Human Cytomegalovirus Tegument Protein pUL71 Is Required for Efficient Virion Egress

ABSTRACT The human cytomegalovirus virion is composed of a DNA genome packaged in an icosahedral capsid, surrounded by a tegument of protein and RNA, all enclosed within a glycoprotein-studded envelope. Achieving this intricate virion architecture requires a coordinated process of assembly and egress. We show here that pUL71, a component of the virion tegument with a previously uncharacterized function, is required for the virus-induced reorganization of host cell membranes, which is necessary for efficient viral assembly and egress. A mutant that did not express pUL71 was able to efficiently accumulate viral genomes and proteins that were tested but was defective for the production and release of infectious virions. The protein localized to vesicular structures at the periphery of the viral assembly compartment, and during infection with a pUL71-deficient virus, these structures were grossly enlarged and aberrantly contained a cellular marker of late endosomes/lysosomes. Mutant virus preparations exhibited less infectivity per unit genome than wild-type virus preparations, due to aggregation of virus particles and their association with membrane fragments. Finally, mutant virus particles accumulated within the cytoplasm of infected cells and were localized to the periphery of large structures with properties of lysosomes, whose formation was kinetically favored in mutant-virus-infected cells. Together, these observations point to a role for pUL71 in the establishment and/or maintenance of a functional viral assembly compartment that is required for normal virion trafficking and egress from infected cells. IMPORTANCE In addition to causing disease in immunocompromised individuals, human cytomegalovirus is the leading known infectious cause of birth defects. To induce these pathologies, the virus must spread from its site of introduction to various organs and tissues in the body. The processes of viral assembly and egress, which underlie the spread of infection, are incompletely understood. We elucidate a role for a virus-coded protein, pUL71, in these processes and demonstrate the importance of maintaining an intricate, virus-induced reorganization of host cell membranes for efficient virus spread.

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Human Cytomegalovirus Envelope Protein gpUL132 Regulates Infectious Virus Production through Formation of the Viral Assembly Compartment

mBio ◽

10.1128/mbio.02044-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Hui Wu ◽

Barbara Kropff ◽

Michael Mach ◽

William J. Britt

Keyword(s):

Human Cytomegalovirus ◽

Envelope Glycoprotein ◽

Envelope Protein ◽

Infectious Virus ◽

Virus Production ◽

Viral Assembly ◽

Mutant Virus ◽

Infected Cells ◽

Assembly Compartment ◽

Infectious Unit

ABSTRACT The human cytomegalovirus (HCMV) UL132 open reading frame encodes a 270-amino-acid type I envelope glycoprotein, gpUL132. The deletion of UL132 (ΔUL132) from the HCMV genome results in a pronounced deficit in virus yield, with an approximately 2-log decrease in the production of infectious virus compared to the wild-type (WT) virus. Characterization of the ΔUL132 mutant virus indicated that it was less infectious with a high particle-to-infectious unit ratio and an altered composition of virion proteins compared to the WT virus. In addition, the viral assembly compartment (AC) failed to form in cells infected with the ΔUL132 mutant virus. The expression of gpUL132 in trans rescued the defects in the morphogenesis of the AC in cells infected with the ΔUL132 mutant virus and in infectious virus production. Furthermore, using cell lines expressing chimeric proteins, we demonstrated that the cytosolic domain of gpUL132 was sufficient to rescue AC formation and WT levels of virus production. Progeny virions from ΔUL132-infected cells expressing the cytosolic domain of gpUL132 exhibited particle-to-infectious unit ratios similar to those of the WT virus. Together, our findings argue that gpUL132 is essential for HCMV AC formation and the efficient production of infectious particles, thus highlighting the importance of this envelope protein for the virus-induced reorganization of intracellular membranes and AC formation in the assembly of infectious virus. IMPORTANCE Following infection of permissive cells, human cytomegalovirus (HCMV) induces the reorganization of intracellular membranes resulting in the formation of a distinctive membranous compartment in the cytoplasm of infected cells. This compartment has been designated the viral assembly compartment (AC) and is thought to be a site for cytoplasmic virion assembly and envelopment. In this study, we have demonstrated that a single virion envelope glycoprotein is essential for AC formation in infected cells, and in its absence, there is a significant decrease in the production of infectious virions. These findings are consistent with those from other studies that have demonstrated the importance of host cell proteins in the formation of the AC and demonstrate a critical role of a single virion protein in AC formation and the efficient assembly of infectious virus.

