scholarly journals Role of Envelope Glycoprotein Complexes in Cell-Associated Spread of Human Cytomegalovirus

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 614
Author(s):  
Nina Weiler ◽  
Caroline Paal ◽  
Kerstin Adams ◽  
Christopher Calcaterra ◽  
Dina Fischer ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Infectious Virus ◽  
Viral Envelope ◽  
Accessory Proteins ◽  
Viral Growth ◽  
Cell Association ◽  
Quantitative Immunoblotting ◽  
Model Virus

The role of viral envelope glycoproteins, particularly the accessory proteins of trimeric and pentameric gH/gL-complexes, in cell-associated spread of human cytomegalovirus (HCMV) is unclear. We aimed to investigate their contribution in the context of HCMV variants that grow in a strictly cell-associated manner. In the genome of Merlin pAL1502, the glycoproteins gB, gH, gL, gM, and gN were deleted by introducing stop codons, and the mutants were analyzed for viral growth. Merlin and recent HCMV isolates were compared by quantitative immunoblotting for expression of accessory proteins of the trimeric and pentameric gH/gL-complexes, gO and pUL128. Isolates were treated with siRNAs against gO and pUL128 and analyzed regarding focal growth and release of infectious virus. All five tested glycoproteins were essential for growth of Merlin pAL1502. Compared with this model virus, higher gO levels were measured in recent isolates of HCMV, and its knockdown decreased viral growth. Knockdown of pUL128 abrogated the strict cell-association and led to release of infectivity, which allowed cell-free transfer to epithelial cells where the virus grew again strictly cell-associated. We conclude that both trimer and pentamer contribute to cell-associated spread of recent clinical HCMV isolates and downregulation of pentamer can release infectious virus into the supernatant.

Cloning and epitope mapping of a functional partial fusion receptor for human cytomegalovirus gH

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Brenda R. Baldwin ◽  
Chen-Ou Zhang ◽  
Susan Keay
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Envelope ◽  
Nuclear Localization Signals ◽  
Synthetic Receptor ◽  
Hydrophilic Region ◽  
Receptor Cdna ◽  
Cell Membrane Protein ◽  
Membrane Spanning ◽  
Fusion Receptor

A cDNA clone encoding a partial putative human cytomegalovirus (HCMV) gH fusion receptor (CMVFR) was previously identified. In this report, the cDNA sequence of CMVFR was determined and the role of this CMVFR in HCMV/cell fusion was confirmed by rendering fusion-incompetent MOLT-4 cells susceptible to fusion following transfection with receptor cDNA. Blocking experiments using recombinant gH or either of two MAbs (against recombinant gH or purified viral gH:gL) provided additional evidence for the role of gH binding to this protein in virus fusion. An HCMV-binding domain of 12 aa in the middle hydrophilic region of CMVFR was identified by fusion blocking studies using synthetic receptor peptides. The 1368 bp cDNA of CMVFR contained a predicted ORF of 345 aa with two potential membrane-spanning domains and several possible nuclear localization signals. A search of sequence databases indicated that CMVFR is a novel protein. Further characterization of this cell membrane protein that confers susceptibility to fusion with the viral envelope should provide important information about the mechanism by which HCMV infects cells.

scholarly journals Human Cytomegalovirus Tropism Modulator UL148 Interacts with SEL1L, a Cellular Factor That Governs Endoplasmic Reticulum-Associated Degradation of the Viral Envelope Glycoprotein gO

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Christopher C. Nguyen ◽  
Mohammed N. A. Siddiquey ◽  
Hongbo Zhang ◽  
Gang Li ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
Host Cells ◽  
Cell Tropism ◽  
Viral Glycoprotein ◽  
Viral Envelope Glycoprotein ◽  
Endoplasmic Reticulum Associated Degradation ◽  
Viral Glycoproteins

