The host exosome pathway underpins biogenesis of the human cytomegalovirus virion

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection. This suggested a common membrane origin, and utilization of host exosome machinery for virion assembly and egress. Thus, we selected a panel of exosome proteins for knock down, and confirmed that loss of 7/9 caused significantly less HCMV production. Saliently, we report that VAMP3 is essential for viral trafficking and release of infectious progeny, in various HCMV strains and cell types. Therefore, we establish that the host exosome pathway is intrinsic for HCMV maturation, and reveal new host regulators involved in viral trafficking, virion envelopment, and release. Our findings underpin future investigation of host exosome proteins as important modulators of HCMV replication with antiviral potential.

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Human Cytomegalovirus Drives WDR5 to the Virion Assembly Compartment to Facilitate Virion Assembly

10.1101/2020.08.03.235630 ◽

2020 ◽

Author(s):

Bo Yang ◽

YongXuan Yao ◽

Hui Wu ◽

Hong Yang ◽

Xue-Hui Ma ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Antiviral Treatment ◽

Critical Role ◽

Virion Assembly ◽

Potential Target ◽

Nuclear Egress ◽

Early Endosomes ◽

Hcmv Replication ◽

Assembly Compartment

AbstractWe previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

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MicroRNA miR-21 Attenuates Human Cytomegalovirus Replication in Neural Cells by Targeting Cdc25a

Journal of Virology ◽

10.1128/jvi.01740-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1070-1082 ◽

Cited By ~ 43

Author(s):

Ya-Ru Fu ◽

Xi-Juan Liu ◽

Xiao-Jun Li ◽

Zhang-zhou Shen ◽

Bo Yang ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Birth Defects ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Viral Gene Expression ◽

Viral Gene ◽

Gene Products ◽

Hcmv Replication

ABSTRACTCongenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily manifesting as neurological disorders. HCMV infection alters expression of cellular microRNAs (miRs) and induces cell cycle arrest, which in turn modifies the cellular environment to favor virus replication. Previous observations found that HCMV infection reduces miR-21 expression in neural progenitor/stem cells (NPCs). Here, we show that infection of NPCs and U-251MG cells represses miR-21 while increasing the levels of Cdc25a, a cell cycle regulator and known target of miR-21. These opposing responses to infection prompted an investigation of the relationship between miR-21, Cdc25a, and viral replication. Overexpression of miR-21 in NPCs and U-251MG cells inhibited viral gene expression, genome replication, and production of infectious progeny, while shRNA-knockdown of miR-21 in U-251MG cells increased viral gene expression. In contrast, overexpression of Cdc25a in U-251MG cells increased viral gene expression and production of infectious progeny and overcame the inhibitory effects of miR-21 overexpression. Three viral gene products—IE1, pp71, and UL26—were shown to inhibit miR-21 expression at the transcriptional level. These results suggest that Cdc25a promotes HCMV replication and elevation of Cdc25a levels after HCMV infection are due in part to HCMV-mediated repression of miR-21. Thus, miR-21 is an intrinsic antiviral factor that is modulated by HCMV infection. This suggests a role for miR-21 downregulation in the neuropathogenesis of HCMV infection of the developing CNS.IMPORTANCEHuman cytomegalovirus (HCMV) is a ubiquitous pathogen and has very high prevalence among population, especially in China, and congenital HCMV infection is a major cause for birth defects. Elucidating virus-host interactions that govern HCMV replication in neuronal cells is critical to understanding the neuropathogenesis of birth defects resulting from congenital infection. In this study, we confirm that HCMV infection downregulates miR-21 but upregulates Cdc25a. Further determined the negative effects of cellular miRNA miR-21 on HCMV replication in neural progenitor/stem cells and U-251MG glioblastoma/astrocytoma cells. More importantly, our results provide the first evidence that miR-21 negatively regulates HCMV replication by targeting Cdc25a, a vital cell cycle regulator. We further found that viral gene products of IE1, pp71, and UL26 play roles in inhibiting miR-21 expression, which in turn causes increases in Cdc25a and benefits HCMV replication. Thus, miR-21 appears to be an intrinsic antiviral factor that represents a potential target for therapeutic intervention.

