scholarly journals Human cytomegalovirus exploits TACC3 to control microtubule dynamics and late stages of infection

2021 ◽  
Author(s):  
Colleen Furey ◽  
Helen Astar ◽  
Derek Walsh
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Coiled Coil ◽  
Microtubule Dynamics ◽  
Replication Cycle ◽  
Microtubule Organizing Center ◽  
Viral Particles ◽  
Organizing Center ◽  
Assembly Compartment ◽  
Late Stages

While it is well established that microtubules (MTs) facilitate various stages of virus replication, how viruses actively control MT dynamics and functions remains less-well understood. Recent work has begun to reveal how several viruses exploit End-Binding (EB) proteins and their associated microtubule plus-end tracking proteins (+TIPs), in particular to enable loading of viral particles onto MTs for retrograde transport during early stages of infection. But distinct from other viruses studied to date, at mid-to-late stages of its unusually protracted replication cycle human cytomegalovirus (HCMV) increases the expression of all three EB family members. This occurs coincident with the formation of a unique structure termed the Assembly Compartment (AC), which serves as a Golgi-derived MT organizing center. Together, the AC and distinct EB proteins enable HCMV to increase the formation of dynamic and acetylated microtubule subsets to regulate distinct aspects of the viral replication cycle. Here, we reveal that HCMV also exploits EB-independent +TIP pathways by specifically increasing the expression of Transforming Acidic Coiled Coil protein 3 (TACC3) to recruit the MT polymerase, chTOG from initial sites of MT nucleation in the AC out into the cytosol, thereby increasing dynamic MT growth. Preventing TACC3 increases or depleting chTOG impaired MT polymerization, resulting in defects in early versus late endosome organization in and around the AC as well as defects in viral trafficking and spread. Our findings provide the first example of a virus that actively exploits EB-independent +TIP pathways to regulate MT dynamics and control late stages of virus replication. Importance Diverse viruses rely on host cell microtubule networks in order to transport viral particles within the dense cytoplasmic environment and to control the broader architecture of the cell to facilitate their replication. Yet precisely how viruses regulate the dynamic behavior and function of microtubule filaments remains poorly defined. We recently showed that the Assembly Compartment (AC) formed by human cytomegalovirus (HCMV) acts as a Golgi-derived microtubule organizing center. Here, we show that at mid-to-late stages of infection, HCMV increases the expression of Transforming Acidic Coiled Coil protein 3 (TACC3) in order to control the localization of the microtubule polymerase, chTOG. This in turn enables HCMV to generate dynamic microtubule subsets that organize endocytic vesicles in and around the AC and facilitate the transport of new viral particles released into the cytosol. Our findings reveal the first instance of viral targeting of TACC3 to control microtubule dynamics and virus spread.

scholarly journals Nuclear Targeting of Adenovirus Type 2 Requires CRM1-mediated Nuclear Export

2005 ◽  
Vol 16 (6) ◽  
pp. 2999-3009 ◽  
Author(s):  
Sten Strunze ◽  
Lloyd C. Trotman ◽  
Karin Boucke ◽  
Urs F. Greber
Keyword(s):  
Nuclear Export ◽  
Adenovirus Type ◽  
Nuclear Pore ◽  
Nuclear Targeting ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Transport ◽  
Viral Particles ◽  
Organizing Center ◽  
Adenovirus Type 2

Incoming adenovirus type 2 (Ad2) and Ad5 shuttle bidirectionally along microtubules, biased to the microtubule-organizing center by the dynein/dynactin motor complex. It is unknown how the particles reach the nuclear pore complex, where capsids disassemble and viral DNA enters the nucleus. Here, we identified a novel link between nuclear export and microtubule-mediated transport. Two distinct inhibitors of the nuclear export factor CRM1, leptomycin B (LMB) and ratjadone A (RJA) or CRM1-siRNAs blocked adenovirus infection, arrested cytoplasmic transport of viral particles at the microtubule-organizing center or in the cytoplasm and prevented capsid disassembly and nuclear import of the viral genome. In mitotic cells where CRM1 is in the cytoplasm, adenovirus particles were not associated with microtubules but upon LMB treatment, they enriched at the spindle poles implying that CRM1 inhibited microtubule association of adenovirus. We propose that CRM1, a nuclear factor exported by CRM1 or a protein complex containing CRM1 is part of a sensor mechanism triggering the unloading of the incoming adenovirus particles from microtubules proximal to the nucleus of interphase cells.

scholarly journals Stathmin Regulates Microtubule Dynamics and Microtubule Organizing Center Polarization in Activated T Cells

