scholarly journals A putative WAVE regulatory complex (WRC) interacting receptor sequence (WIRS) in the cytoplasmic tail of HSV-1 gE does not function in WRC recruitment or neuronal transport

2021 ◽  
Vol 3 (3) ◽  
Author(s):  
Christopher E. Denes ◽  
Timothy P. Newsome ◽  
Monica Miranda-Saksena ◽  
Anthony L. Cunningham ◽  
Russell J. Diefenbach
Keyword(s):  
Plaque Assay ◽  
Cytoplasmic Tail ◽  
Host Protein ◽  
Independent Manner ◽  
Anterograde Transport ◽  
Viral Egress ◽  
Regulatory Complex ◽  
Cell Spread ◽  
Dependent Interaction ◽  
Hsv 1

HSV-1 envelope glycoprotein E (gE) is important for viral egress and cell-to-cell spread but the host protein(s) involved in these functions have yet to be determined. We aimed to investigate a role for the Arp2/3 complex and actin regulation in viral egress based on the identification of a WAVE Regulatory Complex (WRC) Interacting Receptor Sequence (WIRS) in the cytoplasmic tail (CT) of gE. A WIRS-dependent interaction between the gE(CT) and subunits of the WRC was demonstrated by GST-pulldown assay and a role for the Arp2/3 complex in cell-to-cell spread was also observed by plaque assay. Subsequent study of a recombinant HSV-1 gE WIRS-mutant found no significant changes to viral production and release based on growth kinetics studies, or changes to plaque and comet size in various cell types, suggesting no function for the motif in cell-to-cell spread. GFP-Trap pulldown and proximity ligation assays were unable to confirm a WIRS-dependent interaction between gE and the WRC in human cell lines though the WIRS-independent interaction observed in situ warrants further study. Confocal microscopy of infected cells of neuronal origin identified no impairment of gE WIRS-mutant HSV-1 anterograde transport along axons. We propose that the identified gE WIRS motif does not function directly in recruitment of the WRC in human cells, in cell-to-cell spread of virus or in anterograde transport along axons. Further studies are needed to understand how HSV-1 manipulates and traverses the actin cytoskeleton and how gE may contribute to these processes in a WIRS-independent manner.

scholarly journals Two Separate Tyrosine-Based YXXL/Φ Motifs within the Glycoprotein E Cytoplasmic Tail of Bovine Herpesvirus 1 Contribute in Virus Anterograde Neuronal Transport

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1025
Author(s):  
Hocine Yezid ◽  
Christian T. Lay ◽  
Katrin Pannhorst ◽  
Shafiqul I. Chowdhury
Keyword(s):  
Epithelial Cells ◽  
Bovine Herpesvirus ◽  
Cytoplasmic Tail ◽  
Anterograde Transport ◽  
Bovine Herpesvirus 1 ◽  
Glycoprotein E ◽  
Tyrosine Residues ◽  
Neuronal Transport ◽  
Herpesvirus 1 ◽  
Cell Spread

Bovine herpesvirus 1 (BHV-1) causes respiratory infection and abortion in cattle. Following a primary infection, BHV-1 establishes lifelong latency in the trigeminal ganglia (TG). Periodic reactivation of the latent virus in TG neurons results in anterograde virus transport to nerve endings in the nasal mucosa and nasal virus shedding. The BHV-1 glycoprotein E cytoplasmic tail (gE-CT) is necessary for virus cell-to-cell spread in epithelial cells and neuronal anterograde transport. Recently, we identified two tyrosine residues, Y467 and Y563, within the tyrosine-based motifs 467YTSL470 and 563YTVV566, which, together, account for the gE CT-mediated efficient cell-to-cell spread of BHV-1 in epithelial cells. Here, we determined that in primary neuron cultures in vitro, the individual alanine exchange Y467A or Y563A mutants had significantly diminished anterograde axonal spread. Remarkably, the double-alanine-exchanged Y467A/Y563A mutant virus was not transported anterogradely. Following intranasal infection of rabbits, both wild-type (wt) and the Y467A/Y563A mutant viruses established latency in the TG. Upon dexamethasone-induced reactivation, both wt and the mutant viruses reactivated and replicated equally efficiently in the TG. However, upon reactivation, only the wt, not the mutant, was isolated from nasal swabs. Therefore, the gE-CT tyrosine residues Y467 and Y563 together are required for gE CT-mediated anterograde neuronal transport.