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Checks and balances between human cytomegalovirus replication and indoleamine-2,3-dioxygenase

Journal of General Virology ◽

10.1099/vir.0.061994-0 ◽

2014 ◽

Vol 95 (3) ◽

pp. 659-670 ◽

Cited By ~ 9

Author(s):

Albert Zimmermann ◽

Sebastian Hauka ◽

Marco Maywald ◽

Vu Thuy Khanh Le ◽

Silvia K. Schmidt ◽

...

Keyword(s):

Epithelial Cells ◽

Human Cytomegalovirus ◽

Cell Types ◽

Interferon Γ ◽

Counter Measures ◽

Indoleamine 2,3 Dioxygenase ◽

Infected Cells ◽

Hcmv Replication ◽

Ifn Γ ◽

The Impact

Despite a rigorous blockade of interferon-γ (IFN-γ) signalling in infected fibroblasts as a mechanism of immune evasion by human cytomegalovirus (HCMV), IFN-γ induced indoleamine-2,3-dioxygenase (IDO) has been proposed to represent the major antiviral restriction factor limiting HCMV replication in epithelial cells. Here we show that HCMV efficiently blocks transcription of IFN-γ-induced IDO mRNA both in infected fibroblasts and epithelial cells even in the presence of a preexisting IFN-induced antiviral state. This interference results in severe suppression of IDO bioactivity in HCMV-infected cells and restoration of vigorous HCMV replication. Depletion of IDO expression nonetheless substantially alleviated the antiviral impact of IFN-γ treatment in both cell types. These findings highlight the effectiveness of this IFN-γ induced effector gene in restricting HCMV productivity, but also the impact of viral counter-measures.

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The Endothelin Receptor Antagonist Macitentan Inhibits Human Cytomegalovirus Infection

Cells ◽

10.3390/cells10113072 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3072

Author(s):

Natalia Landázuri ◽

Jennifer Gorwood ◽

Ylva Terelius ◽

Fredrik Öberg ◽

Koon Chu Yaiw ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Cell Types ◽

Productive Infection ◽

Endothelin Receptor ◽

Viral Control ◽

Congenital Birth Defects ◽

Specific Antagonist ◽

Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection is an important cause of morbidity and mortality in immunocompromised patients and a major etiological factor for congenital birth defects in newborns. Ganciclovir and its pro-drug valganciclovir are the preferred drugs in use today for prophylaxis and treatment of viremic patients. Due to long treatment times, patients are at risk for developing viral resistance to ganciclovir and to other drugs with a similar mechanism of action. We earlier found that the endothelin receptor B (ETBR) is upregulated during HCMV infection and that it plays an important role in the life cycle of this virus. Here, we tested the hypothesis that ETBR blockade could be used in the treatment of HCMV infection. As HCMV infection is specific to humans, we tested our hypothesis in human cell types that are relevant for HCMV pathogenesis; i.e., endothelial cells, epithelial cells and fibroblasts. We infected these cells with HCMV and treated them with the ETBR specific antagonist BQ788 or ETR antagonists that are approved by the FDA for treatment of pulmonary hypertension; macitentan, its metabolite ACT-132577, bosentan and ambrisentan, and as an anti-viral control, we used ganciclovir or letermovir. At concentrations expected to be relevant in vivo, macitentan, ACT-132577 and BQ788 effectively inhibited productive infection of HCMV. Of importance, macitentan also inhibited productive infection of a ganciclovir-resistant HCMV isolate. Our results suggest that binding or signaling through ETBR is crucial for viral replication, and that selected ETBR blockers inhibit HCMV infection.

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Characteristics of Immediate-Early 2 (IE2) and UL84 Proteins in UL84-Independent Strains of Human Cytomegalovirus (HCMV)

Microbiology Spectrum ◽

10.1128/spectrum.00539-21 ◽

2021 ◽

Author(s):

Salome Manska ◽

Cyprian C. Rossetto

Keyword(s):

Birth Defects ◽

Human Cytomegalovirus ◽

Latent Infection ◽

Lytic Replication ◽

Morbidity And Mortality ◽

Immediate Early ◽

Initial Infection ◽

Congenital Birth Defects

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised individuals and is also the leading viral cause of congenital birth defects. After initial infection, HCMV establishes a lifelong latent infection with periodic reactivation and lytic replication.

