Biology subjects

Glenn, H. L.

Publications and source records attributed to Glenn, H. L..

3 recordsLinked to original sources

bioRxiv2025

Coronavirus membrane protein with a fluorescent protein tag enables tracking of virus particles in live cells

Coronavirus particles assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and exit from host cells via secretory organelles that are not well defined. The interplay between viral components and intracellular transport pathways that facilitate assembly and egress are not fully understood and recent studies suggest that multiple pathways maybe involved. Reverse genetics was used to develop a model system to further understand the assembly and egress processes. Mouse hepatitis coronavirus (MHV-A59) was genetically engineered to express the membrane (M) protein fused to green fluorescent protein (M-GFP), with the chimeric gene cloned in place of the open reading frame (ORF) 4 coding region in the RNA genome. The recovered M-GFP virus also expresses wild-type (WT) M protein (M WT) from its native ORF. The M-GFP virus exhibited morphology and growth properties like WT virus. M-GFP and WT M proteins colocalized early in infection, but less M-GFP trafficked toward the cell surface at later times, suggesting that the fusion protein is incorporated less efficiently into virus particles. M-GFP was stably expressed through at least four virus passages. Early passage virus M-GFP virus particles were visualized by confocal and Total Internal Reflection Fluorescence (TIRF) microscopy in live cells. The fluorescently labeled virus particles represent a new tool for coronavirus intracellular trafficking and egress studies. Such studies can also help provide a more detailed understanding of infection and disease processes to provide new insight for development of new therapeutic strategies. IMPORTANCECoronavirus assembly, intracellular transport, egress, and the virus-host interactions involved in these processes are not fully understood. The M protein is the most abundant virion structural component, which forms the scaffold for assembly of the viral envelope. It facilitates incorporation of the viral nucleocapsid and the other viral membrane proteins, spike (S) and envelope (E). In this study we engineered M protein tagged with GFP, which is expressed along with wild-type (WT) M from the viral genome. M-GFP interacted with WT M protein and co-assembled into virus particles. The resulting fluorescently labeled coronavirus particles open opportunities to use state of the art microscopy to monitor virus assembly, trafficking, and egress in live cells to facilitate deeper understanding of the molecular and cell biology of these processes. The ability to visualize particles also opens opportunities to help further understanding of steps in viral replication and pathogenesis for potential therapeutic targets.

microbiology↗

bioRxiv2023

Herpes Simplex Virus 1 (HSV-1) Uses the Rab6 Post-Golgi Secretory Pathway For Viral Egress

Herpes Simplex Virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles, and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host Rab6 post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and regulates vesicle trafficking and exocytosis at the plasma membrane. HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The Rab6a secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms. IMPORTANCEHSV-1 infects a majority of people. It establishes a life-long latent infection, and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly-assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells, and found that HSV-1 particles use the pre-existing cellular secretory pathway, regulated by the cellular protein, Rab6.

microbiology↗

bioRxiv2023

Nuclear export inhibitor Selinexor targeting XPO1 enhances coronavirus replication.

Nucleocytoplasmic transport of proteins using XPO1 (exportin 1) plays a vital role in cell proliferation and survival. Many viruses also exploit this pathway to promote infection and replication. Thus, inhibiting the XPO1-mediated nuclear export pathway with selective inhibitors has a diverse effect on virus replication by regulating antiviral, proviral, and anti-inflammatory pathways. The XPO1 inhibitor, Selinexor, is an FDA-approved anticancer drug predicted to have antiviral or proviral functions against viruses. Here, we observed that pretreatment of cultured cell lines from human or mouse origin with nuclear export inhibitor Selinexor significantly enhanced protein expression and replication of Mouse Hepatitis Virus (MHV), a mouse coronavirus. Knockdown of cellular XPO1 protein expression also significantly enhanced the replication of MHV in human cells. However, for SARS-CoV-2, selinexor treatment had diverse effects on virus replication in different cell lines. These results indicate that XPO1-mediated nuclear export pathway inhibition might affect coronavirus replication depending on cell types and virus origin.

microbiology↗