scholarly journals Cellular miR-101-1 Reduces Efficiently the Replication of HSV-1 in HeLa Cells

Intervirology ◽  
2021 ◽  
Vol 64 (2) ◽  
pp. 88-95
Author(s):  
Bahar Sadegh Ehdaei ◽  
Ahmad Pirouzmand ◽  
Mehdi Shabani ◽  
Arezoo Mirzaei ◽  
Sharareh Moghim
Keyword(s):  
Hela Cells ◽  
Plaque Assay ◽  
Viral Gene Expression ◽  
Viral Gene ◽  
Herpes Simplex Viruses ◽  
Viral Progeny ◽  
Immunosuppressed Patients ◽  
Health Complications ◽  
Hsv 1

<b><i>Introduction:</i></b> Herpes simplex viruses (HSVs) are widely distributed in the human population. HSV type 1 (HSV-1) is responsible for a spectrum of diseases, ranging from gingivostomatitis to keratoconjunctivitis, and encephalitis. The HSVs establish latent infections in nerve cells, and recurrences are common. Their frequent reactivation in elderly and immunosuppressed patients causes serious health complications. <b><i>Objectives:</i></b> Due to the growing resistance to its main drug, acyclovir, alternative treatments with different mechanisms of action are required. MicroRNAs regulate host and viral gene expression posttranscriptionally. Previous studies reported that mir-101-2 expression has widely participated in the regulation of HSV-1 replication. In this study, we investigate the effect of hsa-miR-101-1 in the replication of HSV-1. <b><i>Methods:</i></b> We found that transfection of miR-101-1 into HeLa cells could reduce effectively HSV-1 replication using plaque assay and real-time PCR methods. <b><i>Results:</i></b> We showed that overexpression of miR-10-1 produced less viral progeny and manifested a weaker cytopathic effect, without affecting cell viability. <b><i>Discussion/Conclusion:</i></b> This result can give us new insights into the control of HSV-1 infections.

scholarly journals Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion

2007 ◽  
Vol 82 (5) ◽  
pp. 2120-2129 ◽  
Author(s):  
Pablo Gastaminza ◽  
Guofeng Cheng ◽  
Stefan Wieland ◽  
Jin Zhong ◽  
Wei Liao ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Buoyant Density ◽  
Infectious Hepatitis ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Independent Manner ◽  
Viral Egress ◽  
Vldl Assembly

ABSTRACT Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.

scholarly journals Functional Relevance of the Transmembrane Domain and Cytoplasmic Tail of the Pseudorabies Virus Glycoprotein H for Membrane Fusion

2018 ◽  
Vol 92 (12) ◽  
Author(s):  
Melina Vallbracht ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Essential Role ◽  
Pseudorabies Virus ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Class Iii ◽  
Complex Needs ◽  
Hsv 1

