Biology subjects

Bobak, C.

Publications and source records attributed to Bobak, C..

4 recordsLinked to original sources

bioRxiv2025

Human cytomegalovirus regulates host DNA repair machinery for viral genome integrity

The DNA damage response (DDR) encompasses a multitude of interconnected pathways that serve as a cellular defense to protect genome integrity. Dysregulation or failure of these pathways results in cancers and genetic disease. DNA viruses, including the herpesvirus cytomegalovirus (CMV), activate DDR signaling during their replicative program. The mechanisms by which they commandeer these responses for replication of their genome is less well understood. Here, we define a viral protein, UL138, that modulates the activity of host DDR pathways. The loss of UL138 results in structural variants, including inversion, deletions, duplication, with signature of homology-directed repair and other DDR pathways. The actions of UL138 are due, in part, to its modulation of pathways regulated by the cellular deubiquitinating complex that targets proliferating cell nuclear antigen (PCNA) and Fanconi Anemia effectors, FANCD2 and FANCI. However, we also show that UL138 accesses pathways independent of USP1-PCNA/FANCD2/FANCI. Disruption of UL138 or these pathways impacts viral genome replication and had consequences for viral genome integrity. This work provides mechanistic insight into the long-standing questions of how DNA viruses recruit, modulate and use cellular DDR pathways. It also puts forth CMV as a model system for further defining these pathways in human cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/683233v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@121e55borg.highwire.dtl.DTLVardef@58d8b7org.highwire.dtl.DTLVardef@fc1628org.highwire.dtl.DTLVardef@12b32a_HPS_FORMAT_FIGEXP M_FIG C_FIG Major findingsO_LIUL138 protects HCMV genome integrity through its interaction with USP1-PCNA/FANCD2/FANCI and repeat sequence-mediated pathways C_LIO_LIUL138 protects HCMV genome integrity in the UL region by mechanisms independent of repeat sequences and USP1-PCNA/FANCD2/FANCI C_LIO_LICollectively, US and UL regions of the HCMV genome have fundamentally different pathways acting on them and UL138 modulates DNA damage and repair pathway choice. C_LI

microbiology↗

bioRxiv2025

Complex roles for proliferating cell nuclear antigen in human cytomegalovirus replication

DNA viruses at once elicit and commandeer host pathways, including DNA repair pathways for virus replication. Despite encoding its own DNA polymerase and processivity factor, human cytomegalovirus (HCMV) recruits the cellular processivity factor, proliferating cell nuclear antigen (PCNA) and specialized host DNA polymerases involved in translesion synthesis (TLS) to replication compartments (RCs) where viral DNA (vDNA) is synthesized. While the recruitment of TLS polymerases is important for viral genome stability, the role of PCNA is poorly understood. PCNA function in DNA repair is regulated by monoubiquitination (mUb) or SUMOylation of PCNA at lysine 164 (K164). We find that mUb-PCNA increases over the course of infection, and modification of K164 is required for PCNA-mediated restriction of virus replication. mUb-PCNA plays important known roles in recruiting TLS polymerases to DNA, which we have shown are important for viral genome integrity and diversity, represented by novel junctions and single nucleotide variants (SNVs), respectively. We find that PCNA drives SNVs on vDNA similar to Y-family TLS polymerases, but that this did not require modification at K164. Unlike TLS polymerases, PCNA was dispensable for preventing large scale rearrangements on vDNA. These striking results suggest separable PCNA-dependent and - independent functions of TLS polymerases on vDNA. By extension, these results imply roles for TLS polymerase beyond their canonical function in TLS in host biology. These findings highlight PCNA as a complex restriction factor for HCMV infection, likely with multiple distinct roles, and provides new insights into the PCNA-mediated regulation of DNA synthesis and repair in viral infection. IMPORTANCEGenome synthesis is a critical step of virus life cycles and a major target of antiviral drugs. Human cytomegalovirus, like other herpesviruses, encodes machinery sufficient for viral DNA synthesis and relies on host factors for efficient replication. We have shown that host DNA repair factors play important roles in HCMV replication, but our understanding of this is incomplete. Building on previous findings that specialized host DNA polymerases contribute to HCMV genome integrity and diversity, we sought to determine the importance of PCNA, the central polymerase regulator. PCNA associates with nascent viral DNA and restricts HCMV replication. While PCNA is dispensable for genome integrity, it contributes to genome diversity. Our findings suggest that host polymerases function on viral genomes by separable PCNA-dependent and - independent mechanisms. Through revealing complex roles for PCNA in HCMV replication, this study expands the repertoire of host DNA synthesis and repair proteins hijacked by this ubiquitous herpesvirus.

microbiology↗

bioRxiv2021

Estimating the Inter- and Intra-Rater Reliability for NASH Fibrosis Staging in the Presence of Bridge Ordinal Ratings with Hierarchical Bridge Category Models

The public health burden of non-alcoholic steatohepatitis (NASH), a liver condition characterized by excessive lipid accumulation and subsequent tissue inflammation and fibrosis, has burgeoned with the spread of western lifestyle habits. Progression of fibrosis into cirrhosis is assessed using histological staging scales (e.g., NASH Clinical Research Network (NASH CRN)). These scales are used to monitor disease progression as well as to evaluate the effectiveness of therapies. However, clinical drug trials for NASH are typically underpowered due to lower than expected inter-/intra-rater reliability, which impacts measurements at screening, baseline, and endpoint. Bridge ratings represent a phenomenon where pathologists assign two adjacent stages simultaneously during assessment and may further complicate these analyses when ad hoc procedures are applied. Statistical techniques, dubbed Bridge Category Models, have been developed to account for bridge ratings, but not for the scenario where multiple pathologists assess biopsies across time points. Here, we develop hierarchical Bayesian extensions for these statistical methods to account for repeat observations and use these methods to assess the impact of bridge ratings on the inter-/intra-rater reliability of the NASH CRN staging scale. We also report on how pathologists may differ in their assignment of bridge ratings to highlight different staging practices. Our findings suggest that Bridge Category Models can capture additional fibrosis staging heterogeneity with greater precision, which translates to potentially higher reliability estimates in contrast to the information lost through ad hoc approaches.

pathology↗

bioRxiv2021

Mixed Effects Machine Learning Models for Colon Cancer Metastasis Prediction using Spatially Localized Immuno-Oncology Markers

Spatially resolved characterization of the transcriptome and proteome promises to provide further clarity on cancer pathogenesis and etiology, which may inform future clinical practice through classifier development for clinical outcomes. However, batch effects may potentially obscure the ability of machine learning methods to derive complex associations within spatial omics data. Profiling thirty-five stage three colon cancer patients using the GeoMX Digital Spatial Profiler, we found that mixed-effects machine learning (MEML) methods{dagger} may provide utility for overcoming significant batch effects to communicate key and complex disease associations from spatial information. These results point to further exploration and application of MEML methods within the spatial omics algorithm development life cycle for clinical deployment.

pathology↗