Cytoplasmic virion assembly compartment of betaherpesviruses

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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Cytomegalovirus miRNAs Target Secretory Pathway Genes to Facilitate Formation of the Virion Assembly Compartment and Reduce Cytokine Secretion

Cell Host & Microbe ◽

10.1016/j.chom.2014.02.004 ◽

2014 ◽

Vol 15 (3) ◽

pp. 363-373 ◽

Cited By ~ 77

Author(s):

Lauren M. Hook ◽

Finn Grey ◽

Robert Grabski ◽

Rebecca Tirabassi ◽

Tracy Doyle ◽

...

Keyword(s):

Secretory Pathway ◽

Cytokine Secretion ◽

Virion Assembly ◽

Assembly Compartment

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Control of Cytoplasmic Maturation Events by Cytomegalovirus Tegument Protein pp150

Journal of Virology ◽

10.1128/jvi.00533-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9433-9444 ◽

Cited By ~ 36

Author(s):

Ritesh Tandon ◽

Edward S. Mocarski

Keyword(s):

Cellular Localization ◽

Cytoplasmic Inclusion ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Reading Frame ◽

Virion Assembly ◽

Recombinant Viruses ◽

Infected Cells ◽

Assembly Compartment

ABSTRACT Cytomegalovirus replication depends upon a betaherpesvirus-conserved 150-kDa tegument phosphoprotein (pp150; encoded by UL32) that supports the final steps in virion maturation at cytoplasmic assembly compartments. Amino acid substitutions were introduced into conserved region 1 (CR1) and CR2 of pp150, affecting a region that may interact with nucleocapsids. Two independent CR2 point mutants (N201A and G207A) failed to support viral replication in evaluations by a transient complementation assay or after reconstruction into recombinant viruses. An assembly compartment-like cytoplasmic inclusion developed in UL32 mutant virus-infected cells that was similar to that of wild-type virus-infected cells. The cellular localization of the trans-Golgi marker Golgin-97 suggested differences in the organization of the assembly compartment compared to that of wild-type virus-infected cells. Replication-defective CR2 point mutants exhibited the same phenotype as that of a virus carrying a complete deletion of the UL32 open reading frame in these assays. Electron micrographs of fibroblasts at 3 or 5 days postinfection with a deletion mutant (ΔUL32) grown on UL32-complementing cells showed a similar number and morphology of capsids in the nucleus, but the cytoplasmic region associated with virion assembly appeared highly vesiculated and contained few recognizable nucleocapsids or complete virus particles. These data demonstrate that the principle role of pp150 is to retain nucleocapsid organization through secondary envelopment at the assembly compartment.

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Arf GTPases Are Required for the Establishment of the Pre-Assembly Compartment in the Early Phase of Cytomegalovirus Infection

Life ◽

10.3390/life11080867 ◽

2021 ◽

Vol 11 (8) ◽

pp. 867

Author(s):

Valentino Pavišić ◽

Hana Mahmutefendić Mahmutefendić Lučin ◽

Gordana Blagojević Blagojević Zagorac ◽

Pero Lučin

Keyword(s):

