Biology subjects

Crawford, L. B.

Publications and source records attributed to Crawford, L. B..

6 recordsLinked to original sources

bioRxiv2026

Human Cytomegalovirus Infects and Remodels 3D-biofabricated Skin Equivalents

Viral infection, including for the prototypical betaherpesvirus human cytomegalovirus (HCMV) is typically studied in two-dimensional monocultures, which provide experimentally tractable systems for measuring viral replication but do not reproduce the biologically accurate multicellular organization or interactions, nor the three-dimensional (3D) architecture of stratified epithelial tissues. Here, we evaluated a biofabricated 3D skin model containing fibroblasts and keratinocytes as a system to study HCMV infection. The model is generated using a fibrin-based matrix and spatially organization deposition of keratinocytes in combination with fibroblasts, followed by cellular differentiation controlled by calcium conditions. Cultures were infected with a GFP-expressing clinical strain of HCMV (TB40/E-GFP) and compared to traditional monolayer cultures or single cell type 3D cultures. HCMV infection was detectable by GFP expression in an MOI-dependent and longitudinal manner. Infectious virus was recovered from both the cellular associated and released into the extracellular space of the model, demonstrating that the model supports productive viral infection. Transit of infectious virus is reduced in both multicellular and single type 3D cultures, suggesting that the matrix composition influences viral kinetics. Treatment with the antiviral Ganciclovir suppressed virus production in both fibroblast monolayer cultures and 3D skin cultures. These findings establish a tractable, longitudinal observable, multicellular 3D system that supports productive HCMV infection and provides a reliable platform for investigating viral replication in a spatially organized tissue context.

microbiology↗

bioRxiv2026

Human cytomegalovirus UL2 is not required for lytic replication or viral latency and reactivation

Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in transplant patients and is a major cause of congenital disease. Understanding the role of viral genes during infection, viral replication, and viral latency establishment and reactivation is key to the development of new antivirals, which are currently limited. The viral RL11 is a conserved, but understudied viral locus, with previously described roles in specific phases of the viral lifecycle. Antisense to and within the RL11 region are two unrelated genes, including UL2. In this study, we investigate the role of UL2 during viral infection and find that UL2 is not required for lytic replication nor required for viral infection, latency establishment (or persistence) and reactivation in either THP-1 monocytes or primary human hematopoietic progenitor cells (HPCs). As viral genes are evolutionarily optimized for viral fitness, this study suggests that HCMV UL2 has a role outside of viral fitness and may be a unique target or contributor to HCMV-mediated processes including immune response or other viral co-factor regulation.

microbiology↗

bioRxiv2026

Human Cytomegalovirus UL4 is Required for Viral Reactivation via Cellular Reprogramming in Latently Infected CD34+ Progenitor Cells and Humanized Mice

Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality after both solid organ and hematopoietic stem cell transplant, due to bone marrow stem cell engraftment failure, myelosuppression, and immunosuppression. While direct infection and viral replication lead to disease, latent infection in the CD34+ hematopoietic progenitor cell (HPC) pool has direct and indirect effects on hematopoiesis. Studies from several laboratories support the hypothesis that HCMV latency and reactivation are intrinsically linked with the state of the cell. We, and others, have previously demonstrated roles for different viral gene products in regulating both cellular differentiation and the balance between viral latency and reactivation, including the viral RL11 proteins UL7 and UL8. In this study, we show that UL4 is expressed during latency and required for reactivation in HPCs, THP-1 monocytes, and humanized mice. describe both a cell-specific and viral lifecycle-specific role for the RL11 gene UL4 during HCMV infection. Additionally, we demonstrate that UL4 plays specific roles in controlling essential cellular functions including aspects of proliferation and differentiation and immune signaling to assist in establishing a virus-favorable environment in progenitor cells. This study identifies a novel viral reactivation factor that has implications for both viral control and alteration of hematopoiesis in transplant patients.

microbiology↗

bioRxiv2026

MarkerScout: A Disease-Agnostic Machine Learning Framework for Biomarker Prediction from Multi-Scale Mechanistic Models