ABSTRACTUL148 is a viral endoplasmic reticulum (ER)-resident glycoprotein that contributes to human cytomegalovirus (HCMV) cell tropism. The influence of UL148 on tropism correlates with its potential to promote the expression of glycoprotein O (gO), a viral envelope glycoprotein that participates in a heterotrimeric complex with glycoproteins H and L that is required for infectivity. In an effort to gain insight into the mechanism, we used mass spectrometry to identify proteins that coimmunoprecipitate from infected cells with UL148. This approach led us to identify an interaction between UL148 and SEL1L, a factor that plays key roles in ER-associated degradation (ERAD). In pulse-chase experiments, gO was less stable in cells infected withUL148-null mutant HCMV than during wild-type infection, suggesting a potential functional relevance for the interaction with SEL1L. To investigate whether UL148 regulates gO abundance by influencing ERAD, small interfering RNA (siRNA) silencing of either SEL1L or its partner, Hrd1, was carried out in the context of infection. Knockdown of these ERAD factors strongly enhanced levels of gO but not other viral glycoproteins, and the effect was amplified in the presence of UL148. Furthermore, pharmacological inhibition of ERAD showed similar results. Silencing of SEL1L during infection also stabilized an interaction of gO with the ER lectin OS-9, which likewise suggests that gO is an ERAD substrate. Taken together, our results identify an intriguing interaction of UL148 with the ERAD machinery and demonstrate that gO behaves as a constitutive ERAD substrate during infection. These findings have implications for understanding the regulation of HCMV cell tropism.IMPORTANCEViral glycoproteins in large part determine the cell types that an enveloped virus can infect and hence play crucial roles in transmission and pathogenesis. The glycoprotein H/L heterodimer (gH/gL) is part of the conserved membrane fusion machinery that all herpesviruses use to enter cells. In human cytomegalovirus (HCMV), gH/gL participates in alternative complexes in virions, one of which is a trimer of gH/gL with glycoprotein O (gO). Here, we show that gO is constitutively degraded during infection by the endoplasmic reticulum-associated degradation (ERAD) pathway and that UL148, a viral factor that regulates HCMV cell tropism, interacts with the ERAD machinery and slows gO decay. Since gO is required for cell-free virus to enter new host cells but dispensable for cell-associated spread that resists antibody neutralization, our findings imply that the posttranslational instability of a viral glycoprotein provides a basis for viral mechanisms to modulate tropism and spread.

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

mBio ◽  
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.

scholarly journals Multimerization of Tegument Protein pp28 within the Assembly Compartment Is Required for Cytoplasmic Envelopment of Human Cytomegalovirus

2008 ◽  
Vol 82 (13) ◽  
pp. 6272-6287 ◽  
Author(s):  
Jun-Young Seo ◽  
William J. Britt
Keyword(s):  
Human Cytomegalovirus ◽  
Potential Role ◽  
Infectious Virus ◽  
The Self ◽  
Tegument Protein ◽  
Recombinant Viruses ◽  
Virus Recombinant ◽  
Protein Amino Acids ◽  
Assembly Compartment

ABSTRACT Human cytomegalovirus (HCMV) UL99-encoded pp28 is an essential tegument protein required for envelopment and production of infectious virus. Nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with the UL99 gene deleted. Previous results have suggested that a key function of pp28 in the envelopment of infectious HCMV is expressed after the protein localizes in the assembly compartment (AC). In this study, we investigated the potential role of pp28 multimerization in the envelopment of the infectious virion. Our results indicated that pp28 multimerized during viral infection and that interacting domains responsible for self-interaction were localized in the amino terminus of the protein (amino acids [aa] 1 to 43). The results from transient-expression and/or infection assays indicated that the self-interaction took place in the AC. A mutant pp28 molecule containing only the first 35 aa failed to accumulate in the AC, did not interact with pp28 in the AC, and could not support virus replication. In contrast, the first 50 aa of pp28 was sufficient for the self-interaction within the AC and the assembly of infectious virus. Recombinant viruses encoding an in-frame deletion of aa 26 to 33 of pp28 were replication competent, whereas infectious virus was not recovered from HCMV BACs lacking aa 26 to 43. These findings suggested that the accumulation of pp28 was a prerequisite for multimerization of pp28 within the AC and that pp28 multimerization in the AC represented an essential step in the envelopment and production of infectious virions.

