Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Ciferri, Claudio; Chandramouli, Sumana; Donnarumma, Danilo; Nikitin, Pavel A.; Cianfrocco, Michael A.; Gerrein, Rachel; Feire, Adam L.; Barnett, Susan W.; Lilja, Anders; Rappuoli, Rino; Norais, Nathalie; Settembre, Ethan C.; Carfı́, Andrea

doi:10.1073/pnas.1424818112

Cited by 141 publications

(216 citation statements)

References 46 publications

Supporting

Mentioning

189

Contrasting

Order By: Relevance

“…7A). The formation of disulfide-linked monomers and homodimers of soluble gH/gL is consistent with what has been described previously (35,54,71). The Strep-tag at the C terminus of UL116 allowed us to isolate gH/UL116-containing complexes from cell supernatants.…”

Section: Resultssupporting

confidence: 87%

“…The gL subunit is not only disulfide linked to gH but also is engaged in disulfide bond formation with gO, in the gH/gL/gO complex, or with UL128 in the pentamer (54). Although the absence of gL from the gH/UL116 dimer suggested that neither gO nor UL128 can associate with this heterodimer, we sought to verify this hypothesis.…”

Section: Resultsmentioning

confidence: 99%

“…The medium then was applied to StrepTrap HP 1-ml columns (GE Life Sciences), and the complex was eluted with the same buffer containing 2.5 mM desthiobiotin. The gH/gL complex harboring the gL-C144A mutation was purified from supernatants of HEK293 cells doubly transfected with plasmids encoding gH-His and untagged gL-C144A as previously described (54). Purified gH/gL-C144A or gH/UL116 was mixed at 1:1.2 with either 3G16 or MSL-109 Fabs.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL

Caló

Cortese

Ciferri³

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied the UL116 gene product, a putative open reading frame identified by in silico analysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of the UL116 gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex. IMPORTANCE HCMV infection can cause severe disease in immunocompromised adults and infants infected in utero.The dissection of the HCMV entry machinery is important to understand the mechanism of viral infection and to identify new vaccine antigens. The gH/gL/gO and gH/gL/UL128/UL130/UL131 (Pentamer) complexes play a key role in HCMV cell entry and tropism. Both complexes are formed by an invariant gH/gL scaffold on which the other subunits assemble. Here, we show that the UL116 gene product is expressed in infected cells and forms a heterodimer with gH. The gH/UL116 complex is carried on the infectious virions, although in smaller amounts than gH/gL complexes. No gH/UL116/gL ternary complex formed in transfected cells, suggesting that the gH/UL116 complex is independent from gL. This new gH-based gL-free complex represents a potential target for a protective HCMV vaccine and opens new perspectives on the comprehension of the HCMV cell entry mechanism and tropism. H uman cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus infecting 40 to 60% of the human population (1).HCMV infection usually is mild or asymptomatic in immunocompetent individuals, while it can cause severe disease in immunocompromised adults. Infants infected in utero can suffer from disseminated HCMV disease and impaired neurological development (2). Transplant patients do not adequately control HCMV infection or reactivation, often resulting in graft rejection and death (3). Preventing primary HCMV infection could significantly decrease the frequency of organ rejection in seronegative recip...

show abstract

Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL

Caló

Cortese

Ciferri³

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…In more detail, gH/gL may be complexed either with pUL128L, giving rise to the PC gH/gL/ pUL128L, or UL74-encoded gO, thus forming the trimeric complex gH/gL/gO, which binds to platelet-derived growth factor receptor a and mediates HCMV entry into HELFs [57][58][59]. Thus, gH/gL/gO and PC were considered to represent two mutually exclusive cell entry complexes, as suggested by mass spectrometry and mutagenesis analysis [60]. However, gO and the UL128-131 gene products were reported to compete for binding of gH/gL, thus influencing the ratio of gH/gL/ gO to gH/gL/UL128-131 within virions as well as the HCMV cell tropism [61,62].…”

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

View full text Add to dashboard Cite

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

show abstract

“…Several lines of evidence indicate that virion cell tropism is forged within the ER. gO and UL128-131 compete within the ER for assembly onto gH/gL; exemplifying this competition, a single cysteine position gL has been identified to form a mutually exclusive disulfide bond with cysteines from either gO or UL128 (Ciferri et al 2015). Interestingly, UL116, a glycoprotein encoded by the gene neighboring gL (UL115), was recently shown to form a complex with gH that lacks gL, gH/UL116, in which UL116 essentially substitutes for gL (Calo et al 2016).…”

Section: Regulation Of Viral Tropismmentioning

confidence: 99%

Recent advances in CMV tropism, latency, and diagnosis during aging

et al. 2017

View full text Add to dashboard Cite

Human cytomegalovirus (CMV) is one of the largest viruses known to cause human diseases. Chronic CMV infection, as defined by anti-CMV IgG serology, increases with age and is highly prevalent in older adults. It has complex biology with significant immunologic and health consequences. This article aims to summarize research findings presented at the 6th International Workshop on CMV and Immunosenescence that relate to advances in the areas of CMV tropism, latency, CMV manipulation of cell metabolism, and T cell memory inflation, as well as novel diagnostic evaluation and translational research of chronic CMV infection in older adults. Information summarized here represents the current state of knowledge in these important fields. Investigators have also identified a number of areas that deserve further and more in-depth investigation, including building more precise parallels between mouse CMV (mCMV) and human CMV (HCMV) research. It is hoped that this article will also stimulate engaging discussion on strategies and direction to advance the science to the next level.

show abstract

Structural and biochemical studies of HCMV gH/gL/gO and Pentamer reveal mutually exclusive cell entry complexes

Cited by 141 publications

References 46 publications

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL

The Human Cytomegalovirus UL116 Gene Encodes an Envelope Glycoprotein Forming a Complex with gH Independently from gL

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

Recent advances in CMV tropism, latency, and diagnosis during aging

Contact Info

Product

Resources

About