Biology subjects

Domanico, L. F.

Publications and source records attributed to Domanico, L. F..

3 recordsLinked to original sources

bioRxiv2025

The producer cell type of HSV-1 alters the proteomic contents and infectious capacity of virions

The cell that a virus replicates in i.e., the producer cell, can alter the macromolecular composition and infectious capacity of the virions that are produced. Herpes Simplex virus type 1 (HSV-1) primarily infects keratinocytes of the epidermis or oral mucosa prior to establishing latency in neurons of the peripheral nervous system, where the virus can persist for the lifetime of the host. Many cell lines that are used to amplify HSV-1 are derived from species and tissue types that are less physiologically relevant to HSV-1 disease. To understand if the producer cell type influences HSV-1 infection, we tested the infectivity of HSV-1 derived from immortalized African green monkey kidney cells (vero), immortalized human keratinocytes (HaCaT), and primary human foreskin fibroblasts (HFF-1). We observed that the producer cell type alters the capacity of HSV-1 to produce viral proteins and infectious virions from infected cells and susceptibility to inhibition of replication by interferon treatment. HaCaT-derived HSV-1 consistently exhibited enhanced replication over HFF-1 or vero-derived virus. To determine if the producer cell type changes the protein composition of virions, we performed an untargeted LC/MS-MS analysis of virions purified from each cell line. Comparison of virion associated proteins revealed quantitative differences in composition of both cellular and viral proteins including ICP0, pUL24 and pUL42. These results highlight the influence that the producer cell-type has on HSV-1 infection outcomes and suggest that cell type specific factors can alter HSV-1 and impact viral replication. ImportanceApproximately 67% of the human population harbors HSV-1 infection. To study HSV-1 infection, laboratories utilize several different cell lines to propagate HSV-1 for downstream experiments. The type of cell used to produce a virus, i.e. the producer cell type, can alter the macromolecular composition, immunogenicity, and infectivity of the virions that are produced across several virus families. We found that the producer cell type of HSV-1 alters virion infectivity and virion protein composition. Therefore, the producer cell type may have implications in the spread of HSV-1 and subsequent disease outcomes in humans. Our results also raise concerns about how the use of different ceil types to propagate HSV-1 may alter the outcome, interpretation, and reproducibility of experimental results.

microbiology↗

bioRxiv2023

Single-cell Herpes Simplex Virus type-1 infection of neurons using drop-based microfluidics reveals heterogeneous replication kinetics

Single-cell analyses of viral infections often reveal heterogeneity that is not detected by traditional population-level studies. This study applies drop-based microfluidics to investigate the dynamics of HSV-1 infection of neurons at the single-cell level. We used micron-scale Matrigel beads, termed microgels, to culture individual murine Superior Cervical ganglia (SCG) neurons or epithelial cells. Microgel-cultured cells are subsequently enclosed in individual media-in-oil droplets with a dual fluorescent-reporter HSV-1, enabling real-time observation of viral gene expression and replication. Infection within drops revealed that the kinetics of initial viral gene expression and replication were dependent on the inoculating dose. Notably, increasing inoculating doses led to earlier onset of viral gene expression and more frequent productive viral replication. These observations provide crucial insights into the complexity of HSV-1 infection in neurons and emphasize the importance of studying single-cell outcomes of viral infection. The innovative techniques presented here for cell culture and infection in drops provide a foundation for future virology and neurobiology investigations.

bioengineering↗

bioRxiv2023

A dual-fluorescent recombinant for live observation of Herpes simplex-type 1 infection outcomes.

Critical stages of lytic Herpes simplex type 1 (HSV-1) replication are marked by the sequential expression of immediate early (IE) to early (E), then late (L) viral genes. HSV-1 also persists in neuronal tissues via a non-replicative, transcriptionally repressed infection called latency. Understanding the regulation of lytic and latent transcriptional profiles provides focused insight into HSV-1 infection and disease. We sought a fluorescence-based approach to observe temporal progression of HSV-1 infection at the single-cell level. We constructed and characterized a novel HSV-1 recombinant that reports IE and L gene expression by fluorescent protein detection. The dual-reporter HSV-1 visualizes IE gene expression by a CMV promotor-driven YFP, and L gene expression by an endogenous mCherry-VP26 fusion. We confirmed that viral gene expression, replication and spread of infection in epithelial cells is not altered by the incorporation of the fluorescent reporters. Interference with viral DNA polymerase activity abolishes VP26-mCherry detection late in HSV-1 infection, visually reporting the failure to complete viral replication. Viral replication in primary neurons is not altered, but retrograde-directed inoculation of the dual-reporter HSV-1 exhibits a modest reduction in titer, compared to unlabeled HSV-1. Low-dose axonal inoculation in the presence of small molecule modulation of neuronal signaling results in divergent outcomes of YFP and mCherry detection, suggesting different states of latent and lytic replication. Rigorous characterization of this dual-reporter HSV-1 recombinant has demonstrated the utility of temporal observation of HSV-1 replication in live cells and the potential for further insight into the dynamics of infection. ImportanceHerpes simplex virus-type 1 (HSV-1) is a prevalent human pathogen that infects approximately 67% of the global population. HSV-1 invades the peripheral nervous system, where latent HSV-1 infection persists within the host for life. Immunological evasion, viral persistence, and herpetic pathologies are determined by regulation of HSV-1 gene expression. Studying HSV-1 gene expression during neuronal infection is challenging but essential for the development of antiviral therapeutics and interventions. We constructed and characterized a dual-fluorescent HSV-1 recombinant that enables visualization of IE and L gene expression. The recombinant HSV-1 is used to observe the progression and outcome of infection. We demonstrate that drug treatments targeting cellular pathways can manipulate latent HSV-1 infection in neurons to achieve divergent outcomes of infection.

microbiology↗