scholarly journals Role of Noncovalent SUMO Binding by the Human Cytomegalovirus IE2 Transactivator in Lytic Growth

2010 ◽  
Vol 84 (16) ◽  
pp. 8111-8123 ◽  
Author(s):  
Eui Tae Kim ◽  
Young-Eui Kim ◽  
Yong Ho Huh ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Human Fibroblasts ◽  
Growth Curves ◽  
Viral Gene Expression ◽  
Lytic Cycle ◽  
Viral Gene ◽  
Dependent Manner ◽  
Viral Growth ◽  
Transactivation Activity

ABSTRACT The 86-kDa immediate-early 2 (IE2) protein of human cytomegalovirus (HCMV) is a promiscuous transactivator essential for viral gene expression. IE2 is covalently modified by SUMO at two lysine residues (K175 and K180) and also interacts noncovalently with SUMO. Although SUMOylation of IE2 has been shown to enhance its transactivation activity, the role of SUMO binding is not clear. Here we showed that SUMO binding by IE2 is necessary for its efficient transactivation function and for viral growth. IE2 bound physically to SUMO-1 through a SUMO-interacting motif (SIM). Mutations in SIM (mSIM) or in both SUMOylation sites and SIM (KR/mSIM), significantly reduced IE2 transactivation effects on viral early promoters. The replication of IE2 SIM mutant viruses (mSIM or KR/mSIM) was severely depressed in normal human fibroblasts. Analysis of viral growth curves revealed that the replication defect of the mSIM virus correlated with low-level accumulation of SUMO-modified IE2 and of viral early and late proteins. Importantly, both the formation of viral transcription domains and the association of IE2 with viral promoters in infected cells were significantly reduced in IE2 SIM mutant virus infection. Furthermore, IE2 was found to interact with the SUMO-modified form of TATA-binding protein (TBP)-associated factor 12 (TAF12), a component of the TFIID complex, in a SIM-dependent manner, and this interaction enhanced the transactivation activity of IE2. Our data demonstrate that the interaction of IE2 with SUMO-modified proteins plays an important role for the progression of the HCMV lytic cycle, and they suggest a novel viral mechanism utilizing the cellular SUMO system.

scholarly journals Mutational Analysis of the Candidate Internal Fusion Peptide of the Avian Leukosis and Sarcoma Virus Subgroup A Envelope Glycoprotein

1998 ◽  
Vol 72 (4) ◽  
pp. 3259-3267 ◽  
Author(s):  
Lorraine D. Hernandez ◽  
Judith M. White
Keyword(s):  
Envelope Glycoprotein ◽  
Mutational Analysis ◽  
Fusion Peptide ◽  
Viral Envelope ◽  
Hydrophobic Residues ◽  
Virus Particles ◽  
Sarcoma Virus ◽  
A Cell ◽  
Hydrophobic Amino Acids