2012 ◽  
Vol 188 (11) ◽  
pp. 5421-5427 ◽  
Author(s):  
Erin L. Filbert ◽  
Marie Le Borgne ◽  
Joseph Lin ◽  
John E. Heuser ◽  
Andrey S. Shaw
Keyword(s):  
T Cells ◽  
Microtubule Dynamics ◽  
Microtubule Organizing Center ◽  
Activated T Cells ◽  
Organizing Center

scholarly journals Centrosome-dependent microtubule organization sets the conditions for axon formation

2020 ◽  
Author(s):  
Durga Praveen Meka ◽  
Oliver Kobler ◽  
Souhaila Wuesthoff ◽  
Birgit Schwanke ◽  
Christoph Krisp ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Growth Speed ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Axon Elongation ◽  
Axon Development ◽  
Organizing Center ◽  
Cytoskeleton Remodeling ◽  
Axon Specification ◽  
Developing Neurons

AbstractMicrotubule remodeling is critical during axon development when the more stable microtubules populate the axon. It is not completely understood, however, how this local cytoskeleton remodeling is coordinated. The centrosome, the main microtubule-organizing center (MTOC), has been suggested to be crucial for axon specification 1–5. Conversely, it was proposed that axon elongation is independent of centrosomal functions 6. Here we report that microtubule dynamics in early neurons follow a radial organization which establishes the conditions for the axon formation. Using high-resolution microscopy of early developing neurons, we demonstrate that few somatic acetylated microtubules are restricted near the centrosome. At later stages, however, acetylated microtubules spread out in the soma and concentrate in the growing axon. Furthermore, live-imaging of the microtubule plus-end binding protein EB3 in early differentiating neurons shows that growing microtubules have increased length and growth speed near the MTOC, suggesting local differences that might favor axon selection. Importantly, due to the lack of somatic stable/acetylated microtubules in early developing neurons, disruption of the F-actin cytoskeleton does not induce multiple axons, as it does at later stages of differentiation. Finally, we demonstrate that overexpression of the centrosomal protein 120 (Cep120), known for promoting microtubule acetylation and stabilization, induces multiple axons, while its downregulation decreases the content of proteins regulating microtubule dynamics and stability, hence hampering axon formation. Collectively, our data show that early centrosome-dependent microtubule organization contributes to axon formation.

scholarly journals LARG and mDia1 Link Gα12/13 to Cell Polarity and Microtubule Dynamics

2008 ◽  
Vol 19 (1) ◽  
pp. 30-40 ◽  
Author(s):  
Polyxeni Goulimari ◽  
Helga Knieling ◽  
Ulrike Engel ◽  
Robert Grosse
Keyword(s):  
Cell Polarity ◽  
Glycogen Synthase ◽  
Cell Polarization ◽  
Microtubule Dynamics ◽  
Microtubule Organizing Center ◽  
Embryonic Fibroblasts ◽  
Extracellular Signals ◽  
Receptor Signal ◽  
Organizing Center ◽  
G Protein Coupled

Regulation of cell polarity is a process observed in all cells. During directed migration, cells orientate their microtubule cytoskeleton and the microtubule-organizing-center (MTOC), which involves integrins and downstream Cdc42 and glycogen synthase kinase-3β activity. However, the contribution of G protein-coupled receptor signal transduction for MTOC polarity is less well understood. Here, we report that the heterotrimeric Gα12 and Gα13 proteins are necessary for MTOC polarity and microtubule dynamics based on studies using Gα12/13-deficient mouse embryonic fibroblasts. Cell polarization involves the Gα12/13-interacting leukemia-associated RhoGEF (LARG) and the actin-nucleating diaphanous formin mDia1. Interestingly, LARG associates with pericentrin and localizes to the MTOC and along microtubule tracks. We propose that Gα12/13 proteins exert essential functions linking extracellular signals to microtubule dynamics and cell polarity via RhoGEF and formin activity.

scholarly journals Accumulation of Virion Tegument and Envelope Proteins in a Stable Cytoplasmic Compartment during Human Cytomegalovirus Replication: Characterization of a Potential Site of Virus Assembly

2000 ◽  
Vol 74 (2) ◽  
pp. 975-986 ◽  
Author(s):  
Veronica Sanchez ◽  
Kenneth D. Greis ◽  
Elizabeth Sztul ◽  
William J. Britt
Keyword(s):  
Human Cytomegalovirus ◽  
Virus Assembly ◽  
Human Fibroblasts ◽  
Cell Protein ◽  
Cell Fractionation ◽  
Microtubule Organizing Center ◽  
Tegument Proteins ◽  
Organizing Center ◽  
Processed Form ◽  
Replicative Cycle