ABSTRACTHerpesvirus membrane fusion depends on the core fusion machinery, comprised of glycoproteins B (gB) and gH/gL. Although gB structurally resembles autonomous class III fusion proteins, it strictly depends on gH/gL to drive membrane fusion. Whether the gH/gL complex needs to be membrane anchored to fulfill its function and which role the gH cytoplasmic (CD) and transmembrane domains (TMD) play in fusion is unclear. While the gH CD and TMD play an important role during infection, soluble gH/gL of herpes simplex virus 1 (HSV-1) seems to be sufficient to mediate cell-cell fusion in transient assays, arguing against an essential contribution of the CD and TMD. To shed more light on this apparent discrepancy, we investigated the role of the CD and TMD of the related alphaherpesvirus pseudorabies virus (PrV) gH. For this purpose, we expressed C-terminally truncated and soluble gH and replaced the TMD with a glycosylphosphatidylinositol (gpi) anchor. We also generated chimeras containing the TMD and/or CD of PrV gD or HSV-1 gH. Proteins were characterized in cell-based fusion assays and during virus infection. Although truncation of the CD resulted in decreased membrane fusion activity, the mutant proteins still supported replication of gH-negative PrV, indicating that the PrV gH CD is dispensable for viral replication. In contrast, PrV gH lacking the TMD, membrane-anchored via a lipid linker, or comprising the PrV gD TMD were nonfunctional, highlighting the essential role of the gH TMD for function. Interestingly, despite low sequence identity, the HSV-1 gH TMD could substitute for the PrV gH TMD, pointing to functional conservation.IMPORTANCEEnveloped viruses depend on membrane fusion for virus entry. While this process can be mediated by only one or two proteins, herpesviruses depend on the concerted action of at least three different glycoproteins. Although gB has features of bona fide fusion proteins, it depends on gH and its complex partner, gL, for fusion. Whether gH/gL prevents premature fusion or actively triggers gB-mediated fusion is unclear, and there are contradictory results on whether gH/gL function requires stable membrane anchorage or whether the ectodomains alone are sufficient. Our results show that in pseudorabies virus gH, the transmembrane anchor plays an essential role for gB-mediated fusion while the cytoplasmic tail is not strictly required.

scholarly journals The cytoplasmic tail of human mannosidase Man1b1 contributes to catalysis-independent quality control of misfolded alpha1-antitrypsin

2020 ◽  
Vol 117 (40) ◽  
pp. 24825-24836 ◽  
Author(s):  
Ashlee H. Sun ◽  
John R. Collette ◽  
Richard N. Sifers
Keyword(s):  
Secretory Pathway ◽  
Cytoplasmic Tail ◽  
Cellular Functions ◽  
Independent Manner ◽  
Accelerated Degradation ◽  
Naturally Occurring ◽  
Regulatory Capacity ◽  
Alpha1 Antitrypsin ◽  
Catalytic Removal ◽  
Insight Into

The failure of polypeptides to achieve conformational maturation following biosynthesis can result in the formation of protein aggregates capable of disrupting essential cellular functions. In the secretory pathway, misfolded asparagine (N)-linked glycoproteins are selectively sorted for endoplasmic reticulum-associated degradation (ERAD) in response to the catalytic removal of terminal alpha-linked mannose units. Remarkably, ER mannosidase I/Man1b1, the first alpha-mannosidase implicated in this conventional N-glycan-mediated process, can also contribute to ERAD in an unconventional, catalysis-independent manner. To interrogate this functional dichotomy, the intracellular fates of two naturally occurring misfolded N-glycosylated variants of human alpha1-antitrypsin (AAT), Null Hong Kong (NHK), and Z (ATZ), in Man1b1 knockout HEK293T cells were monitored in response to mutated or truncated forms of transfected Man1b1. As expected, the conventional catalytic system requires an intact active site in the Man1b1 luminal domain. In contrast, the unconventional system is under the control of an evolutionarily extended N-terminal cytoplasmic tail. Also, N-glycans attached to misfolded AAT are not required for accelerated degradation mediated by the unconventional system, further demonstrating its catalysis-independent nature. We also established that both systems accelerate the proteasomal degradation of NHK in metabolic pulse-chase labeling studies. Taken together, these results have identified the previously unrecognized regulatory capacity of the Man1b1 cytoplasmic tail and provided insight into the functional dichotomy of Man1b1 as a component in the mammalian proteostasis network.

scholarly journals UL36p Is Required for Efficient Transport of Membrane-Associated Herpes Simplex Virus Type 1 along Microtubules

