Dual Role of Herpes Simplex Virus 1 pUS9 in Virus Anterograde Axonal Transport and Final Assembly in Growth Cones in Distal Axons

ABSTRACTThe herpes simplex virus type 1 (HSV-1) envelope protein pUS9 plays an important role in virus anterograde axonal transport and spread from neuronal axons. In this study, we used both confocal microscopy and transmission electron microscopy (TEM) to examine the role of pUS9 in the anterograde transport and assembly of HSV-1 in the distal axon of human and rat dorsal root ganglion (DRG) neurons using US9 deletion (US9−), repair (US9R), and wild-type (strain F, 17, and KOS) viruses. Using confocal microscopy and single and trichamber culture systems, we observed a reduction but not complete block in the anterograde axonal transport of capsids to distal axons as well as a marked (∼90%) reduction in virus spread from axons to Vero cells with the US9 deletion viruses. Axonal transport of glycoproteins (gC, gD, and gE) was unaffected. Using TEM, there was a marked reduction or absence of enveloped capsids, in varicosities and growth cones, in KOS strain and US9 deletion viruses, respectively. Capsids (40 to 75%) in varicosities and growth cones infected with strain 17, F, and US9 repair viruses were fully enveloped compared to less than 5% of capsids found in distal axons infected with the KOS strain virus (which also lacks pUS9) and still lower (<2%) with the US9 deletion viruses. Hence, there was a secondary defect in virus assembly in distal axons in the absence of pUS9 despite the presence of key envelope proteins. Overall, our study supports a dual role for pUS9, first in anterograde axonal transport and second in virus assembly in growth cones in distal axons.IMPORTANCEHSV-1 has evolved mechanisms for its efficient transport along sensory axons and subsequent spread from axons to epithelial cells after reactivation. In this study, we show that deletion of the envelope protein pUS9 leads to defects in virus transport along axons (partial defect) and in virus assembly and egress from growth cones (marked defect). Virus assembly and exit in the neuronal cell body are not impaired in the absence of pUS9. Thus, our findings indicate that pUS9 contributes to the overall HSV-1 anterograde axonal transport, including a major role in virus assembly at the axon terminus, which is not essential in the neuronal cell body. Overall, our data suggest that the process of virus assembly at the growth cones differs from that in the neuronal cell body and that HSV-1 has evolved different mechanisms for virus assembly and exit from different cellular compartments.

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Herpes Simplex Virus Utilizes the Large Secretory Vesicle Pathway for Anterograde Transport of Tegument and Envelope Proteins and for Viral Exocytosis from Growth Cones of Human Fetal Axons

Journal of Virology ◽

10.1128/jvi.01579-08 ◽

2009 ◽

Vol 83 (7) ◽

pp. 3187-3199 ◽

Cited By ~ 63

Author(s):

Monica Miranda-Saksena ◽

Ross A. Boadle ◽

Anupriya Aggarwal ◽

Bibing Tijono ◽

Frazer J. Rixon ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Axonal Transport ◽

Virus Assembly ◽

Growth Cones ◽

Secretory Vesicle ◽

Envelope Proteins ◽

Anterograde Axonal Transport ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Axonal transport of herpes simplex virus (HSV-1) is essential for viral infection and spread in the peripheral nervous system of the host. Therefore, the virus probably utilizes existing active transport and targeting mechanisms in neurons for virus assembly and spread from neurons to skin. In the present study, we used transmission immnunoelectron microscopy to investigate the nature and origin of vesicles involved in the anterograde axonal transport of HSV-1 tegument and envelope proteins and of vesicles surrounding partially and fully enveloped capsids in growth cones. This study aimed to elucidate the mechanism of virus assembly and exit from axons of human fetal dorsal root ganglia neurons. We demonstrated that viral tegument and envelope proteins can travel in axons independently of viral capsids and were transported to the axon terminus in two types of transport vesicles, tubulovesicular membrane structures and large dense-cored vesicles. These vesicles and membrane carriers were derived from the trans-Golgi network (TGN) and contained key proteins, such as Rab3A, SNAP-25, GAP-43, and kinesin-1, involved in the secretory and exocytic pathways in axons. These proteins were also observed on fully and partially enveloped capsids in growth cones and on extracellular virions. Our findings provide further evidence to the subassembly model of separate transport in axons of unenveloped capsids from envelope and tegument proteins with final virus assembly occurring at the axon terminus. We postulate that HSV-1 capsids invaginate tegument- and envelope-bearing TGN-derived vesicles and utilize the large secretory vesicle pathway of exocytosis for exit from axons.

