Entry of herpesviruses into mammalian cells

147

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We report observations of stochastic collisions of murine cytomegalovirus (MCMV) on ultramicroelectrodes (UMEs), extending the observation of discrete collision events on UMEs to biologically relevant analytes. Adsorption of an antibody specific for a virion surface glycoprotein allowed differentiation of MCMV from MCMV bound by antibody from the collision frequency decrease and current magnitudes in the electrochemical collision experiments, which shows the efficacy of the method to size viral samples. To add selectivity to the technique, interactions between MCMV, a glycoprotein-specific primary antibody to MCMV, and polystyrene bead "anchors," which were functionalized with a secondary antibody specific to the Fc region of the primary antibody, were used to affect virus mobility. Bead aggregation was observed, and the extent of aggregation was measured using the electrochemical collision technique. Scanning electron microscopy and optical microscopy further supported aggregate shape and extent of aggregation with and without MCMV. This work extends the field of collisions to biologically relevant antigens and provides a novel foundation upon which qualitative sensor technology might be built for selective detection of viruses and other biologically relevant analytes.collisions | cytomegalovirus | electrochemistry | murine cytomegalovirus | virus O ver the past decade, the study of discrete collision events on ultramicroelectrodes (UMEs) has gained attention due to the interest in understanding stochastic phenomena by electrochemistry. By observing the collisions of small particles, there is the possibility that information can be deduced that is not available in ensemble measurements. The electrochemical study of single collision events has been applied to a wide range of hard nanoparticles (NPs), which include metal, metal oxide, and organic NPs [platinum (1), silver (2), gold (3), nickel (4), copper (5), iridium oxide (6), cerium oxide (7), titanium oxide (8), silicon oxide (9), indigo (10), polystyrene (11), and relatively large aggregates of fullerene (12)]. Recently, collisions of soft particles have been investigated, such as toluene droplets (13) and liposomes (14). Also, collisions of toluene and tri-n-propylamine droplets were observed simultaneously by both electrochemical and electrogenerated chemiluminescent (ECL) measurements (15).Similarly, a variety of techniques have been developed to observe these collision events. The interested reader can consult the references for a discussion on each of the techniques used to observe stochastic events electrochemically: blocking (9, 13), electrocatalytic amplification (1), open circuit potential (16), droplet blocking/reactor (13,14), and ECL (15,17,18). The simplest and most reproducible method of observing collisions is a technique termed blocking, which is so named because particles, which are brought to the electrode by a diffusion-limited flux and/or electrophoretic migration, irreversibly adsorb (1) to the electrode surface, blocking the flux of red...

Section: Electrochemical Detection Of Discrete CMV Collisionsmentioning

confidence: 99%

Electrochemical detection of a single cytomegalovirus at an ultramicroelectrode and its antibody anchoring

Dick

Hilterbrand

Boika

et al. 2015

147

132

“…Similar to other herpesviruses, the envelope glycoproteins gB and gH/gL form the conserved fusion machinery required for viral entry (5,6). Recent structural and mutagenesis analysis suggested that gB is responsible for mediating virus and host membrane fusion during viral entry (7,8).…”

mentioning

confidence: 99%

“…It has been proposed that gH/gL is involved in the entry process through activation of gB (12). In addition to gB and gH/gL, most herpesviruses encode additional glycoproteins that are able to interact with gH/gL and are capable of either mediating binding to specific cellular receptors or regulating the activity of the gH/gL-gB complex (5,6).…”