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Human Cytomegalovirus pUS24 Is a Virion Protein That Functions Very Early in the Replication Cycle

Journal of Virology ◽

10.1128/jvi.00399-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8371-8378 ◽

Cited By ~ 22

Author(s):

Xuyan Feng ◽

Jörg Schröer ◽

Dong Yu ◽

Thomas Shenk

Keyword(s):

Human Cytomegalovirus ◽

Mutant Virus ◽

Replication Cycle ◽

Immediate Early ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Virion Protein ◽

Viral Dna ◽

Infected Cells

ABSTRACT We have characterized the function of the human cytomegalovirus US24 gene, a US22 gene family member. Two US24-deficient mutants (BADinUS24 and BADsubUS24) exhibited a 20- to 30-fold growth defect, compared to their wild-type parent (BADwt), after infection at a relatively low (0.01 PFU/cell) or high (1 PFU/cell) input multiplicity. Representative virus-encoded proteins and viral DNA accumulated with normal kinetics to wild-type levels after infection with mutant virus when cells received equal numbers of mutant and wild-type infectious units. Further, the proteins were properly localized and no ultrastructural differences were found by electron microscopy in mutant-virus-infected cells compared to wild-type-virus-infected cells. However, virions produced by US24-deficient mutants had a 10-fold-higher genome-to-PFU ratio than wild-type virus. When infections were performed using equal numbers of input virus particles, the expression of immediate-early, early, and late viral proteins was substantially delayed and decreased in the absence of US24 protein. This delay is not due to inefficient virus entry, since two tegument proteins and viral DNA moved to the nucleus equally well in mutant- and wild-type-virus-infected cells. In summary, US24 is a virion protein and virions produced by US24-deficient viruses exhibit a block to the human cytomegalovirus replication cycle after viral DNA reaches the nucleus and before immediate-early mRNAs are transcribed.

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Reduction of phospholipase D activity during coxsackievirus infection

Journal of General Virology ◽

10.1099/vir.0.83169-0 ◽

2007 ◽

Vol 88 (11) ◽

pp. 3027-3030 ◽

Cited By ~ 1

Author(s):

Daniël Duijsings ◽

Els Wessels ◽

Sjenet E. van Emst-de Vries ◽

Willem J. G. Melchers ◽

Peter H. G. M. Willems ◽

...

Keyword(s):

Host Cell ◽

Phospholipase D ◽

Cell Membranes ◽

Ectopic Expression ◽

Rna Replication ◽

Enterovirus Infection ◽

Defective Mutant ◽

Infected Cells ◽

Host Cell Membranes ◽

Adp Ribosylation Factor

During enterovirus infection, host cell membranes are rigorously rearranged and modified. One ubiquitously expressed lipid-modifying enzyme that might contribute to these alterations is phospholipase D (PLD). Here, we investigated PLD activity in coxsackievirus-infected cells. We show that PLD activity is not required for efficient coxsackievirus RNA replication. Instead, PLD activity rapidly decreased upon infection and upon ectopic expression of the viral 3A protein, which inhibits the PLD activator ADP-ribosylation factor 1. However, similar decreases were observed during infection with coxsackieviruses carrying defective mutant 3A proteins. Possible causes for the reduction of PLD activity and the biological consequences are discussed.

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Host membrane lipids are trafficked to membranes of intravacuolar bacteriumEhrlichia chaffeensis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921619117 ◽

2020 ◽

Vol 117 (14) ◽

pp. 8032-8043 ◽

Cited By ~ 4

Author(s):

Mingqun Lin ◽

Giovanna Grandinetti ◽

Lisa M. Hartnell ◽

Donald Bliss ◽

Sriram Subramaniam ◽

...