ABSTRACT The assembly of human cytomegalovirus (HCMV) is thought to be similar to that which has been proposed for alphaherpesviruses and involve envelopment of tegumented subviral particles at the nuclear membrane followed by export from the cell by a poorly defined pathway. However, several studies have shown that at least two tegument virion proteins remain in the cytoplasm during the HCMV replicative cycle, thereby suggesting that HCMV cannot acquire its final envelope at the nuclear envelope. We investigated the assembly of HCMV by determining the intracellular trafficking of the abundant tegument protein pp150 (UL32) in productively infected human fibroblasts. Our results indicated that pp150 remained within the cytoplasm throughout the replicative cycle of HCMV and accumulated in a stable, juxtanuclear structure late in infection. Image analysis using a variety of cell protein-specific antibodies indicated that the pp150-containing structure was not a component of the endoplasmic reticulum, (ER), ER-Golgi intermediate compartment, cis or medial Golgi, or lysosomes. Partial colocalization of the structure was noted with thetrans-Golgi network, and it appeared to lie in close proximity to the microtubule organizing center. Two additional tegument proteins (pp28 and pp65) and three envelope glycoproteins (gB, gH, and gp65) localized in this same structure late infection. This compartment appeared to be relatively stable since pp150, pp65, and the processed form of gB could be coisolated following cell fractionation. Our findings indicated that pp150 was expressed exclusively within the cytoplasm throughout the infectious cycle of HCMV and that the accumulation of the pp150 in this cytoplasmic structure was accompanied by at least five other virion proteins. These results suggested the possibility that this virus-induced structure represented a cytoplasmic site of virus assembly.

scholarly journals Mutated in Colorectal Cancer (MCC): a centrosomal protein that relocalizes to the ncMTOC during intestinal cell differentiation

2021 ◽  
Author(s):  
Lucian B. Tomaz ◽  
Bernard A. Liu ◽  
Sheena L.M. Ong ◽  
Ee Kim Tan ◽  
Meroshini M ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Intestinal Epithelium ◽  
Coiled Coil ◽  
Human Colon ◽  
Intestinal Cell ◽  
Microtubule Organizing Center ◽  
Human Colon Cancer ◽  
Protein Levels ◽  
Organizing Center ◽  
Casein Kinases

Mutated in Colorectal Cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, and that Mcc protein is distinctly associated with the centrosome in these cells. Upon intestinal cellular differentiation, Mcc is redeployed to the non-centrosomal microtubule organizing center (ncMTOC) at the apical domain of villus cells. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by Casein Kinases 1δ/ε, which are critical modulators of WNT signaling. Together, our findings support a putative role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

scholarly journals Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport

2008 ◽  
Vol 82 (13) ◽  
pp. 6492-6500 ◽  
Author(s):  
Jason G. Smith ◽  
Aurelia Cassany ◽  
Larry Gerace ◽  
Robert Ralston ◽  
Glen R. Nemerow
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Cell Entry ◽  
Adenovirus Infection ◽  
Membrane Penetration ◽  
Microtubule Organizing Center ◽  
Cytoplasmic Transport ◽  
Organizing Center ◽  
Dependent Transport ◽  
Late Stages

ABSTRACT Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

scholarly journals The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system

2005 ◽  
Vol 86 (2) ◽  
pp. 297-306 ◽  
Author(s):  
Detlef Michel ◽  
Irena Milotić ◽  
Markus Wagner ◽  
Bianca Vaida ◽  
Jens Holl ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Ex Vivo ◽  
Stop Codon ◽  
Artificial Chromosome ◽  
Replication Cycle ◽  
Coding Region ◽  
C Terminus ◽  
Translation Stop Codon

The human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of this study was to investigate the UL78 gene and to clarify whether it is essential for replication. UL78 transcription was activated early after infection, was inhibited by cycloheximide but not by phosphonoacetic acid, and resulted in a 1·7 kb mRNA. Later in the replication cycle, a second mRNA of 4 kb evolved, comprising the UL77 and UL78 ORFs. The 5′ end of the UL78 mRNA initiated 48 bp upstream of the translation start and the polyadenylated tail started 268 bp downstream of the UL78 translation stop codon within the UL79 ORF. By using bacterial artificial chromosome technology, a recombinant HCMV lacking most of the UL78 coding region was constructed. Successful reconstitution of the UL78-deficient virus proved that the gene was not essential for virus replication in fibroblasts. The deletion also did not reduce virus replication in ex vivo-cultured sections of human renal arteries. Analysis of viral proteins at different stages of the replication cycle confirmed these results. Among clinical HCMV isolates, the predicted UL78 protein was highly conserved. However, an accumulation of different single mutations could be found in the N-terminal region and at the very end of the C terminus. Due to the absence of an in vivo HCMV model, the role of UL78 in the pathogenesis of HCMV infection in humans remains unclear.