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Herpes Simplex Virus gE/gI and US9 Promote both Envelopment and Sorting of Virus Particles in the Cytoplasm of Neurons, Two Processes That Precede Anterograde Transport in Axons

Journal of Virology ◽

10.1128/jvi.00050-17 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 17

Author(s):

Grayson DuRaine ◽

Todd W. Wisner ◽

Paul Howard ◽

Melissa Williams ◽

David C. Johnson

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Membrane Proteins ◽

Axonal Transport ◽

Neuronal Cell ◽

Anterograde Transport ◽

Peripheral Tissues ◽

Virus Particles ◽

Anterograde Axonal Transport ◽

Simplex Virus

ABSTRACT Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE− US9− double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE− US9− mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific. IMPORTANCE Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde transport) or from cell bodies to axon tips (anterograde transport). This transit in axons is essential for alphaherpesviruses to establish latency in ganglia and then to reactivate and move back to peripheral tissues for spread to other hosts. Anterograde transport of HSV requires two membrane proteins: gE/gI and US9. Our studies reveal new mechanisms for how gE/gI and US9 initiate anterograde axonal transport. HSV mutants lacking both gE and US9 fail to properly assemble enveloped virus particles in the cytoplasm, which blocks anterograde transport of enveloped particles. In addition, there are defects in the sorting of virus particles such that particles, when formed, do not enter proximal axons.

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Insights into herpesvirus assembly from the structure of the pUL7:pUL51 complex

eLife ◽

10.7554/elife.53789 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 6

Author(s):

Benjamin G Butt ◽

Danielle J Owen ◽

Cy M Jeffries ◽

Lyudmila Ivanova ◽

Chris H Hill ◽

...

Keyword(s):

Virus Assembly ◽

Direct Role ◽

The Core ◽

Structural Framework ◽

Golgi Membranes ◽

Infected Cells ◽

Simplex Virus ◽

Human Herpesviruses ◽

Hsv 1

Herpesviruses acquire their membrane envelopes in the cytoplasm of infected cells via a molecular mechanism that remains unclear. Herpes simplex virus (HSV)−1 proteins pUL7 and pUL51 form a complex required for efficient virus envelopment. We show that interaction between homologues of pUL7 and pUL51 is conserved across human herpesviruses, as is their association with trans-Golgi membranes. We characterized the HSV-1 pUL7:pUL51 complex by solution scattering and chemical crosslinking, revealing a 1:2 complex that can form higher-order oligomers in solution, and we solved the crystal structure of the core pUL7:pUL51 heterodimer. While pUL7 adopts a previously-unseen compact fold, the helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B and pUL51 forms ESCRT-III–like filaments, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. Our results provide a structural framework for understanding the role of the conserved pUL7:pUL51 complex in herpesvirus assembly.

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The Basic Domain of Herpes Simplex Virus 1 pUS9 Recruits Kinesin-1 To Facilitate Egress from Neurons

Journal of Virology ◽

10.1128/jvi.03041-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 2102-2111 ◽

Cited By ~ 27

Author(s):

Russell J. Diefenbach ◽

April Davis ◽

Monica Miranda-Saksena ◽

Marian A. Fernandez ◽

Barbara J. Kelly ◽

...

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Axonal Transport ◽

Herpes Simplex Virus 1 ◽

Basic Domain ◽

Anterograde Axonal Transport ◽

Arginine Residues ◽

Hiv Acquisition ◽

Simplex Virus ◽

Hsv 1

ABSTRACTThe alphaherpesviral envelope protein pUS9 has been shown to play a role in the anterograde axonal transport of herpes simplex virus 1 (HSV-1), yet the molecular mechanism is unknown. To address this, we used anin vitropulldown assay to define a series of five arginine residues within the conserved pUS9 basic domain that were essential for binding the molecular motor kinesin-1. The mutation of these pUS9 arginine residues to asparagine blocked the binding of both recombinant and native kinesin-1. We next generated HSV-1 with the same pUS9 arginine residues mutated to asparagine (HSV-1pUS9KBDM) and then restored them being to arginine (HSV-1pUS9KBDR). The two mutated viruses were analyzed initially in a zosteriform model of recurrent cutaneous infection. The primary skin lesion scores were identical in severity and kinetics, and there were no differences in viral load at dorsal root ganglionic (DRG) neurons at day 4 postinfection (p.i.) for both viruses. In contrast, HSV-1pUS9KBDM showed a partial reduction in secondary skin lesions at day 8 p.i. compared to the level for HSV-1pUS9KBDR. The use of rat DRG neuronal cultures in a microfluidic chamber system showed both a reduction in anterograde axonal transport and spread from axons to nonneuronal cells for HSV-1pUS9KBDM. Therefore, the basic domain of pUS9 contributes to anterograde axonal transport and spread of HSV-1 from neurons to the skin through recruitment of kinesin-1.IMPORTANCEHerpes simplex virus 1 and 2 cause genital herpes, blindness, encephalitis, and occasionally neonatal deaths. There is also increasing evidence that sexually transmitted genital herpes increases HIV acquisition, and the reactivation of HSV increases HIV replication and transmission. New antiviral strategies are required to control resistant viruses and to block HSV spread, thereby reducing HIV acquisition and transmission. These aims will be facilitated through understanding how HSV is transported down nerves and into skin. In this study, we have defined how a key viral protein plays a role in both axonal transport and spread of the virus from nerve cells to the skin.