Early Phase ◽

Cytomegalovirus Infection ◽

Intracellular Localization ◽

Host Cells ◽

Small Interfering Rnas ◽

Virion Assembly ◽

Early Endosomes ◽

Arf Gtpases ◽

Golgi Network ◽

Assembly Compartment

Shortly after entering the cells, cytomegaloviruses (CMVs) initiate massive reorganization of cellular endocytic and secretory pathways, which results in the forming of the cytoplasmic virion assembly compartment (AC). We have previously shown that the formation of AC in murine CMV- (MCMV) infected cells begins in the early phase of infection (at 4–6 hpi) with the pre-AC establishment. Pre-AC comprises membranes derived from the endosomal recycling compartment, early endosomes, and the trans-Golgi network, which is surrounded by fragmented Golgi cisterns. To explore the importance of Arf GTPases in the biogenesis of the pre-AC, we infected Balb 3T3 cells with MCMV and analyzed the expression and intracellular localization of Arf proteins in the early phases (up to 16 hpi) of infection and the development of pre-AC in cells with a knockdown of Arf protein expression by small interfering RNAs (siRNAs). Herein, we show that even in the early phase, MCMVs cause massive reorganization of the Arf system of the host cells and induce the over-recruitment of Arf proteins onto the membranes of pre-AC. Knockdown of Arf1, Arf3, Arf4, or Arf6 impaired the establishment of pre-AC. However, the knockdown of Arf1 and Arf6 also abolished the establishment of infection. Our study demonstrates that Arf GTPases are required for different steps of early cytomegalovirus infection, including the establishment of the pre-AC.

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Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis

Journal of Virology ◽

10.1128/jvi.01827-21 ◽

2022 ◽

Author(s):

Bo Yang ◽

YongXuan Yao ◽

Han Cheng ◽

Xian-Zhang Wang ◽

Yue-peng Zhou ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Host Protein ◽

Endomembrane System ◽

Virion Assembly ◽

Repeat Domain ◽

Virion Morphogenesis ◽

Membrane Reorganization ◽

Golgi Network ◽

Assembly Compartment

Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 200 predicted open reading frames and exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was up-regulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and co-localized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside of the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. Importance During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for anti-viral treatment.

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

Journal of Virology ◽

10.1128/jvi.03416-14 ◽

2014 ◽

Vol 89 (6) ◽

pp. 3049-3061 ◽

Cited By ~ 33

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.

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Phosphorylation of Golgi Peripheral Membrane Protein Grasp65 Is an Integral Step in the Formation of the Human Cytomegalovirus Cytoplasmic Assembly Compartment

mBio ◽

10.1128/mbio.01554-16 ◽

2016 ◽

Vol 7 (5) ◽

Cited By ~ 13

Author(s):

G. Michael Rebmann ◽

Robert Grabski ◽

Veronica Sanchez ◽

William J. Britt

Keyword(s):

Infected Cell ◽

Human Cytomegalovirus ◽

Infectious Virus ◽

Viral Proteins ◽

Structural Proteins ◽

Golgi Membrane ◽

Cellular Membranes ◽

Virion Assembly ◽

Intracellular Membranes ◽

Assembly Compartment

ABSTRACT Human cytomegalovirus (HCMV) is the largest member of the Herpesviridae and represents a significant cause of disease. During virus replication, HCMV alters cellular functions to facilitate its replication, including significant reorganization of the secretory and endocytic pathways of the infected cell. A defining morphologic change of the infected cell is the formation of a membranous structure in the cytoplasm that is designated the virion assembly compartment (AC), which consists of virion structural proteins surrounded by cellular membranes. The loss of normal Golgi compartment morphology and its relocalization from a juxtanuclear ribbonlike structure to a series of concentric rings on the periphery of the AC represents a readily recognized reorganization of cellular membranes in the HCMV-infected cell. Although trafficking of viral proteins to this compartment is required for the assembly of infectious virions, the functional significance of the reorganization of intracellular membranes like the Golgi membranes into the AC in the assembly of infectious virus remains understudied. In this study, we determined that Golgi membrane ribbon fragmentation increased during the early cytoplasmic phase of virion assembly and that Golgi membrane fragmentation in infected cells was dependent on the phosphorylation of an integral cis- Golgi protein, Grasp65. Inhibition of Golgi membrane fragmentation and of its reorganization into the AC resulted in decreased production of infectious particles and alteration of the incorporation of an essential protein into the envelope of the mature virion. These results demonstrated the complexity of the virus-host cell interactions required for efficient assembly of this large DNA virus. IMPORTANCE The human cytomegalovirus (HCMV)-induced reorganization of intracellular membranes that is required for the formation of the viral assembly compartment (AC) has been an area of study over the last 20 years. The significance of this virus-induced structure has been evinced by the results of several studies which showed that relocalization of viral proteins to the AC was required for efficient assembly of infectious virus. In this study, we have identified a mechanism for the fragmentation of the Golgi ribbon in the infected cell en route to AC morphogenesis. Identification of this fundamental process during HCMV replication allowed us to propose that the functional role of Golgi membrane reorganization during HCMV infection was the concentration of viral structural proteins and subviral structures into a single intracellular compartment in order to facilitate efficient protein-protein interactions and the virion protein trafficking required for the assembly of this large and structurally complex virus.

