Herzog, N. L. | bioRxiv Science

bioRxiv2025

Transcriptional consequences of herpes simplex virus 1 ICP4 inducible expression in uninfected cells

Successful viral infections reflect the balanced outcome of a tightly regulated program of viral gene expression and manipulation of the host cell environment to favor production of new infectious particles. The productive (lytic) replication cycle of herpes simplex virus 1 (HSV-1) is dependent on the essential transcription factor ICP4 encoded by one of five immediate-early genes. At different steps in the HSV-1 temporal cascade, ICP4 either positively or negatively regulates transcription of immediate-early, early, and late HSV-1 genes, largely through sequence-specific binding to cis-acting regulatory elements. In contrast, the direct regulation of host transcription by ICP4 is less well understood. In this study we exogenously expressed doxycycline-inducible wild-type and mutant ICP4 proteins in uninfected primary human fibroblasts and performed RNA-Seq to identify ICP4-driven changes to the host transcriptome. Cross-referencing our findings to a published dataset of ICP4-dependent changes to the host transcriptome in HSV-1 infected cells provided validation for a subset of differentially-expressed genes regulated by ICP4. Furthermore, disrupting the ICP4 DNA-binding domain was sufficient to alter the cellular gene transcriptional program responsive to ICP4. This indicates that the DNA-binding domain of ICP4, which is required for site-specific DNA binding to the virus genome, may also regulate binding to the host genome. Together, these data provide a comprehensive transcriptomic analysis of how wild-type and mutant ICP4 expression impact cellular gene expression in uninfected cells. IMPORTANCEHerpes simplex virus 1 (HSV-1) is a widespread human pathogen responsible for a variety of mild to severe disease states. The viral lifecycle is driven by the essential viral transcription factor ICP4, which ensures the coordinated expression of most viral genes. Expression of ICP4-regulated genes and viral lifecycle progression impacts host gene expression; therefore, determining the specific impact of ICP4 on host gene expression has been challenging. Here, we provide a more direct analysis, examining changes in the transcriptome of primary fibroblasts when ICP4 is expressed alone. Differentially expressed genes were identified and cross-referenced to published data from infections with wild-type and ICP4-null viruses. We show that mutations in ICP4 that disrupt interactions with the host transcriptional machinery or interfere with site-specific DNA binding further modified this ICP4-driven remodeling of the host transcription. These data provide a comprehensive transcriptomic analysis of ICP4-driven dysregulation of host gene expression in uninfected cells.

microbiology↗

bioRxiv2025

Herpes simplex virus-1 fluidizes the nucleus enabling condensate formation

Molecular processes are profoundly influenced by the biophysical properties of the cell interior. However, the mechanisms that control these physical properties, and the processes they impact remain poorly understood, especially in the nucleus. We hypothesized that some viruses might change the biophysical properties of the nucleus to favor virus survival and replication and found that herpes simplex virus 1 (HSV-1) increases the mesoscale fluidity of the nucleus. The HSV-1 protein ICP4 caused fluidization and enabled growth of synthetic nuclear condensates. Conversely, conditions that decreased nuclear fluidity inhibited the growth of viral replication compartment condensates and reduced infectious virus production. Together, our data suggest that ICP4 increases nuclear fluidity to promote the formation of condensates that drive the progression of the HSV-1 life cycle. We speculate that a key function of ICP4 is to overcome the crowding and elastic confinement within cell nuclei that are a fundamental barrier to virus replication.

cell biology↗

bioRxiv2021

nucGEMs probe the biophysical properties of the nucleoplasm

The cell interior is highly crowded and far from thermodynamic equilibrium. This environment can dramatically impact molecular motion and assembly, and therefore influence subcellular organization and biochemical reaction rates. These effects depend strongly on length-scale, with the least information available at the important mesoscale (10-100 nanometers), which corresponds to the size of crucial regulatory molecules such as RNA polymerase II. It has been challenging to study the mesoscale physical properties of the nucleoplasm because previous methods were labor-intensive and perturbative. Here, we report nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs). Introduction of a single gene leads to continuous production and assembly of protein-based bright fluorescent nanoparticles of 40 nm diameter. We implemented nucGEMs in budding and fission yeast and in mammalian cell lines. We found differences in particle motility between the nucleus and the cytosol at the mesoscale, that mitotic chromosome condensation ejects nucGEMs from the nucleus, and that nucGEMs are excluded from heterochromatin and the nucleolus. nucGEMs enable hundreds of nuclear rheology experiments per hour, and allow evolutionary comparison of the physical properties of the cytosol and nucleoplasm.

biophysics↗