Identifying robust biomarkers from high-dimensional biomedical data is a central challenge in translational research, but candidate rankings produced by any single feature-selection or classification method depend on algorithmic choices and rarely reproduce across pipelines. We present a disease-agnostic machine-learning framework that addresses this dependence by systematically benchmarking 25 (feature-selection x classifier) pipelines under five-fold stratified cross-validation, aggregating per-feature evidence by two independent methods (a weighted-selection consensus score and Robust Rank Aggregation), and characterizing the direction of each candidate using Cohens d. We demonstrate the framework on immune-response measurements from two clinical phases: SARS-CoV-2 hospitalization and intensive-care admission; obtaining cross-validated mean F1 above 0.99 with balanced classification errors and producing tiered, direction-aware biomarker lists per phase. Interleukin-18 (IL-18) reached the strongest tier in both phases with consistent direction. The framework generalizes to any binary clinical classification problem and supports principled, reproducible biomarker prioritization.

bioinformatics↗

bioRxiv2020

Human cytomegalovirus UL7, miR-US5-1 and miR-UL112-3p inactivation of FOXO3a protects CD34+ hematopoietic progenitor cells from apoptosis

Human cytomegalovirus (HCMV) infection of myeloid-lineage cells, such as CD34+ hematopoietic progenitor cells (HPCs) or monocytes results in the upregulation of anti-apoptotic cellular proteins that protect the newly infected cells from programmed cell death. The mechanisms used by HCMV to regulate pro-apoptotic cellular proteins upon infection of CD34+ HPCs has not been fully explored. Here we show that HCMV utilizes pUL7, a secreted protein that signals through the FLT3 receptor, and miR-US5-1 and miR-UL112-3p to reduce the abundance and activity of the pro-apoptotic transcription factor FOXO3a at early times after infection of CD34+ HPCs. Regulation of FOXO3a by pUL7, miR-US5-1 and miR-UL112 results in reduced expression of the pro-apoptotic BCL2L11 transcript and protection of CD34+ HPCs from virus-induced apoptosis. These data highlight the importance of both viral proteins and miRNAs in protecting CD34+ HPCs from apoptosis at early times post-infection, allowing for the establishment of latency and maintenance of viral genome-containing cells. ImportanceHuman cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34+ hematopoietic progenitor cells (HPCs) and periodically reactivate to cause disease in the absence of an intact immune system. What viral gene products are required for successful establishment of latency are still not fully understood. Here we show that both a viral protein and viral miRNAs are required to prevent apoptosis after infection of CD34+ HPCs. HCMV pUL7 and miRNAs miR-US5-1 and miR-UL112-3p act to limit the expression and activation of the transcription factor FOXO3a which in turn reduces expression of pro-apoptotic gene BCL2L11 and prevents virus-induced apoptosis in CD34+ HPCs.

microbiology↗

bioRxiv2019

HCMV miR-US22 down-regulation of EGR-1 regulates CD34+ hematopoietic progenitor cell proliferation and viral reactivation

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation during latency. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus.\n\nAuthor summaryHuman cytomegalovirus (HCMV) is a widespread herpesvirus that persists in the host and remains a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is complex, and the molecular mechanisms for establishment, maintenance, and reactivation from latency are poorly understood.\n\nQuiescent stem cells in the bone marrow represent a critical reservoir of latent HCMV, and the mobilization and differentiation of these cells is closely linked to viral reactivation from latency. HCMV encodes small regulatory RNAs, called miRNAs that play important roles in the regulation of viral and cellular gene expression. In this study, we show that HCMV miR-US22 targets Early growth response gene 1 (EGR-1) a host transcription factor that is necessary for stem cell quiescence and self-renewal in the bone marrow. Expression of this miR-US22 down-regulates expression of EGR-1 that reduces CD34+ HPCs proliferation and total hematopoietic colony formation. An HCMV miR-US22 mutant is unable to reactivate from latency suggesting that the ability of the miRNA to disrupt CD34+ HPC renewal in the bone marrow niche to initiate a differentiation pathway is critical for viral reactivation.

microbiology↗