Biology subjects

Hogue, I. B.

Publications and source records attributed to Hogue, I. B..

6 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

Live-cell Fluorescence Microscopy of HSV-1 Cellular Egress by Exocytosis

1.1The human pathogen Herpes Simplex Virus 1 (HSV-1) produces a lifelong infection in the majority of the worlds population. While the generalities of alpha herpesvirus assembly and egress pathways are known, the precise molecular and spatiotemporal details remain unclear. In order to study this aspect of HSV-1 infection, we engineered a recombinant HSV-1 strain expressing a pH-sensitive reporter, gM-pHluorin. Using a variety of fluorescent microscopy modalities, we can detect individual virus particles undergoing intracellular transport and exocytosis at the plasma membrane. We show that particles exit from epithelial cells individually, not bulk release of many particles at once, as has been reported for other viruses. In multiple cell types, HSV-1 particles accumulate over time at the cell periphery and cell-cell contacts. We show that this accumulation effect is the result of individual particles undergoing exocytosis at preferential sites and that these egress sites can contribute to cell-cell spread. We also show that the viral membrane proteins gE, gI, and US9, which have important functions in intracellular transport in neurons, are not required for preferential egress and clustering in non-neuronal cells. Importantly, by comparing HSV-1 to a related alpha herpesvirus, pseudorabies virus, we show that this preferential exocytosis and clustering effect is cell type-dependent, not virus dependent. This preferential egress and clustering appears to be the result of the arrangement of the microtubule cytoskeleton, as virus particles co-accumulate at the same cell protrusions as an exogenous plus end-directed kinesin motor. ImportanceAlpha herpesviruses produce lifelong infections of their human and animal hosts. The majority of people in the world are infected with Herpes Simplex Virus 1 (HSV-1), which typically causes recurrent oral or genital lesions. However, HSV-1 can also spread to the central nervous system, causing severe encephalitis, and might also contribute to the development of neurodegenerative diseases. Many of the steps of how these viruses infect and replicate inside host cells are known in depth, but the final step, exiting from the infected cell, is not fully understood. In this study, we engineered a novel variant of HSV-1 that allows us to visualize how individual virus particles exit from infected cells. With this imaging assay, we investigated preferential egress site formation in certain cell types and their contribution to cell-cell spread of HSV-1.

microbiology↗

bioRxiv2023

Viral attachment blocking chimera composed of DNAorigami and nanobody inhibits Pseudorabies Virusinfection in vitro

Antivirals are indispensable tools that can be targeted at viral domains directly or indirectly at cellular domains to obstruct viral infections and reduce pathogenicity. Despite their transformative use in healthcare, antivirals have been clinically approved to treat only 10 of the more than 200 known pathogenic human viruses. Additionally, many virus functions are intimately coupled with host cellular processes, which present challenges in antiviral development due to the limited number of clear targets per virus, necessitating an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. We hypothesize that a Viral Attachment Blocking Chimera (VirABloC) composed of a viral binder and a bulky scaffold that sterically blocks interactions between a viral particle and a host cell may be suitable for the development of antivirals agnostic to the extravirion epitope that is being bound. We test this hypothesis by modifying a nanobody that specifically recognizes a non-essential epitope presented on the extra virion surface of Pseudorabies virus strain 486 with a 3-dimensional wireframe DNA origami structure [~]100 nm in diameter. The nanobody switches from having no inhibitory properties (tested up to 50 {micro}M) to 4.2 {+/-} 0.9 nM IC50 when conjugated with the DNA origami scaffold. Mechanistic studies support that inhibition is mediated by the non-covalent attachment of the DNA origami scaffold to the virus particle, which obstructs the attachment of the viruses onto host cells. These results support the potential of VirABloC as a generalizable approach to developing antivirals.

biophysics↗

bioRxiv2019

Alpha herpesvirus egress and spread from neurons uses constitutive secretory mechanisms independent of neuronal firing activity

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system causing severe deadly or debilitating disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous firing, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. To address this hypothesis, we used a compartmentalized primary neuron culture system to measure egress and spread of pseudorabies virus (PRV), pharmacological and optogenetics approaches to modulate neuronal firing activity, and a live-cell fluorescence microscopy assay to directly visualize the exocytosis of individual virus particles from infected neurons. Using tetrodotoxin to silence neuronal activity, we observed no inhibition of virus spread, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread. Using a live-cell fluorescence microscopy method to directly measure virus egress from infected neurons, we observed no association between virus particle exocytosis and intracellular Ca2+ signaling. Finally, we observed virus particle exocytosis occurs in association with constitutive secretory Rab GTPases, Rab6a and Rab8a, not Rab proteins that are associated with the Ca2+-regulated secretory pathway in neurons, Rab3a and Rab11a. Therefore, we conclude that alpha herpesvirus egress and spread is independent of neuronal activity and Ca2+ signaling because virus particle exocytosis uses constitutive secretory mechanisms in neurons.\n\nAuthor SummaryAlpha herpesviruses, including important human pathogens Herpes Simplex Virus 1 and 2, and Varicella-Zoster Virus, are among the very few viruses that naturally infect the nervous system. These viruses cause recurrent herpetic and zosteriform lesions, peripheral neuropathies, and deadly or debilitating central nervous system diseases. Many of the molecular and cellular mechanisms of viral egress and spread remain unknown, particularly in the context of specialized neuronal cell biology. Our results indicate that elevated firing activity of infected neurons is not functionally or mechanistically linked to virus egress and spread; therefore, therapies targeting peripheral neuropathic symptoms, elevated neuronal activity, and synaptic vesicle secretory mechanisms are unlikely to affect virus spread in the nervous system.

microbiology↗

bioRxiv2019

A Kinesin-3 Recruitment Complex Facilitates Axonal Sorting of Enveloped Alpha Herpesvirus Capsids

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.\n\nAuthor SummaryAlpha-herpesviruses represent a group of large, enveloped DNA viruses that are capable to establish a quiescent (also called latent) but reactivatable form of infection in the peripheral nervous system of their hosts. Following reactivation of latent genomes, virus progeny are formed in the soma of neuronal cells and depend on sorting into the axon for anterograde spread of infection to mucosal sites and potentially new host. We studied two alpha-herpesviruses (the veterinary pathogen pseudorabies virus (PRV) and human herpes simplex virus 1 (HSV-1)) and found viral membrane proteins Us7, Us8, and Us9 to form a complex, which is able to recruit kinsin-3 motors. Motor recruitment facilitates axonal sorting and subsequent transport to distal egress sites. Complex formation occurs at the trans-Golgi network and mediates efficiency of axonal sorting and motility characteristics of egressing capsids. We also used an artificial kinesin-3 recruitment system, which allows controlled induction of axonal sorting and transport for virus mutants lacking Us7, Us8, and Us9. Overall, these data contribute to our understanding of anterograde alpha-herpesvirus spread and kinesin-mediated sorting of vesicular axonal cargoes.

microbiology↗