Kevin P. Bohannon

The Herpesvirus VP1/2 Protein Is an Effector of Dynein-Mediated Capsid Transport and Neuroinvasion

Microtubule transport of herpesvirus capsids from the cell periphery to the nucleus is imperative for viral replication and, in the case of many alphaherpesviruses, transmission into the nervous system. Using the neuroinvasive herpesvirus, pseudorabies virus (PRV), we show that the viral protein 1/2 (VP1/2) tegument protein associates with the dynein/dynactin microtubule motor complex and promotes retrograde microtubule transport of PRV capsids. Functional activation of VP1/2 requires binding to the capsid protein pUL25 or removal of the capsid-binding domain. A proline-rich sequence within VP1/2 is required for the efficient interaction with the dynein/dynactin microtubule motor complex as well as for PRV virulence and retrograde axon transport in vivo. Additionally, in the absence of infection, functionally active VP1/2 is sufficient to move large surrogate cargoes via the dynein/dynactin microtubule motor complex. Thus, VP1/2 tethers PRV capsids to dynein/dynactin to enhance microtubule transport, neuroinvasion, and pathogenesis.

Alphaherpesviruses and the Cytoskeleton in Neuronal Infections

Following infection of exposed peripheral tissues, neurotropic alphaherpesviruses invade nerve endings and deposit their DNA genomes into the nuclei of neurons resident in ganglia of the peripheral nervous system. The end result of these events is the establishment of a life-long latent infection. Neuroinvasion typically requires efficient viral transmission through a polarized epithelium followed by long-distance transport through the viscous axoplasm. These events are mediated by the recruitment of the cellular microtubule motor proteins to the intracellular viral particle and by alterations to the cytoskeletal architecture. The focus of this review is the interplay between neurotropic herpesviruses and the cytoskeleton.

Differential protein partitioning within the herpesvirus tegument and envelope underlies a complex and variable virion architecture

Significance Neuroinvasive herpesviruses cause diseases in humans ranging from cold sores to central nervous system infections. Unlike most icosahedral viruses, herpesvirus capsids are surrounded by protein layers that lack polyhedral architecture. The outer layers are critical for herpesvirus infectivity. Although the disorganized layers are visible by electron microscopy, the protein topography of these layers remains unclear. We fused fluorophores to virus proteins and pinpointed their positions within virions at nanometer resolution. The findings reveal a complex and variable asymmetric architecture, shed light on herpesvirus assembly, and provide a foundation for visualizing viral particle dynamics during herpesvirus infection. The herpesvirus virion is a multilayered structure consisting of a DNA-filled capsid, tegument, and envelope. Detailed reconstructions of the capsid are possible based on its icosahedral symmetry, but the surrounding tegument and envelope layers lack regular architecture. To circumvent limitations of symmetry-based ultrastructural reconstruction methods, a fluorescence approach was developed using single-particle imaging combined with displacement measurements at nanoscale resolution. An analysis of 11 tegument and envelope proteins defined the composition and plasticity of symmetric and asymmetric elements of the virion architecture. The resulting virion protein map ascribes molecular composition to density profiles previously acquired by traditional ultrastructural methods, and provides a way forward to examine the dynamics of the virion architecture during infection.

Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection.

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP-pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP-pUL25 and FP-pUL36 preserved wild-type properties better than traditional FP-pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

Refractive Index Imaging of Cells with Variable-Angle Near-Total Internal Reflection (TIR) Microscopy

The refractive index in the interior of single cells affects the evanescent field depth in quantitative studies using total internal reflection (TIR) fluorescence, but often that index is not well known. We here present method to measure and spatially map the absolute index of refraction in a microscopic sample, by imaging a collimated light beam reflected from the substrate/buffer/cell interference at variable angles of incidence. Above the TIR critical angle (which is a strong function of refractive index), the reflection is 100%, but in the immediate sub-critical angle zone, the reflection intensity is a very strong ascending function of incidence angle. By analyzing the angular position of that edge at each location in the field of view, the local refractive index can be estimated. In addition, by analyzing the steepness of the edge, the distance-to-substrate can be determined. We apply the technique to liquid calibration samples, silica beads, cultured Chinese hamster ovary cells, and primary culture chromaffin cells. The optical technique suffers from decremented lateral resolution, scattering, and interference artifacts. However, it still provides reasonable results for both refractive index (~1.38) and for distance-to-substrate (~150 nm) for the cells, as well as a lateral resolution to about 1 µm.