2008 ◽  
Vol 82 (15) ◽  
pp. 7388-7394 ◽  
Author(s):  
Sara K. Shanda ◽  
Duncan W. Wilson
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Anterograde Transport ◽  
Tegument Proteins ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Microtubule-mediated anterograde transport is essential for the transport of herpes simplex virus type 1 (HSV-1) along axons, yet little is known regarding the mechanism and the machinery required for this process. Previously, we were able to reconstitute anterograde transport of HSV-1 on microtubules in an in vitro microchamber assay. Here we report that the large tegument protein UL36p is essential for this trafficking. Using a fluorescently labeled UL36 null HSV-1 strain, KΔUL36GFP, we found that it is possible to isolate a membrane-associated population of this virus. Although these viral particles contained normal amounts of tegument proteins VP16, vhs, and VP22, they displayed a 3-log decrease in infectivity and showed a different morphology compared to UL36p-containing virions. Membrane-associated KΔUL36GFP also displayed a slightly decreased binding to microtubules in our microchamber assay and a two-thirds decrease in the frequency of motility. This decrease in binding and motility was restored when UL36p was supplied in trans by a complementing cell line. These findings suggest that UL36p is necessary for HSV-1 anterograde transport.

scholarly journals Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production

2016 ◽  
Vol 90 (22) ◽  
pp. 10351-10361 ◽  
Author(s):  
Dmitry V. Chouljenko ◽  
Nithya Jambunathan ◽  
Vladimir N. Chouljenko ◽  
Misagh Naderi ◽  
Michal Brylinski ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Infectious Virus ◽  
Virus Production ◽  
Herpes Simplex Virus 1 ◽  
Anterograde Transport ◽  
Tyrosine Residues ◽  
Glycoprotein K ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTThe herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment attrans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927–5935, 2014,http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production.IMPORTANCEThe HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.

scholarly journals Cellular Cholesterol Facilitates the Postentry Replication Cycle of Herpes Simplex Virus 1

2017 ◽  
Vol 91 (14) ◽  
Author(s):  
George A. Wudiri ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex ◽  
Viral Genome ◽  
Herpes Simplex Virus 1 ◽  
Cellular Cholesterol ◽  
Viral Capsids ◽  
Simplex Virus ◽  
Spread Of Infection ◽  
Cell Spread ◽  
Replicative Cycle ◽  
Hsv 1

ABSTRACT Cholesterol is an essential component of cell membranes and is required for herpes simplex virus 1 (HSV-1) entry (1–3). Treatment of HSV-1-infected Vero cells with methyl beta-cyclodextrin from 2 to 9 h postentry reduced plaque numbers. Transport of incoming viral capsids to the nuclear periphery was unaffected by the cholesterol reduction, suggesting that cell cholesterol is important for the HSV-1 replicative cycle at a stage(s) beyond entry, after the arrival of capsids at the nucleus. The synthesis and release of infectious HSV-1 and cell-to-cell spread of infection were all impaired in cholesterol-reduced cells. Propagation of HSV-1 on DHCR24−/− fibroblasts, which lack the desmosterol-to-cholesterol conversion enzyme, resulted in the generation of infectious extracellular virions (HSVdes) that lack cholesterol and likely contain desmosterol. The specific infectivities (PFU per viral genome) of HSVchol and HSVdes were similar, suggesting cholesterol and desmosterol in the HSV envelope support similar levels of infectivity. However, infected DHCR24−/− fibroblasts released ∼1 log less infectious HSVdes and ∼1.5 log fewer particles than release of cholesterol-containing particles (HSVchol) from parental fibroblasts, suggesting that the hydrocarbon tail of cholesterol facilitates viral synthesis. Together, the results suggest multiple roles for cholesterol in the HSV-1 replicative cycle. IMPORTANCE HSV-1 infections are associated with a wide range of clinical manifestations that are of public health importance. Cholesterol is a key player in the complex interaction between viral and cellular factors that allows HSV-1 to enter host cells and establish infection. Previous reports have demonstrated a role for cellular cholesterol in the entry of HSV-1 into target cells. Here, we employed both chemical treatment and cells that were genetically defined to synthesize only desmosterol to demonstrate that cholesterol is important at stages following the initial entry and transport of viral capsids to the nucleus. Viral protein expression, encapsidation of the viral genome, and the release of mature virions were impacted by the reduction of cellular cholesterol. Cholesterol was also critical for cell-to-cell spread of infection. These findings provide new insights into the cholesterol dependence of HSV-1 replication.