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Cytomegalovirus Strain TB40/E Restrictions and Adaptations to Growth in ARPE-19 Epithelial Cells

Microorganisms ◽

10.3390/microorganisms8040615 ◽

2020 ◽

Vol 8 (4) ◽

pp. 615

Author(s):

Mai Vo ◽

Alexis Aguiar ◽

Michael A. McVoy ◽

Laura Hertel

Keyword(s):

Epithelial Cells ◽

Cell Types ◽

Virion Assembly ◽

Genomic Changes ◽

Bodily Fluids ◽

Post Entry ◽

Assembly Compartment ◽

Mixed Stock ◽

Entry Efficiency

Despite displaying broad tropism in vivo, human cytomegalovirus (CMV) contained in bodily fluids replicates inefficiently in most cultured cell types except fibroblasts. As propagation in fibroblasts leads to the accumulation of genomic changes, a number of strains were generated by serial passaging on endothelial cells. One of these, TB40/E, was shown to contain a mixture of genetically distinct virus variants, and to retain tropism for fibroblasts, endothelial and epithelial cells. Cloning of an endotheliotropic subpopulation produced the TB40-BAC4 variant, extensively used in CMV tropism studies. Because TB40-BAC4 represents only one of the different variants comprising TB40/E, we generated a series of epithelial-cell adapted stocks derived from a TB40/E mixed stock, rather than from TB40-BAC4. Within two passages on ARPE-19 cells, virus populations were produced with the ability to enter and initiate replication with similar efficiencies in both epithelial cells and fibroblasts. Although the ability to release progeny also increased, cell-free virus yields from ARPE-19 cells remained consistently two to three-logs lower than from fibroblasts, hinting at the existence of a post-entry and post-genome synthesis block in epithelial cells. Multinucleated syncytia also rapidly appeared exclusively in ARPE-19 cell cultures, where their numbers and dimensions increased with virus passage. Irrespective of the number of infected nuclei comprising each syncytium, however, only one cytoplasmic virion assembly compartment was consistently observed, leading us to speculate that improvements in entry efficiency associated with ARPE-19 cell adaptation lead to the development of syncytia, which may negatively affect progeny release by limiting the amount of resources available to maturing virions.

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Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly

Journal of Virology ◽

10.1128/jvi.02101-20 ◽

2021 ◽

Author(s):

Bo Yang ◽

YongXuan Yao ◽

Hui Wu ◽

Hong Yang ◽

Xue-Hui Ma ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Late Phase ◽

Viral Gene ◽

Virion Assembly ◽

Nuclear Egress ◽

Early Endosomes ◽

Virion Morphogenesis ◽

Wd Repeat ◽

Assembly Compartment

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, 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 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis. Importance Human 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 reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.

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Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection

Open Biology ◽

10.1098/rsob.160298 ◽

2017 ◽

Vol 7 (11) ◽

pp. 160298 ◽

Cited By ~ 7

Author(s):

Jonathan Pavelin ◽

Dominique McCormick ◽

Stephen Chiweshe ◽

Saranya Ramachandran ◽

Yao-Tang Lin ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Virion Assembly ◽

Complex Process ◽

Novel Host ◽

Infected Cells ◽

Human Cytomegalovirus Infection ◽

Infection Inhibition ◽

Or Gene ◽

Assembly Compartment

Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy.

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