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MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.00621-16 ◽

2016 ◽

Vol 90 (19) ◽

pp. 8621-8633 ◽

Cited By ~ 14

Author(s):

Elizabeth Sloan ◽

Anne Orr ◽

Roger D. Everett

Keyword(s):

Immune Response ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Hcmv Infection ◽

Nuclear Bodies ◽

Herpes Simplex Virus 1 ◽

Simplex Virus ◽

Antiviral Role ◽

Hsv 1

ABSTRACTWe previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of herpes simplex virus 1 (HSV-1) ICP0-mediated degradation (E. Sloan, et al., PLoS Pathog11:e1005059, 2015,http://dx.doi.org/10.1371/journal.ppat.1005059). Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1 and are also degraded or inactivated by ICP0, here we further investigate the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild-type (wt) human cytomegalovirus (HCMV) infection, as its plaque-forming efficiency increased in MORC3-depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3, and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx, and γH2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response.IMPORTANCEHerpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML NB proteins, which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells and is a target for HSV-1-mediated degradation, we investigated the role of MORC3 during HSV-1 infection. We found that MORC3 is also recruited to viral HSV-1 genomes, and importantly it contributes to the fully efficient recruitment of PML, hDaxx, Sp100, and γH2AX to these sites. Depletion of MORC3 resulted in an increase in ICP0-null HSV-1 and wt HCMV replication and plaque formation; therefore, this study reveals that MORC3 is an antiviral factor which plays an important role during HSV-1 and HCMV infection.

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Separation of a process from the neuronal cell body using a thin aluminum foil separator

Brain Research Protocols ◽

10.1016/s1385-299x(98)00014-2 ◽

1998 ◽

Vol 2 (4) ◽

pp. 352-356 ◽

Cited By ~ 1

Author(s):

Kosuke Noda ◽

Keita Jimbo ◽

Kazuo Suzuki ◽

Kentaro Yoda

Keyword(s):

Cell Body ◽

Neuronal Cell ◽

Aluminum Foil ◽

Neuronal Cell Body ◽

Thin Aluminum ◽

Thin Aluminum Foil

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Differences in splicing defects between the grey and white matter in myotonic dystrophy type 1

10.1101/819433 ◽

2019 ◽

Author(s):

Masamitsu Nishi ◽

Takashi Kimura ◽

Mitsuru Furuta ◽

Koichi Suenaga ◽

Tsuyoshi Matsumura ◽

...

Keyword(s):

White Matter ◽

Myotonic Dystrophy ◽

Cell Body ◽

Neuronal Cell ◽

Neuronal Cell Body ◽

Myotonic Dystrophy Type 1 ◽

Myotonic Dystrophy Type ◽

And Control ◽

The Brain

AbstractMyotonic dystrophy type 1 (DM1) is a multi-system disorder caused by CTG repeats in the myotonic dystrophy protein kinase (DMPK) gene. This leads to sequestration of the splicing factor, muscleblind-like 2 (MBNL2), and aberrant splicing, mainly in the central nervous system. We investigated the splicing patterns of MBNL1/2 and genes controlled by MBNL2 in several regions of the brain and between the grey matter (GM) and white matter (WM) in DM1 patients using RT-PCR. Compared with the control, the percentage of spliced-in parameter (PSI) for most of the examined exons were significantly altered in most of the brain regions of DM1 patients, except for the cerebellum. The splicing of many genes was differently regulated between the GM and WM in both DM1 and control. The level of change in PSI between DM1 and control was higher in the GM than in the WM. The differences in alternative splicing between the GM and WM may be related to the effect of DM1 on the WM of the brain. We hypothesize that in DM1, aberrantly spliced isoforms in the neuronal cell body of the GM may not be transported to the axon. This might affect the WM as a consequence of Wallerian degeneration secondary to cell body damage. Our findings may have implications for analysis of the pathological mechanisms and exploring potential therapeutic targets.