scholarly journals Determination of Minimum Herpes Simplex Virus Type 1 Components Necessary To Localize Transcriptionally Active DNA to ND10

2003 ◽  
Vol 77 (10) ◽  
pp. 5821-5828 ◽  
Author(s):  
Qiyi Tang ◽  
Luge Li ◽  
Alexander M. Ishov ◽  
Valerie Revol ◽  
Alberto L. Epstein ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Host Protein ◽  
Transcript Accumulation ◽  
Viral Dna ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT DNA viruses such as herpes simplex virus type 1 (HSV-1) appear to start their replicative processes at specific nuclear domains known as ND10. In analyses to determine the minimum viral components needed for transcript accumulation at ND10, we find that a specific viral DNA sequence, OriS, and the viral immediate-early proteins ICP4 and ICP27 are sufficient for a reporter gene placed in cis to the OriS sequence to transcribe at ND10. A chromatin immunoprecipitation assay demonstrated expected critical intermediates in retaining the minimal genome at ND10 for the HSV-1 replication origin through direct or indirect binding to the host protein Daxx. Coimmunoprecipitation assays with antibodies to Daxx and ICP4, ICP27, and ICP8 showed that the respective proteins interact, possibly forming a complex. A potential complex between the origin, early viral DNA-binding protein ICP8 and Daxx did not result in transcription at ND10. Thus, the deposition of transcriptionally active HSV-1 genomes at ND10 is most likely a consequence of retention at ND10 through the interaction of viral genome-bound ICP4 and ICP27 with Daxx. Such a complex might be more likely immobilized at the outside of ND10 by the PML-interacting Daxx than at other nuclear sites.

scholarly journals Listeria monocytogenes Isolates from Foods and Humans Form Distinct but Overlapping Populations

2004 ◽  
Vol 70 (10) ◽  
pp. 5833-5841 ◽  
Author(s):  
Michael J. Gray ◽  
Ruth N. Zadoks ◽  
Esther D. Fortes ◽  
Belgin Dogan ◽  
Steven Cai ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Plaque Assay ◽  
Virulence Gene ◽  
The United States ◽  
Polymorphism Analysis ◽  
Genetic Lineages ◽  
The Public ◽  
Cell Spread ◽  
Specific Food

ABSTRACT A total of 502 Listeria monocytogenes isolates from food and 492 from humans were subtyped by EcoRI ribotyping and PCR-restriction fragment length polymorphism analysis of the virulence gene hly. Isolates were further classified into genetic lineages based on subtyping results. Food isolates were obtained through a survey of selected ready-to-eat food products in Maryland and California in 2000 and 2001. Human isolates comprised 42 isolates from invasive listeriosis cases reported in Maryland and California during 2000 and 2001 as well as an additional 450 isolates from cases that had occurred throughout the United States, predominantly from 1997 to 2001. Assignment of isolates to lineages and to the majority of L. monocytogenes subtypes was significantly associated with the isolate source (food or human), although most subtypes and lineages included both human and food isolates. Some subtypes were also significantly associated with isolation from specific food types. Tissue culture plaque assay characterization of the 42 human isolates from Maryland and California and of 91 representative food isolates revealed significantly higher average infectivity and cell-to-cell spread for the human isolates, further supporting the hypothesis that food and human isolates form distinct populations. Combined analysis of subtype and cytopathogenicity data showed that strains classified into specific ribotypes previously linked to multiple human listeriosis outbreaks, as well as those classified into lineage I, are more common among human cases and generate larger plaques than other subtypes, suggesting that these subtypes may represent particularly virulent clonal groups. These data will provide a framework for prediction of the public health risk associated with specific L. monocytogenes subtypes.

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