mentioning

confidence: 99%

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

Ciferri

Chandramouli

Donnarumma

et al. 2015

138

189

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading viral cause of birth defects after congenital infection. The glycoprotein complexes gH/gL/gO and gH/gL/UL128/UL130/UL131A (Pentamer) are key targets of the human humoral response against HCMV and are required for HCMV entry into fibroblasts and endothelial/epithelial cells, respectively. We expressed and characterized soluble forms of gH/gL, gH/gL/gO, and Pentamer. Mass spectrometry and mutagenesis analysis revealed that gL-Cys144 forms disulfide bonds with gO-Cys351 in gH/gL/gO and with UL128-Cys162 in the Pentamer. Notably, Pentamer harboring the UL128-Cys162Ser/gL-Cys144Ser mutations had impaired syncytia formation and reduced interference of HCMV entry into epithelial cells. Electron microscopy analysis showed that HCMV gH/gL resembles HSV gH/gL and that gO and UL128/UL130/UL131A bind to the same site at the gH/gL N terminus. These data are consistent with gH/gL/gO and Pentamer forming mutually exclusive cell entry complexes and reveal the overall location of gH/gL-, gH/gL/ gO-, and Pentamer-specific neutralizing antibody binding sites. Our results provide, to our knowledge, the first structural view of gH/gL/ gO and Pentamer supporting the development of vaccines and antibody therapeutics against HCMV.human cytomegalovirus | HCMV | Pentamer complex | gH/gL/gO | virus entry

“…herpes simplex virus | glycoprotein | gH | gL | virus entry E ntry of herpesviruses into the cell is an orchestrated process that necessitates a multipartite glycoprotein system, rather than onetwo glycoproteins, as is the case for the vast majority of viruses (1)(2)(3)(4). For herpes simplex virus (HSV), the entry-fusion apparatus consists of four essential glycoproteins -gD, the heterodimer gH/gL, and gB-plus cognate cellular receptors.…”

mentioning

confidence: 99%

Dissociation of HSV gL from gH by αvβ6- or αvβ8-integrin promotes gH activation and virus entry

Gianni

Massaro

Campadelli-Fiume

2015

Herpes simplex virus (HSV) is an important human pathogen. It enters cells through an orchestrated process that requires four essential glycoproteins, gD, gH/gL, and gB, activated in cascade fashion by receptor-binding and signaling. gH/gL heterodimer is conserved across the Herpesviridae family. HSV entry is enabled by gH/gL interaction with αvβ6-or αvβ8-integrin receptors. We report that the interaction of virion gH/gL with integrins resulted in gL dissociation and its release in the medium. gL dissociation occurred if all components of the entry apparatus-receptor-bound gD and gB-were present and was prevented if entry was blocked by a neutralizing monoclonal antibody to gH or by a mutation in gH. We propose that (i) gL dissociation from gH/gL is part of the activation of HSV glycoproteins, critical for HSV entry; and (ii) gL is a functional inhibitor of gH and maintains gH in an inhibited form until receptor-bound gD and integrins signal to gH/gL.herpes simplex virus | glycoprotein | gH | gL | virus entry E ntry of herpesviruses into the cell is an orchestrated process that necessitates a multipartite glycoprotein system, rather than onetwo glycoproteins, as is the case for the vast majority of viruses (1-4). For herpes simplex virus (HSV), the entry-fusion apparatus consists of four essential glycoproteins -gD, the heterodimer gH/gL, and gB-plus cognate cellular receptors. gD is the major determinant of HSV tropism. Structurally, it exhibits an Ig-folded core with N-and C-terminal extensions (5, 6). The structure of the gH/gL heterodimer does not resemble that of any known protein (7-9). The binding site of gL in gH maps to the N-terminal domain I. Whether gH/gL has a profusion activity in itself or is only an intermediate between gD and gB in the chain of activation of the glycoproteins is an open question (10-13). gB is considered the fusogenic glycoprotein, based on structural features, including a trimeric fold and a bipartite fusion loop (14, 15). gH, gL, and gB constitute the conserved-entry glycoproteins across the Herpesviridae family. The ability of gH to heterodimerize with gL is also conserved across the family, highlighting that the heterodimeric structure is critical to the function of two glycoproteins.The prevailing model of HSV entry envisions that following a first virion attachment to cells mediated by heparan sulfate glycosaminoglycans, the interaction of gD with one of its receptors, nectin1 and HVEM (herpesvirus entry mediator), results in conformational changes to gD, in particular to the ectodomain C terminus, which harbors the profusion domain (5,6,16,17). The activated gD recruits gH/gL, which, in turn, recruits gB. gB executes the virus-cell fusion (2-4, 18, 19). We observed that the glycoproteins are already in complex in resting virions (17,18). In contrast with the view that glycoproteins are stepwise-recruited to a complex, we favor the view that the process of activation of the viral glycoproteins results from the interaction of preassembled glycoproteins' complexes with cellu...