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Human Cytomegalovirus Replication Is Inhibited by the Autophagy-Inducing Compounds Trehalose and SMER28 through Distinctively Different Mechanisms

Journal of Virology ◽

10.1128/jvi.02015-17 ◽

2017 ◽

Vol 92 (6) ◽

Cited By ~ 4

Author(s):

Alex E. Clark ◽

Maite Sabalza ◽

Philip L. S. M. Gordts ◽

Deborah H. Spector

Keyword(s):

Glucose Uptake ◽

Human Cytomegalovirus ◽

Cell Types ◽

Genome Replication ◽

Cell Type ◽

Early Protein ◽

Mechanism Of Inhibition ◽

New Strategy ◽

Viral Genome Replication ◽

Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is the top viral cause of birth defects worldwide, and current therapies have high toxicity. We previously reported that the mTOR-independent autophagy-inducing disaccharide trehalose inhibits HCMV replication in multiple cell types. Here, we examine the mechanism of inhibition and introduce the autophagy inducer SMER28 as an additional inhibitor of HCMV acting through a different mechanism. We find that trehalose induces vacuolation and acidification of vacuoles and that debris, including debris with an appearance consistent with that of abnormal virions, is present in multivesicular bodies. Trehalose treatment increased the levels of Rab7, a protein required for lysosomal biogenesis and fusion, and slightly decreased the levels of Rab11, which is associated with recycling endosomes. We also present evidence that trehalose can promote autophagy without altering cellular glucose uptake. We show that SMER28 inhibits HCMV at the level of early protein production and interferes with viral genome replication in a cell type-dependent fashion. Finally, we show that SMER28 treatment does not cause the vacuolation, acidification, or redistribution of Rab7 associated with trehalose treatment and shows only a modest and cell type-dependent effect on autophagy. We propose a model in which the reciprocal effects on Rab7 and Rab11 induced by trehalose contribute to the redirection of enveloped virions from the plasma membrane to acidified compartments and subsequent degradation, and SMER28 treatment results in decreased expression levels of early and late proteins, reducing the number of virions produced without the widespread vacuolation characteristic of trehalose treatment. IMPORTANCE There is a need for less toxic HCMV antiviral drugs, and modulation of autophagy to control viral infection is a new strategy that takes advantage of virus dependence on autophagy inhibition. The present study extends our previous work on trehalose by showing a possible mechanism of action and introduces another autophagy-inducing compound, SMER28, that is effective against HCMV in several cell types. The mechanism by which trehalose induces autophagy is currently unknown, although our data show that trehalose does not inhibit cellular glucose uptake in cells relevant for HCMV replication but instead alters virion degradation by promoting acidic vacuolization. The comparison of our cell types and those used by others highlights the cell type-dependent nature of studying autophagy.

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Regulation of the Transcription and Replication Cycle of Human Cytomegalovirus Is Insensitive to Genetic Elimination of the Cognate NF-κB Binding Sites in the Enhancer

Journal of Virology ◽

10.1128/jvi.00640-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9899-9904 ◽

Cited By ~ 34

Author(s):

Montse Gustems ◽

Eva Borst ◽

Chris A. Benedict ◽

Carmen Pérez ◽

Martin Messerle ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Binding Sites ◽

Regulatory Network ◽

Cell Types ◽

Response Elements ◽

Multiple Cell ◽

Hcmv Replication ◽

Major Immediate Early Promoter ◽

Transcription And Replication

ABSTRACT The role of NF-κB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-κB response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEP-enhancer that, although this region is essential for HCMV growth, none of the four NF-κB response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.

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Human Cytomegalovirus Persistently Infects Aortic Endothelial Cells

Journal of Virology ◽

10.1128/jvi.72.7.5661-5668.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5661-5668 ◽

Cited By ~ 88

Author(s):