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Construction and optimization of herpes simplex virus vectors for central nervous system gene delivery based on CRISPR/Cas9-mediated genome editing

Current Gene Therapy ◽

10.2174/1566523219666210618154326 ◽

2021 ◽

Vol 21 ◽

Author(s):

Xinwei Huang ◽

Xiuqing Li ◽

Lijuan Yang ◽

Pengfei Wang ◽

Jingyuan Yan ◽

...

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Transgene Expression ◽

Mouse Hippocampus ◽

Simplex Virus ◽

Hsv 1

Aims: We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Background: Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood. Objective: This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. Method: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model. Result: The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.

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Form and Function in Cells of the Brain

The Neuron ◽

10.1093/med/9780199773893.003.0002 ◽

2015 ◽

pp. 23-38

Author(s):

Irwin B. Levitan ◽

Leonard K. Kaczmarek

Keyword(s):

Axonal Transport ◽

Molecular Motors ◽

Critical Role ◽

Neuronal Cell ◽

Cell Types ◽

Neuronal Cell Body ◽

Long Distance ◽

Form And Function ◽

And Function ◽

The Brain

This chapter examines unique mechanisms that the neuron has evolved to establish and maintain the form required for its specialized signaling functions. Unlike some other organs, the brain contains a variety of cell types including several classes of glial cells, which play a critical role in the formation of the myelin sheath around axons and may be involved in immune responses, synaptic transmission, and long-distance calcium signaling in the brain. Neurons share many features in common with other cells (including glia), but they are distinguished by their highly asymmetrical shapes. The neuronal cytoskeleton is essential for establishing this cell shape during development and for maintaining it in adulthood. The process of axonal transport moves vesicles and other organelles to regions remote from the neuronal cell body. Proteins such as kinesin and dynein, called molecular motors, make use of the energy released by hydrolysis of ATP to drive axonal transport.

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Herpes Simplex Virus 1 Strains 17syn+ and KOS(M) Differ Greatly in Their Ability To Reactivate from Human Neurons In Vitro

Journal of Virology ◽

10.1128/jvi.00796-20 ◽

2020 ◽

Vol 94 (15) ◽

Author(s):

Tristan R. Grams ◽

Terri G. Edwards ◽

David C. Bloom

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Latent Infection ◽

Neuronal Cell ◽

Herpes Simplex Virus 1 ◽

Viral Genomes ◽

Human Neuronal Cell ◽

Cell Line Model ◽

Simplex Virus ◽

Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) establishes a lifelong latent infection in peripheral nerve ganglia. Periodically, the virus reactivates from this latent reservoir and is transported to the original site of infection. Strains of HSV-1 have been noted to vary greatly in their virulence and reactivation efficiencies in animal models. While HSV-1 strain 17syn+ can be readily reactivated, strain KOS(M) shows little to no reactivation in the mouse and rabbit models of induced reactivation. Additionally, 17syn+ is markedly more virulent in vivo than KOS. This has raised questions regarding potential strain-specific differences in neuroinvasion and neurovirulence and their contribution to differences in the establishment of latency (or ability to spread back to the periphery) and to the reactivation phenotype. To determine if any difference in the ability to reactivate between strains 17syn+ and KOS(M) is manifest at the level of neurons, we utilized a recently characterized human neuronal cell line model of HSV latency and reactivation (LUHMES). We found that KOS(M) established latency with a higher number of viral genomes than strain 17syn+. Strikingly, we show that the KOS(M) viral genomes have a higher burden of heterochromatin marks than strain 17syn+. The increased heterochromatin profile for KOS(M) correlates with the reduced expression of viral lytic transcripts during latency and impaired induced reactivation compared to that of 17syn+. These results suggest that genomes entering neurons from HSV-1 infections with strain KOS(M) are more prone to rapid heterochromatinization than those of 17syn+ and that this results in a reduced ability to reactivate from latency. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes a lifelong infection in neuronal cells. The virus periodically reactivates and causes recurrent disease. Strains of HSV-1 vary greatly in their virulence and potential to reactivate in animal models. Although these differences are phenotypically well defined, factors contributing to the strains’ abilities to reactivate are largely unknown. We utilized a human neuronal cell line model of HSV latency and reactivation (LUHMES) to characterize the latent infection of two HSV-1 wild-type strains. We find that strain-specific differences in reactivation are recapitulated in LUHMES. Additionally, these differences correlate with the degree of heterochromatinization of the latent genomes. Our data suggest that the epigenetic state of the viral genome is an important determinant of reactivation that varies in a strain-specific manner. This work also shows the first evidence of strain-specific differences in reactivation outside the context of the whole animal at a human neuronal cell level.

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