ABSTRACT The transmembrane subunit (TM) of the avian leukosis and sarcoma virus (ALSV) envelope glycoprotein (Env) contains a stretch of conserved hydrophobic amino acids internal to its amino terminus (residues 21 to 42). By analogy with similar sequences in other viral envelope glycoproteins, this region has been proposed to be a fusion peptide. We investigated the role of this region by changing each of three hydrophobic residues (Ile-21, Val-30, and Ile-39) to glutamatic acid and lysine in the ALSV subgroup A Env. Like wild-type (wt) Env, all six mutant Env proteins were proteolytically processed, oligomerized, and expressed at the cell surface in a form that bound Tva, the ALSV subgroup A receptor. Like wt Env, Ile21Glu, Ile21Lys, Val30Glu, and Val30Lys changed conformation upon binding Tva, as assayed by sensitivity to thermolysin. Ile39Glu and Ile39Lys were cleaved by thermolysin in both the absence and presence of Tva. Although incorporated into virus particles at approximately equal levels, all mutant Envs were compromised in their ability to support infection. The mutants at residues 21 and 30 showed levels of infection 2 to 3 orders of magnitude lower than that of wt Env. The mutants at residue 39 were noninfectious. Furthermore, none of the mutants displayed activity in a cell-cell fusion assay. Our results support the contention that residues 21 to 42 of ALSV subgroup A Env constitute its fusion peptide.

scholarly journals Human cytomegalovirus tropism modulator UL148 interacts with SEL1L, a cellular factor that governs ER-associated degradation of the viral envelope glycoprotein, gO

2018 ◽  
Author(s):  
Christopher C. Nguyen ◽  
Mohammed N. Siddiquey ◽  
Hongbo Zhang ◽  
Jeremy P. Kamil
Keyword(s):  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
Cell Tropism ◽  
Viral Envelope Glycoprotein ◽  
Viral Glycoproteins ◽  
Infected Cells ◽  
Sirna Silencing ◽  
Functional Relevance ◽  
Type Infection

ABSTRACTUL148 is a viral endoplasmic reticulum (ER)-resident glycoprotein that contributes to human cytomegalovirus (HCMV) cell tropism. The influence of UL148 on tropism correlates with its potential to promote the expression of glycoprotein O (gO), a viral envelope glycoprotein that participates in a heterotrimeric complex with glycoproteins H and L that is required for infectivity. In an effort to gain insight into mechanism, we used mass spectrometry to identify proteins that co-immunoprecipitate from infected cells with UL148. This approach led us to identify an interaction between UL148 and SEL1L, a factor that plays key roles in ER-associated degradation (ERAD). In pulse-chase experiments, gO was less stable in cells infected with aUL148-null mutant HCMV than during wild-type infection, suggesting a potential functional relevance for the interaction with SEL1L. To investigate whether UL148 regulates gO abundance by influencing ERAD, siRNA silencing of either SEL1L or its partner, Hrd1, was carried out in the context of infection. Knockdown of these ERAD factors strongly enhanced levels of gO, but not other viral glycoproteins, and the effect was amplified in the presence of UL148. Furthermore, pharmacological inhibition of ERAD showed similar results. Silencing of SEL1L during infection also stabilized an interaction of gO with the ER lectin OS-9, which likewise suggests that gO is an ERAD substrate. Taken together, our results identify an intriguing interaction of UL148 with the ERAD machinery, and demonstrate that gO behaves as a constitutive ERAD substrate during infection. These findings have implications for understanding the regulation of HCMV cell tropism.

scholarly journals Viral Regulation of Cell Tropism in Human Cytomegalovirus

2015 ◽  
Vol 90 (2) ◽  
pp. 626-629 ◽  
Author(s):  
Gang Li ◽  
Jeremy P. Kamil
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Cytomegalovirus ◽  
Envelope Glycoprotein ◽  
Cell Types ◽  
Viral Envelope ◽  
Cell Entry ◽  
Cell Tropism ◽  
Enveloped Viruses ◽  
Viral Envelope Glycoprotein ◽  
Viral Glycoproteins

The viral glycoproteins that decorate enveloped viruses play crucial roles in cell entry and in large part dictate the spectrum of cell types that a virus can infect. The identification in human cytomegalovirus (HCMV) of a viral endoplasmic reticulum (ER)-resident glycoprotein that regulates the composition of alternative viral envelope glycoprotein complexes raises the intriguing possibility that certain viruses might actively regulate the tropism of progeny virions to improve their fitness or to navigate through the host.

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