Kenneth N. Fish ◽

Cecilia Soderberg-Naucler ◽

Lisa K. Mills ◽

Stephan Stenglein ◽

Jay A. Nelson

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Antigen Expression ◽

Cell Types ◽

Aortic Endothelial Cells ◽

Phenotypic Differences ◽

Extracellular Virus ◽

Viral Particles ◽

Infected Cells ◽

Hcmv Replication

ABSTRACT Endothelial cells (EC) have been implicated as constituting an important cell type in the pathogenesis of human cytomegalovirus (HCMV). Microvascular and macrovascular EC exhibit different biochemical and functional properties depending on the organ of origin. Phenotypic differences between microvascular and macrovascular EC may alter the ability of these cells to support HCMV replication. In this study, we compared the replication of HCMV in primary macrovascular aortic EC (AEC) with that in brain microvascular EC (BMVEC). An examination of IE72, pp65, and gB viral antigen expression in BMVEC and AEC by immunoflourescence revealed similar frequencies of infected cells. Intracellular production of virus was 3 log units greater in BMVEC than in AEC, while equal quantities of extracellular virus were produced in both cell types. HCMV infection of BMVEC resulted in rapid cellular lysis, while the virus was nonlytic and continuously released from HCMV-infected AEC for the life span of the culture. An examination of infected cells by electron microscopy revealed the formation of abundant nucleocapsids in both AEC and BMVEC. However, significant amounts of mature viral particles were only detected in the cytoplasm of BMVEC. These observations indicate that levels of HCMV replication in EC obtained from different organs are distinct and suggest that persistently infected AEC may serve as a reservoir of virus.

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The Skull’s Girder: A Brief Review of the Cranial Base

Journal of Developmental Biology ◽

10.3390/jdb9010003 ◽

2021 ◽

Vol 9 (1) ◽

pp. 3

Author(s):

Shankar Rengasamy Venugopalan ◽

Eric Van Otterloo

Keyword(s):

Birth Defects ◽

Vertebral Column ◽

Endochondral Ossification ◽

Cranial Base ◽

Facial Skeleton ◽

The Core ◽

Skull Vault ◽

Congenital Birth Defects ◽

Intimate Association ◽

The Brain

The cranial base is a multifunctional bony platform within the core of the cranium, spanning rostral to caudal ends. This structure provides support for the brain and skull vault above, serves as a link between the head and the vertebral column below, and seamlessly integrates with the facial skeleton at its rostral end. Unique from the majority of the cranial skeleton, the cranial base develops from a cartilage intermediate—the chondrocranium—through the process of endochondral ossification. Owing to the intimate association of the cranial base with nearly all aspects of the head, congenital birth defects impacting these structures often coincide with anomalies of the cranial base. Despite this critical importance, studies investigating the genetic control of cranial base development and associated disorders lags in comparison to other craniofacial structures. Here, we highlight and review developmental and genetic aspects of the cranial base, including its transition from cartilage to bone, dual embryological origins, and vignettes of transcription factors controlling its formation.

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Early human cytomegalovirus replication after transplantation is associated with a decreased relapse risk: evidence for a putative virus-versus-leukemia effect in acute myeloid leukemia patients

Blood ◽

10.1182/blood-2010-08-304121 ◽

2011 ◽

Vol 118 (5) ◽

pp. 1402-1412 ◽

Cited By ~ 198

Author(s):

Ahmet H. Elmaagacli ◽

Nina K. Steckel ◽

Michael Koldehoff ◽

Yael Hegerfeldt ◽

Rudolf Trenschel ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Human Cytomegalovirus ◽

Myeloid Leukemia ◽

Homogeneous Population ◽

Relapse Risk ◽

Pp65 Antigenemia ◽

Hcmv Replication ◽

The Impact ◽

Early Human ◽

Acute Myeloid

Abstract The impact of early human cytomegalovirus (HCMV) replication on leukemic recurrence was evaluated in 266 consecutive adult (median age, 47 years; range, 18-73 years) acute myeloid leukemia patients, who underwent allogeneic stem cell transplantation (alloSCT) from 10 of 10 high-resolution human leukocyte Ag-identical unrelated (n = 148) or sibling (n = 118) donors. A total of 63% of patients (n = 167) were at risk for HCMV reactivation by patient and donor pretransplantation HCMV serostatus. In 77 patients, first HCMV replication as detected by pp65-antigenemia assay developed at a median of 46 days (range, 25-108 days) after alloSCT. Taking all relevant competing risk factors into account, the cumulative incidence of hematologic relapse at 10 years after alloSCT was 42% (95% confidence interval [CI], 35%-51%) in patients without opposed to 9% (95% CI, 4%-19%) in patients with early pp65-antigenemia (P < .0001). A substantial and independent reduction of the relapse risk associated with early HCMV replication was confirmed by multivariate analysis using time-dependent covariate functions for grades II to IV acute and chronic graft-versus-host disease, and pp65-antigenemia (hazard ratio = 0.2; 95% CI, 0.1-0.4, P < .0001). This is the first report that demonstrates an independent and substantial reduction of the leukemic relapse risk after early replicative HCMV infection in a homogeneous population of adult acute myeloid leukemia patients.

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