scholarly journals Human Cytomegalovirus Exploits Interferon-Induced Transmembrane Proteins To Facilitate Morphogenesis of the Virion Assembly Compartment

2014 ◽  
Vol 89 (6) ◽  
pp. 3049-3061 ◽  
Author(s):  
Maorong Xie ◽  
Baoqin Xuan ◽  
Jiaoyu Shan ◽  
Deng Pan ◽  
Yamei Sun ◽  
...  
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Virus Entry ◽  
Transmembrane Proteins ◽  
Host Cells ◽  
Lung Fibroblasts ◽  
Type I ◽  
Dna Virus ◽  
Virion Assembly ◽  
Assembly Compartment

ABSTRACTRecently, interferon-induced transmembrane proteins (IFITMs) have been identified to be key effector molecules in the host type I interferon defense system. The invasion of host cells by a large range of RNA viruses is inhibited by IFITMs during the entry step. However, the roles of IFITMs in DNA virus infections have not been studied in detail. In this study, we report that human cytomegalovirus (HCMV), a large human DNA virus, exploits IFITMs to facilitate the formation of the virion assembly compartment (vAC) during infection of human fibroblasts. We found that IFITMs were expressed constitutively in human embryonic lung fibroblasts (MRC5 cells). HCMV infection inhibited IFITM protein accumulation in the later stages of infection. Overexpression of an IFITM protein in MRC5 cells slightly enhanced HCMV production and knockdown of IFITMs by RNA interference reduced the virus titer by about 100-fold on day 8 postinfection, according to the findings of a virus yield assay at a low multiplicity of infection. Virus gene expression and DNA synthesis were not affected, but the typical round structure of the vAC was not formed after the suppression of IFITMs, thereby resulting in defective virion assembly and the production of less infectious virion particles. Interestingly, the replication of herpes simplex virus, a human herpesvirus that is closely related to HCMV, was not affected by the suppression of IFITMs in MRC5 cells. These results indicate that IFITMs are involved in a specific pathway required for HCMV replication.IMPORTANCEHCMV is known to repurpose the interferon-stimulated genes (ISGs) viperin and tetherin to facilitate its replication. Our results expand the range of ISGs that can be exploited by HCMV for its replication. This is also the first report of a proviral function of IFITMs in DNA virus replication. In addition, whereas previous studies showed that IFITMs modulate virus entry, which is a very early stage in the virus life cycle, we identified a new function of IFITMs during the very late stage of virus replication, i.e., virion assembly. Virus entry and assembly both involve vesicle transport and membrane fusion; thus, a common biochemical activity of IFITMs is likely to be involved. Therefore, our findings may provide a new platform for dissecting the molecular mechanism of action of IFITMs during the blocking or enhancement of virus infection, which are under intense investigation in this field.

scholarly journals Human Cytomegalovirus TRS1 Protein Is Required for Efficient Assembly of DNA-Containing Capsids

2004 ◽  
Vol 78 (19) ◽  
pp. 10221-10229 ◽  
Author(s):  
Joan E. Adamo ◽  
Jörg Schröer ◽  
Thomas Shenk
Keyword(s):  
Virus Replication ◽  
Human Cytomegalovirus ◽  
Dna Cleavage ◽  
Late Phase ◽  
Mutant Virus ◽  
Replication Cycle ◽  
Tegument Protein ◽  
Deficient Mutant ◽  
Infected Cells ◽  
Virus Replication Cycle

ABSTRACT The human cytomegalovirus tegument protein, pTRS1, appears to function at several discrete stages of the virus replication cycle. We previously demonstrated that pTRS1 acts during the late phase of infection to facilitate the production of infectious virions. We now have more precisely identified the late pTRS1 function by further study of a mutant virus lacking the TRS1 region, ADsubTRS1. We observed a significant reduction in the production of capsids, especially DNA-containing C-capsids, in mutant virus-infected cells. ADsubTRS1 exhibited normal cleavage of DNA concatemers, so the defect in C-capsid production must occur after DNA cleavage and before DNA is stably inserted into a capsid. Further, the normal virus-induced morphological reorganization of the nucleus did not occur after infection with the pTRS1-deficient mutant.

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