Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

Vanarsdall, Adam L.; Howard, Paul W.; Wisner, Todd W.; Johnson, David C.

doi:10.1371/journal.ppat.1005564

Cited by 72 publications

(83 citation statements)

References 46 publications

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“…Previous work has shown that HCMV gB and/or gH interacts with THY-1 (29) and integrins (30,104). In addition, a gH/gL/gB complex is present in HCMV particles (10). Therefore, THY-1 and integrins might cooperatively mediate HCMV internalization by bridging virus attachment with macropinocytosis.…”

Section: Discussionmentioning

confidence: 98%

“…The pentamer and trimer glycoprotein complexes interact with specific cellular receptors in a cell typedependent manner (8), which has been postulated to lead to the recruitment of gB and to trigger membrane fusion (9). However, a recent study showed that HCMV gH/gL and gB form stable gBgH/gL complexes that are incorporated into virions prior to receptor binding or membrane fusion and that only small amounts of gH/gL/gO and gH/gL/UL128-131A complexes are associated with gB (10).…”

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confidence: 99%

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Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

Fischer

Cohen

2016

J Virol

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Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of phosphatidylinositol 3-kinase (PI3K)/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt dependent, and is a clathrin-independent endocytic process, we determined whether THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1-dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. 5-(N-Ethyl-Nisopropyl)amiloride (EIPA) and soluble THY-1 blocked HCMV infection in the cell lines by >80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na ؉ /H ؉ exchange, PI3K, and Pak1 kinase, which are critical for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin as determined by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1-associated virus entry. These results suggest that HCMV has adapted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles, to facilitate efficient entry into certain cell types by a macropinocytosis-like process. IMPORTANCE Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects.The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells is controversial. In this study, we show that THY-1, a cell surface protein that is critical for the early stage of entry of HCMV into certain cell types, contributes to virus entry by macropinocytosis. Our findings suggest that HCMV has adapted to utilize THY-1 to facilitate entry of HCMV into macropinosomes in certain cell types. Further knowledge about the mechanism of HCMV entry into cells may facilitate the development of novel inhibitors of virus infection. Human cytomegalovirus (HCMV) infects over half of the general population. While the infection is often asymptomatic when occurring in young children, it poses a serious health threat in transplant recipients and in immunocompromised individuals. HCMV is the most common infectious cause of birth defects. The virus infects a variety of host cells in vivo, including epithelial, endothelial, and muscle cells as well as fibroblasts. HCMV encodes multiple glycoproteins, some of which are not shared with other human herpesviruses, such as UL128, UL130, and UL131A. Other glycoproteins ar...

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Section: Discussionmentioning

confidence: 98%

mentioning

confidence: 99%

Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

Fischer

Cohen

2016

J Virol

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“…Recently, the HCMV gH/gL complex has been shown to form a stable complex with the fusion protein gB in virions including up to 50 % of total gH/gL, suggesting that gH/gL interacts with gB to mediate membrane fusion [65].…”

Section: Genetic Determinants Of Endothelial Cells and Leukocyte Tropismmentioning

confidence: 99%

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

Gerna

Revello

Baldanti

et al. 2017

Journal of General Virology

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Between the 1980s and 1990s, three assays were developed for diagnosis of human cytomegalovirus (HCMV) infections: leuko (L)-antigenemia, L-viremia and L-DNAemia, detecting viral protein pp65, infectious virus and viral DNA, respectively, in circulating leukocytes Repeated initial attempts to reproduce the three assays in vitro using laboratory-adapted strains and infected cell cultures were consistently unsuccessful. Results were totally reversed when wild-type HCMV strains were used to infect either fibroblasts or endothelial cells. Careful analysis and sequencing of plaque-purified viruses from recent clinical isolates drew attention to the ULb¢ region of the HCMV genome. Using bacterial artificial chromosome technology, it was shown by both gain-of-function and loss-of-function experiments that UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes. In addition, a number of clinical isolates passaged in human fibroblasts had lost both properties (leuko-tropism and endothelial cell-tropism) when displaying a mutation in the UL131-128 locus

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“…Three of the viral glycoproteins are essential for fusion of herpesvirus envelopes with host cell membranes: the fusion protein gB and the glycoproteins gH and gL, presumably functioning as a trigger of gB fusion activity (7)(8)(9). In support of this model, Varnarsdall et al demonstrated recently that HCMV gH/gL and gB interact within the virus envelope (10). In contrast to gH/gL of herpes simplex virus (HSV) or Kaposi's sarcoma-associated herpesvirus (KSHV), HCMV gH/gL is stably bound to accessory proteins which are crucial for infection of different host cells.…”

mentioning

confidence: 94%

Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex

Stegmann¹,

Abdellatif

Sampaio³

et al. 2017

J Virol

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The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection.IMPORTANCE Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational approach we analyzed these amino acids to elucidate their role in the function of gO. All conserved amino acids contributed either to formation of the trimeric complex or to cell-free infection. Notably, these two phenotypes were not inevitably linked as the mutation of a charged cluster in the center of gO abrogated cell-free infection while trimeric complexes were still being formed. Cysteine 343 was essential for covalent binding of gO to gH/gL; however, noncovalent complex formation in the absence of cysteine 343 also allowed for cell-free infectivity.

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Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

Cited by 72 publications

References 46 publications

Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

The pentameric complex of human Cytomegalovirus: cell tropism, virus dissemination, immune response and vaccine development

Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex

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