Biology subjects

Mohr, I.

Publications and source records attributed to Mohr, I..

11 recordsLinked to original sources

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↗

bioRxiv2023

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1) and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent virus is also present in immune cells recovered from ganglia in a mouse model used for studying latency. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for this conclusion. Unexpectedly, off-target priming in 3 scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained by inaccuracies in the data analyses.

microbiology↗

bioRxiv2023

Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN5

Small GTPases function by conformational switching ability between GDP- and GTP-bound states in rapid cell signaling events. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. Two TTN5 variants were included in the study with point mutations at conserved residues, suspected to be functional for nucleotide exchange and GTP hydrolysis, TTN5T30N and TTN5Q70L. We found that TTN5 had a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism. TTN5 acted as a non-classical small GTPase with a remarkably low GTP hydrolysis activity, suggesting it is likely present in GTP-loaded active form in the cell. We analyzed signals from yellow fluorescent protein (YFP)-tagged TTN5 and from in situ immunolocalization of hemagglutine-tagged HA3-TTN5 in Arabidopsis seedlings and in a transient expression system. Together with colocalization using endomembrane markers and pharmacological treatments the microscopic analysis suggests that TTN5 can be present at the plasma membrane and dynamically associated with membranes of vesicles, Golgi stacks and multivesicular bodies. While the TTN5Q70L variant showed similar GTPase activities and localization behavior as wild-type TTN5, the TTN5T30N mutant differed in some aspects. Hence, the unusual capacity of rapid nucleotide exchange activity of TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system. HighlightsO_LIThe small ARF-like GTPase TTN5 has a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism C_LIO_LIBiochemical data classified TTN5 as a non-classical small GTPase, likely present in GTP-loaded active form in the cell C_LIO_LIYFP-TTN5 is dynamically associated with vesicle transport and different processes of the endomembrane system, requiring the active form of TTN5 C_LI

plant biology↗

bioRxiv2023

Small ADP-ribosylation factor-like GTPase TITAN5 (TTN5/ARL2) is linked with the dynamic regulation of IRON-REGULATED TRANSPORTER1

Iron acquisition is crucial for plants. The abundance of IRON-REGULATED TRANSPORTER 1 (IRT1) at the plasma membrane is controlled through endomembrane trafficking. Vesicular trafficking requires small ARF-like GTPases, one of them is TITAN 5 (TTN5). Its physiological functions during the life cycle and cellular targets remain unknown. Little is known how the vesicular trafficking mechanism affects IRT1 localization. We show that TTN5 interacts with the large cytoplasmic variable region and protein-regulatory platform of IRT1. ttn5-1+/- plants have a reduced activity of root iron reductase, needed for iron uptake via IRT1. Fluorescent fusion proteins of TTN5 and IRT1 colocalize at the plasma membrane and in endosomes/multivesicular bodies, where IRT1 sorting and cycling between the plasma membrane and the vacuole are coordinated. Colocalization at the plasma membrane depends partly on the interaction ability of TTN5. TTN5 can also interact with peripheral membrane proteins that are components of the IRT1 regulation machinery, like the trafficking factor SNX1, the C2 domain protein EHB1 and the SEC14-GOLD protein PATL2. Hence, this work links iron acquisition and vesicular trafficking involving a small GTPase of the ARF family. This opens up the possibility to study the involvement of TTN5 in nutritional cell biology in the endomembrane system. HighlightsO_LITTN5 interacts with the large intracellular loop and variable region of IRON-REGULATED TRANSPORTER 1 (IRT1) C_LIO_LITTN5 has a positive effect on root iron (Fe) reductase activity. C_LIO_LITTN5 and IRT1 colocalize at the plasma membrane and in the endomembrane system related to vesicle transport C_LIO_LITTN5 can interact with peripheral membrane proteins of the IRT1 interactome, EHB1, PATL2 and SNX1 suggesting a coordinating role in IRT1 regulation C_LI One-sentence summaryTTN5, a small ARF-like GTPase, is connected to the dynamic regulation of IRON-REGULATED TRANSPORTER 1 (IRT1) in the vesicular trafficking system through direct protein interaction and colocalization, linking with various peripheral membrane proteins of the IRT1 interactome and iron reductase activity.

plant biology↗

bioRxiv2021

Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing

The mRNA 5 cap structure serves both to protect transcripts from degradation and promote their translation. Cap removal is thus an integral component of mRNA turnover that is carried out by cellular decapping enzymes, whose activity is tightly regulated and coupled to other stages of the mRNA decay pathway. The poxvirus vaccinia virus (VACV) encodes its own decapping enzymes, D9 and D10, that act on cellular and viral mRNA, but may be regulated differently than their cellular counterparts. Here, we evaluated the targeting potential of these viral enzymes using RNA sequencing from cells infected with wild-type and decapping mutant versions of VACV as well as in uninfected cells expressing D10. We found that D9 and D10 target an overlapping subset of viral transcripts but that D10 plays a dominant role in depleting the vast majority of human transcripts, although not in an indiscriminate manner. Unexpectedly, the splicing architecture of a gene influences how robustly its corresponding transcript is targeted by D10, as transcripts derived from intronless genes are less susceptible to enzymatic decapping by D10. As all VACV genes are intronless, preferential decapping of transcripts from intron-encoding genes provides an unanticipated mechanism for the virus to disproportionately deplete host transcripts and remodel the infected cell transcriptome.