Keyword(s):

Host Cell ◽

Cell Membranes ◽

Membrane Lipids ◽

Ion Beam ◽

Multivesicular Bodies ◽

Bacterial Membranes ◽

Bacterial Proliferation ◽

Early Endosomes ◽

Infected Cells ◽

Host Cell Membranes

Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here thatE. chaffeensisis dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation.E. chaffeensiscannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3′-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked toEhrlichiainclusion and bacterial membranes, but DiI-prelabeledEhrlichiamembranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes toEhrlichiainclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes toEhrlichia. In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by theE. chaffeensistype IV secretion system effector Etf-1, which traffic to and fuse withEhrlichiainclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion thatEhrlichiainclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

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Exon 3 of the Human Cytomegalovirus Major Immediate-Early Region Is Required for Efficient Viral Gene Expression and for Cellular Cyclin Modulation

Journal of Virology ◽

10.1128/jvi.79.12.7438-7452.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7438-7452 ◽

Cited By ~ 14

Author(s):

Elizabeth A. White ◽

Deborah H. Spector

Keyword(s):

Human Cytomegalovirus ◽

Cyclin B1 ◽

Mutant Virus ◽

Immediate Early ◽

Wild Type Virus ◽

Viral Gene ◽

Protein Accumulation ◽

Wild Type ◽

Altered Expression ◽

Infected Cells

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early (IE) proteins share an 85-amino-acid N-terminal domain specified by exons 2 and 3 of the major IE region, UL122-123. We have constructed IE Δ30-77, a recombinant virus that lacks the majority of IE exon 3 and consequently expresses smaller forms of both IE1 72- and IE2 86-kDa proteins. The mutant virus is viable but growth impaired at both high and low multiplicities of infection and exhibits a kinetic defect that is not rescued by growth in fibroblasts expressing IE1 72-kDa protein. The kinetics of mutant IE2 protein accumulation in IE Δ30-77 virus-infected cells are approximately normal compared to wild-type virus-infected cells, but the IE Δ30-77 virus is delayed in expression of early viral genes, including UL112-113 and UL44, and does not sustain expression of mutant IE1 protein as the infection progresses. Additionally, cells infected with IE Δ30-77 exhibit altered expression of cellular proteins compared to wild-type HCMV-infected cells. PML is not dispersed but is retained at ND10 sites following infection with IE Δ30-77 mutant virus. While the deletion mutant retains the ability to mediate the stabilization of cyclin B1, cdc6, and geminin in infected cells, its capacity to upregulate the expression of cyclin E has been reduced. These data indicate that the activity of one or both of the HCMV major IE proteins is required in vivo for the modulation of cell cycle proteins observed in cells infected with wild-type HCMV.

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The Extended Intermediate of the SARS-CoV-2 Spike Protein uses Extreme Reach and Flexibility to Capture Host Cell Membranes

Biophysical Journal ◽

10.1016/j.bpj.2020.11.2027 ◽

2021 ◽

Vol 120 (3) ◽

pp. 321a

Author(s):

Rui Su ◽

Jin Zeng ◽

Sathish Thiyagarajan ◽

Ben O'Shaughnessy

Keyword(s):

Host Cell ◽

Cell Membranes ◽

Spike Protein ◽

Host Cell Membranes

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Involvement of the N-Terminal Deubiquitinating Protease Domain of Human Cytomegalovirus UL48 Tegument Protein in Autoubiquitination, Virion Stability, and Virus Entry

Journal of Virology ◽

10.1128/jvi.02766-15 ◽

2016 ◽

Vol 90 (6) ◽

pp. 3229-3242 ◽

Cited By ~ 11

Author(s):

Young-Eui Kim ◽

Se Eun Oh ◽

Ki Mun Kwon ◽

Chan Hee Lee ◽

Jin-Hyun Ahn

Keyword(s):

Human Cytomegalovirus ◽

Virus Entry ◽

Terminal Region ◽

Mutant Virus ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Viral Growth ◽

Internal Region ◽

Deubiquitinating Protease

ABSTRACTHuman cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360–1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360–1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation intrans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360–1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.IMPORTANCEHCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.

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Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Journal of Virology ◽

10.1128/jvi.02256-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2597-2609 ◽

Cited By ~ 38

Author(s):

Brent J. Ryckman ◽

Marie C. Chase ◽

David C. Johnson

Keyword(s):

Endothelial Cells ◽

Human Cytomegalovirus ◽

Null Mutant ◽

Virus Particles ◽

Extracellular Virus ◽

Biochemical Studies ◽

Infected Cells ◽

Low Passage ◽

Er Export ◽

Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.

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