionally active, expressing a large number of genes that can be grouped into immediate-early (IE), early, and late classes according to the time course of their expression. The mechanisms that regulate the balance between lytic and latent infection are of intense interest because latency ensures that infected individuals retain the virus for life, allowing periodic episodes of reactivation and the ensuing clinical symptoms.Transcription of the HSV-1 genome is controlled primarily by three viral proteins. VP16 is a virion component that activates the expression of IE genes, IE protein ICP4 is essential for the transcription of E and L genes, and IE protein ICP0 is very important in certain cell types for initiation of the viral transcription program. Viruses with mutations that inactivate VP16 and/or ICP0 do not enter productive replication after low-multiplicity infection of human fibroblasts and certain other cultured cells but instead are re...

Probing PML body function in ALT cells reveals spatiotemporal requirements for telomere recombination

“…Nonetheless, these interactions display morphological characteristics that strongly resemble depicted associations of PML with foreign viral DNA (1,30) or with hypomethylated heterochromatic DNA sequences (14). In these cases, and in contrast to telomeres clusters in APBs, the PML protein engulfs the DNA rather than the latter being associated with the surface of the PML body.…”

Promyelocytic Leukemia-Nuclear Body Proteins: Herpesvirus Enemies, Accomplices, or Both?

“…Herpesviral genomes are found to be associated with PML-NBs in the absence of viral gene expression and at early times after infection [10,22,23]. Only HCMV genomes located near PML-NBs appeared to be transcriptionally active, leading to the hypothesis that PML-NBs represented positively acting, pre-existing, sub-nuclear sites that provided a structural environment conducive for viral gene expression [22], and perhaps viral DNA replication as well.…”

“…However, using quiescent viral genomes, which lack functional VP16, ICP0 and ICP4, the role of PML-NBs during quiescence in fibroblasts has been investigated [23]. These three viral proteins are the principal viral transcriptional transactivators necessary for the initiation of lytic viral replication.…”

“…Infection of fibroblasts with the quiescent virus leads to the assembly of new PML-NBs around the infecting viral genome, which last for many days postinfection [23]. In the absence of PML, Sp100 and Daxx initially co-localize with the viral genome, but do not remain associated with it for extended periods of time postinfection [23]. A caveat of the HSV-1 quiescence experiments as a model for the establishment of latency is the absence of three functional viral transactivators, especially VP16, which stimulates IE gene expression through a mechanism that may not be related to PML-NB proteins.…”

“…These three viral proteins are the principal viral transcriptional transactivators necessary for the initiation of lytic viral replication. Infection of fibroblasts with the quiescent virus leads to the assembly of new PML-NBs around the infecting viral genome, which last for many days postinfection [23]. In the absence of PML, Sp100 and Daxx initially co-localize with the viral genome, but do not remain associated with it for extended periods of time postinfection [23].…”