Drosophila Schneider 2 (S2) cells: a novel tool for studying HSV-induced membrane fusion.

Drosophila S2 cells and mammalian CHO-K1 cells were used to investigate the requirements for HSV-1 cell fusion. Infection assays indicated S2 cells were not permissive for HSV-1. HVEM and nectin-1 mediated cell fusion between CHO-K1 cells and S2 cells when either CHO-K1 or S2 cells were used as target cells. Interestingly, PILRα did not mediate fusion between CHO-K1 or S2 cells due to a glycosylation defect of PILRα and gB in S2 cells. Fusion activity was not detected for any receptor tested when S2 cells were used both as target cells and effector cells indicating S2 cells may lack a key cellular factor present in mammalian cells that is required for cell fusion. Thus, insect cells may provide a novel tool to study the interaction of HSV-1 glycoproteins and cellular factors required for fusion, as well as a means to identify unknown cellular factors required for HSV replication.

Slow fusion pore expansion creates a unique reaction chamber for co-packaged cargo

A lumenal secretory granule protein, tissue plasminogen activator (tPA), greatly slows fusion pore dilation and thereby slows its own discharge. We investigated another outcome of the long-lived narrow fusion pore: the creation of a nanoscale chemical reaction chamber for granule contents in which the pH is suddenly neutralized upon fusion. Bovine adrenal chromaffin cells endogenously express both tPA and its primary protein inhibitor, plasminogen activator inhibitor 1 (PAI). We found by immunocytochemistry that tPA and PAI are co-packaged in the same secretory granule. It is known that PAI irreversibly and covalently inactivates tPA at neutral pH. We demonstrate with zymography that the acidic granule lumen protects tPA from inactivation by PAI. Immunocytochemistry, total internal reflection fluorescence (TIRF) microscopy, and polarized TIRF microscopy demonstrated that co-packaged PAI and tPA remain together in granules for many seconds in the nanoscale reaction chamber, more than enough time to inhibit tPA and create a new secreted protein species.

A pUL25 dimer interfaces the pseudorabies virus capsid and tegument

Inside the virions of α-herpesviruses, tegument protein pUL25 anchors the tegument to capsid vertices through direct interactions with tegument proteins pUL17 and pUL36. In addition to promoting virion assembly, both pUL25 and pUL36 are critical for intracellular microtubule-dependent capsid transport. Despite these essential roles during infection, the stoichiometry and precise organization of pUL25 and pUL36 on the capsid surface remain controversial due to the insufficient resolution of existing reconstructions from cryo-electron microscopy (cryoEM). Here, we report a three-dimensional (3D) icosahedral reconstruction of pseudorabies virus (PRV), a varicellovirus of the α-herpesvirinae subfamily, obtained by electron-counting cryoEM at 4.9 Å resolution. Our reconstruction resolves a dimer of pUL25 forming a capsid-associated tegument complex with pUL36 and pUL17 through a coiled coil helix bundle, thus correcting previous misinterpretations. A comparison between reconstructions of PRV and the γ-herpesvirus Kaposis sarcoma-associated herpesvirus (KSHV) reinforces their similar architectures and establishes important subfamily differences in the capsid-tegument interface.

Identification of β-synuclein on secretory granules in chromaffin cells and the effects of α- and β-synuclein on BDNF discharge following fusion

Synuclein is strongly implicated in the pathogenesis of Parkinson’s disease as well as in other neurodegenerative diseases. However, its normal function in cells is not understood. The N-termini of α-, β-, and γ-synuclein are comprised of seven 11-amino acid repeats that are predicted to form amphipathic helices. α-Synuclein binds to negatively charged lipids, especially small vesicles and tubulates and vesiculates lipids. The membrane-binding and membrane-curving abilities raise the possibility that synuclein could alter cellular processes that involve highly curved structures. In the present study we examined the localization of endogenous synuclein in bovine chromaffin cells by immunocytochemistry and its possible function to control protein discharge upon fusion of the granule with the plasma membrane by regulating the fusion pore. We found with quantitative immunocytochemistry that endogenous β-synuclein associates with secretory granules. Endogenous α-synuclein only rarely is found on secretory granules. Overexpression of α-synuclein but not β-synuclein quickened the median duration of the post-fusion discharge of BDNF-pHluorin by 30%, consistent with α-synuclein speeding fusion pore expansion.