microbiology↗

bioRxiv2020

Subjugating Translational Inhibition in Response to Ribosomal Protein Insufficiency by a Herpesvirus Ribosome-Associated Protein

In addition to being required for protein synthesis, ribosomes and ribosomal proteins (RPs) also regulate mRNA translation in uninfected and virus-infected cells. By individually depleting 85 RPs using RNAi, we found overall protein synthesis in uninfected primary fibroblasts was more sensitive to RP-depletion than those infected with herpes simplex virus-1 (HSV-1). Although representative RP-depletion (uL3, uS4, uL5) inhibited protein synthesis in cells infected with other DNA viruses, HSV-1-infected cell protein synthesis unexpectedly endured and required a single virus-encoded gene product, VP22. During individual RP-insufficiency, VP22-expressing HSV-1 replicated better than a VP22-deficient variant. Furthermore, VP22 cosedimented with ribosomes and polyribosomes in infected cells. This identifies VP22 as a virus-encoded, polyribosome-associated protein that compensates for RP-insufficiency to support viral protein synthesis and replication. Moreover, it reveals an unanticipated class of virus-encoded, ribosome-associated effectors that reduce the dependence of protein synthesis upon RPs and broadly support translation during physiological stress such as infection.

microbiology↗

bioRxiv2019

The cohesin loader NIPBL interacts with pre-ribosomal RNA and treacle to regulate ribosomal RNA synthesis

NIPBL is an essential loader of cohesin to mediate sister chromatid cohesion and chromatin loop organization. NIPBL mutations cause Cornelia de Lange Syndrome. How NIPBLs genomic localization is specified is not fully understood. We found that NIPBL localizes to the nucleolus in an RNA-dependent manner and binds directly to ribosomal RNA (rRNA). We identified two RNA binding domains in NIPBL in vitro, both of which are required for efficient rRNA binding in vivo. NIPBL binds to ribosomal DNA (rDNA) in an RNA-stimulated manner, recruits PAF1 and promotes pre-rRNA transcription. Stress that inhibits rRNA synthesis displaces NIPBL from the nucleolus and rDNA. Interestingly, treacle, mutated in Treacher Collins syndrome, tightly binds to and recruits NIPBL to the nucleolus, nucleolar organizer regions, and the stress-induced nucleolar cap. The results reveal that a subpopulation of NIPBL is recruited to the nucleolus through its interaction with RNA and treacle and regulates pre-rRNA transcription.

molecular biology↗

bioRxiv2019

Regulation of HMGB2 integrates ribosome biogenesis and innate immune responses to DNA

Ribosomes are universally important in biology and their production is dysregulated by developmental disorders, cancer, and virus infection. Although presumed required for protein synthesis, how ribosome biogenesis impacts virus reproduction and cell-intrinsic immune responses remains untested. Surprisingly, we find that restricting ribosome biogenesis stimulated human cytomegalovirus (HCMV) replication without suppressing translation. Interfering with ribosomal RNA (rRNA) accumulation triggered nucleolar stress and repressed expression of High Mobility Group Box 2 (HMGB2), a chromatin-associated protein that facilitates cytoplasmic double-stranded (ds) DNA-sensing by cGAS. Furthermore, it reduced cytoplasmic HMGB2 abundance and impaired induction of interferon beta (IFNB1) mRNA, which encodes a critical anti-proliferative, proinflammatory cytokine, in response to HCMV or dsDNA in uninfected cells. This establishes that rRNA accumulation regulates innate immune responses to dsDNA by controlling HMGB2 abundance. Moreover, it reveals that rRNA accumulation and/or nucleolar activity unexpectedly regulate dsDNA-sensing to restrict virus reproduction and regulate inflammation.

microbiology↗

bioRxiv2019

Topoisomerase 2-dependent nuclear DNA damage shapes extracellular growth factor responses through AKT phosphorylation dynamics to control virus latency

The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2{beta}-DNA cleavage complex (TOP2{beta}cc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA repair pathways that remove TOP2{beta}cc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.

molecular biology↗

bioRxiv2018

Native RNA sequencing on nanopore arrays redefines the transcriptional complexity of a viral pathogen

Viral genomes exhibit a higher gene density and more diversified transcriptome than the host cell. Coding potential is maximized through the use of multiple reading frames, placement of genes on opposing strands, inefficient or modified use of termination signals, and the deployment of complex alternative splicing patterns. As a consequence, detailed characterization of viral transcriptomes by conventional methods can be challenging. Full length native RNA sequencing (nRNA-seq) using nanopore arrays offers an exciting alternative. Individual transcripts are sequenced directly, without the biases inherent to the recoding or amplification steps included in other sequencing methodologies. nRNA-seq simplifies the detection of variation brought about by RNA splicing, use of alternative transcription initiation and termination sites, and other RNA modifications. Here we use nRNA-seq to profile the herpes simplex virus type 1 transcriptome during early and late stages of productive infection of primary cells. We demonstrate the effectiveness of the approach and identify a novel class of intergenic transcripts, including an mRNA that accumulates late in infection that codes for a novel fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L.

microbiology↗